PHYSIOLOGICAL REVIEWS
Vol. 81, No. 1, January 2001
Printed in U.S.A.
Small GTP-Binding Proteins
YOSHIMI TAKAI, TAKUYA SASAKI, AND TAKASHI MATOZAKI
Department of Molecular Biology and Biochemistry, Osaka University Graduate School
of Medicine/Faculty of Medicine, Suita, Japan
http://physrev.physiology.org
0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society
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I. Introduction
II. General Properties
A. Structure
B. A role as molecular switches
C. Localization
III. Ras Proteins as Regulators of Gene Expression
A. Outline
B. Ras protein cycle: activation/inactivation
C. Raf protein kinase activation by Ras proteins
D. Modifiers of the Ras protein-induced Raf protein kinase activation
E. Other effectors of Ras proteins
F. Transport of newly synthesized Ras proteins from the endoplasmic reticulum
to the plasma membrane
G. Ras proteins and cancer
H. Rap proteins
I. Ral proteins
J. Other Ras family members
IV. Rho/Rac/Cdc42 Proteins as Regulators of Both Cytoskeletal Reorganization and Gene Expression
A. Outline
B. Reorganization of the actin cytoskeleton
C. Rho/Rac/Cdc42 protein cycle: cyclical activation/inactivation
D. Mode of action of Rho proteins in cytoskeletal reorganization
E. Mode of action of Rac/Cdc42 proteins in cytoskeletal reorganization
F. Mode of action of Rho/Rac/Cdc42 proteins in gene expression
G. Other functions of Rho/Rac/Cdc42 proteins
V. Rab Proteins as Regulators of Vesicle Trafficking
A. Outline
B. Vesicle trafficking
C. Localization of Rab proteins
D. Rab protein cycle: cyclical activation/inactivation and translocation
E. SNAREs and tethering proteins in vesicle targeting/docking/fusion
F. Mode of action of Rab proteins in vesicle targeting/docking/fusion
G. Rab3A in Ca2⫹-dependent exocytosis
H. Rab proteins and cytoskeleton
I. Rab proteins in vesicle budding
VI. Sar1/Arf Proteins as Regulators of Vesicle Budding
A. Outline
B. Coat proteins and vesicle budding
C. Arf protein cycle: cyclical activation/inactivation and translocation
D. Arf proteins in vesicle budding
E. Arf6 in endocytic recycling and cytoskeletal reorganization
F. Sar1 as a regulator of vesicle budding
VII. Ran Function in Nucleocytoplasmic Transport and Microtubule Organization
A. Outline
B. Nucleocytoplasmic transport
C. Ran cycle: cyclical activation/inactivation and translocation
D. Mode of action of Ran in nucleocytoplasmic transport
E. A role for Ran in microtubule organization
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VIII. Small G Protein Cascades and Cross-talks
A. Small G protein cascades
B. Cross-talk between small G proteins
IX. Conclusions and Perspectives
A. Roles in two types of cell regulation
B. A role as biotimers rather than as molecular switches
C. A role as spatial determinants
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I. INTRODUCTION
Small GTP-binding proteins (G proteins) are monomeric G proteins with molecular masses of 20 – 40 kDa.
The Ha-Ras and Ki-Ras genes were first discovered as the
v-Ha-Ras and v-Ki-Ras oncogenes of sarcoma viruses
around 1980 (111, 660). Their cellular oncogenes were
then identified in humans, and their mutations were furthermore found in some human carcinomas (146, 252,
499, 561, 626, 661). The mutated forms were subsequently
shown to stimulate proliferation and transformation of
cultured cells (71, 84, 182, 681). Moreover, the mutated
forms were shown to induce cell differentiation in neuronal cells (41, 249, 523). These findings drew the attention
of many scientists not only in the cancer research field but
also in many other fields. Finally, these Ras proteins were
shown to be related to the heterotrimeric G proteins, such
as Gs and Gi, and G proteins involved in protein synthesis,
such as elongation factor Tu (EF-Tu) (222, 644, 659).
The Rho gene was discovered as a homolog of the
Ras gene in Aplysia in 1985 (421); the YPT1 gene, which
had been discovered as an open reading frame between
the actin and tubulin genes in the yeast Saccharomyces
cerevisiae (S. cerevisiae) in 1983 (208), was identified to
encode a small G protein in 1986 (641); Arf protein, which
was purified as a cofactor for the cholera toxin-catalyzed
ADP-ribosylation of Gs in 1984 (326), was identified to
encode a small G protein in 1986 (327). The SEC4 gene,
which had been isolated as a gene involved in secretion in
the yeast in 1980 (527), was identified to encode a small G
protein in 1987 (623). These results suggested the presence of a big family of Ras-like small G proteins. Actually,
many small G proteins were systematically isolated by
molecular biological (100, 101, 105, 551, 573) and biochemical (291, 337, 344, 534, 535, 793, 797) methods.
Now, more than 100 small G proteins have been
identified in eukaryotes from yeast to human, and they
comprise a superfamily (60, 250, 701). The members of
this superfamily are structurally classified into at least five
families: the Ras, Rho, Rab, Sar1/Arf, and Ran families
(Table 1 and Fig. 1). In the yeast S. cerevisiae, sequence
analysis against complete genomic sequence has revealed
that there are 4 Ras family members, 6 Rho family members, 11 Rab family members, 7 Sar1/Arf family members,
and 2 Ran family members (210, 383). The functions of
many small G proteins have recently been elucidated: the
Ras subfamily members (Ras proteins) of the Ras family
mainly regulate gene expression, the Rho/Rac/Cdc42 subfamily members (Rho/Rac/Cdc42 proteins) of the Rho
family regulate both cytoskeletal reorganization and gene
expression, the Rab and Sar1/Arf family members (Rab
and Sar1/Arf proteins) regulate intracellular vesicle trafficking, and the Ran family members (Ran) regulate nucleocytoplasmic transport during the G1, S, and G2 phases
of the cell cycle and microtubule organization during the
M phase. Many upstream regulators and downstream effectors of small G proteins have been identified, and
modes of activation and actions have gradually been elucidated. In this review, functions of small G proteins and
their modes of activation and action are described. However, this review may not cover all detailed information
regarding each small G protein; readers may refer to other
recent excellent reviews (3, 49, 58, 80, 82, 139, 325, 417,
420, 428, 483, 491, 519, 536, 630, 713, 744, 758).
As to nomenclature, the term small GTPases is often
used, but “small G proteins” is used here because small G
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Takai, Yoshimi, Takuya Sasaki, and Takashi Matozaki. Small GTP-Binding Proteins. Physiol Rev 81: 153–208,
2001.—Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the
Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers
(biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation
of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of
specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates
nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of
small G proteins have been isolated, and their modes of activation and action have gradually been elucidated.
Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins
and their modes of activation and action are described.
TABLE
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SMALL G PROTEINS
January 2001
1. The small G protein superfamily
Ras Family
Ha-Ras
Ki-Ras
N-Ras
R-Ras
M-Ras
RalA
RalB
Rap1A
Rap1B
Rap2A
Rap2B
Tc21
Rit
Rin
Rad
Kir/Gem
Yeast
Ras1
Ras2
Rsr1
Yct7
Rheb
␬B-Ras1
␬B-Ras2
RhoA
RhoB
RhoC
RhoD
RhoE/
Rnd3/
Rho8
RhoG
RhoH/
TTF
Rac1
Rac2
Rac3
Cdc42
Rnd1/
Rho6
Rnd2/
Rho7
Tc10
Rho1
Rho2
Rho3
Rho4
Cdc42
Yns0
Rab Family
Rab1A
Rab1B
Rab2
Rab3A
Rab3B
Rab3C
Rab3D
Rab4
Rab5A
Rab5B
Rab5C
Rab6
Rab7
Rab8
Rab9
Rab10
Rab11A
Rab11B
Rab12
Rab13
Rab14
Rab15
Rab16
Rab17
Rab18
Rab19
Rab20
Rab21
Rab22
Rab23
Rab24
Rab25
Ypt1
Sec4
Ypt31/
Ypt8
Ypt32/
Ypt9
Ypt51/
Vps21
Ypt52
Ypt53
Ypt6
Ypt7
Ypt10
Ypt11
Rab26
Rab27A
Rab27B
Rab28
Rab29
Rab30
Rab31
Rab32
Rab33A
Rab33B
Sar1/Arf
Family
Ran
Family
Arf1
Arf2
Arf3
Arf4
Arf5
Arf6
Sar1a
Sar1b
Arl1
Arl2
Arl3
Arl4
Arl5
Arl6
Arl7
Ard1
Ran
Arf1
Arf2
Arf3
Sar1
Arl1
Arl2
Cin4
Gsp1
Gsp2
See References 251a and 810a.
proteins have both GDP/GTP-binding and GTPase activities. In many cases, the GTPase activity is necessary for
the termination of the functions of small G proteins, but
not essential for them to perform their functions. From
this point of view, the term GTPase is misleading. “G
proteins” represent heterotrimeric G proteins and “small
GTP-binding proteins” should be used, but just for simplicity “small G proteins” is used here. G proteins used
here include heterotrimetric G proteins (223), G proteins
involved in protein synthesis (338), and small G proteins.
Guanine nucleotide exchange factor (GEF) is often used,
but guanine nucleotide exchange protein (GEP) is used
here, because all GEFs thus far found are proteins and
“GEPs” is a more correct term.
II. GENERAL PROPERTIES
A. Structure
A comparison of the amino acid sequences of Ras
proteins from various species has revealed that they are
conserved in primary structures and are 30 –55% homologous to each other. Among Ras proteins, each protein
shares relatively high (50 –55%) amino acid identity,
whereas Rab and Rho/Rac/Cdc42 proteins share ⬃30%
amino acid identity with Ras proteins (250, 742). Nevertheless, like other G proteins, all small G proteins have
consensus amino acid sequences responsible for specific
interaction with GDP and GTP and for GTPase activity,
which hydrolyzes bound GTP to GDP and Pi (61, 701, 742)
(Fig. 2A). Moreover, they have a region interacting with
downstream effectors. In addition, small G proteins belonging to Ras, Rho/Rac/Cdc42, and Rab proteins have
sequences at their COOH termini that undergo posttranslational modifications with lipid, such as farnesyl, geranylgeranyl, palmitoyl, and methyl moieties, and proteolysis (89, 228, 427, 701, 811) (Fig. 3). Arf proteins have an
NH2-terminal Gly residue that is modified with myristic
acid (493). Sar1 and Ran do not have such sequences to
direct posttranslational modifications.
Crystallographic and NMR analyses of some small G
proteins, including Ha-Ras, N-Ras, Rap2A, RhoA, Rac1,
Rab3A, Rab7, Arf1, and Ran, have revealed that all GDP/
GTP-binding domains have a common topology (219)
(Fig. 2B). By comparison of the structure of Ha-Ras in the
GTP-bound conformation and the GDP-bound conformation, two highly flexible regions surrounding the ␥-phosphate of GTP have been established (471, 560): the switch
I region within loop L2 and ␤2 (the effector region) and
the switch II region within loop L4 and helix ␣2. A twostate model for the movement of the effector loop in the
GTP-bound form of Ha-Ras has been established; the
flexibility of the loop can conveniently be monitored by a
large shift of Tyr-32 relative to the phosphate groups,
because the hydroxyl group of Tyr-32 forms hydrogen
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Mammal
Rho Family
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1. Dendrogram of the small G protein superfamily. [Modified from Garcia-Ranea and Valencia (210).]
bond with the ␥-phosphate of GTP. Binding of c-Raf-1, an
effector of Ras proteins (see below), stabilizes the effector loop in the active conformation (459).
The COOH-terminal regions are classified into at
least four groups: 1) Cys-A-A-X (A, aliphatic acid; X, any
amino acid); 2) Cys-A-A-Leu/Phe; 3) Cys-X-Cys; and 4)
Cys-Cys (89, 228, 427, 701) (Fig. 3). The Cys-A-A-X struc-
ture is furthermore subclassified into two groups: one has
an additional Cys residue upstream of the Cys residue of
the Cys-A-A-X structure (1a), and the other has a polybasic region (1b). In the case of the Cys-A-A-X structure,
Ha-Ras and Ki-Ras are first farnesylated at the Cys residue
followed by the proteolytic removal of the A-A-X portion
and the carboxylmethylation of the exposed Cys residue
FIG. 2. Structure of small G proteins. A: consensus
amino acid sequences responsible for specific interaction
with GDP and GTP and for GTPase activity. B: crystallographic structure of small G proteins. The crystallographic
structure of Ha-Ras is representatively shown. A, Ala; D,
Asp; E, Glu; G, Gly; K, Lys; N, Asn; S, Ser; X, any amino
acid. [Modified from Pai et al. (560).]
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FIG.
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SMALL G PROTEINS
(90, 201, 247, 256, 313). Ha-Ras has an additional Cys
residue that is further palmitoylated (256). The Cys-A-ALeu structure of Rap1 is first geranylgeranylated followed
by the same modifications (336). Both Cys residues of the
Cys-X-Cys structure of Rab3A are geranylgeranylated, and
the COOH-terminal Cys residue is carboxylmethylated
(177). Both Cys residues of the Cys-Cys structure of Rab1
are geranylgeranylated, but the COOH-terminal Cys residue is not carboxylmethylated (672). The lipid modifications of these small G proteins are necessary for their
binding to membranes and regulators and for their activation of downstream effectors as described below (89,
228, 257, 427, 701, 702, 811).
The farnesyl moiety is derived from farnesyl pyrophosphate, an intermediate product of the mevalonate
pathway which produces cholesterol from mevalonate
(231). Mevalonate is produced from 3-hydroxy-3-methyglutaryl-CoA by the action of 3-hydroxy-3-methyglutarylcoenzyme reductase. A specific inhibitor for this enzyme,
named pravachol, is used as a very effective drug for
arteriosclerosis (171, 231). The geranylgeranyl moiety is
derived from geranylgeranyl pyrophosphate, which is an
intermediate product for the synthesis of dolichol and
ubiquinone (220, 231). The palmitoyl moiety is derived
from palmitoyl CoA. The methyl moiety is derived from
S-adenosyl-methionine. The enzymes that transfer the
prenyl moieties have been isolated and characterized
(811). The farnesylation of the Cys-A-A-X structure is
catalyzed by farnesyltransferase, the geranylgeranylation
of the Cys-A-A-Leu structure is catalyzed by geranylgeranyltrasnferase I, and the prenylation of the Cys-X-Cys and
Cys-Cys structures is catalyzed by geranylgeranyltransferase II. Farnesyltransferase and geranylgeranyltransferase I consist of two subunits, ␣ and ␤ subunits, and the
␣-subunits of both enzymes are identical (648). Geranylgeranyltransferase II consists of three subunits, originally termed component A but recently renamed Rab
escort protein I (Rep1), and ␣- and ␤-subunits (289, 645,
646, 672). Rep1 binds unprenylated Rab proteins, presents
them to the catalytic ␣␤-subunits, and remains bound to
Rab proteins after the geranylgeranyl transfer reaction
(20). In cells, Rab GDP dissociation inhibitor (GDI) (see
below) may dissociate this product from Rep1, allowing
multiple cycles of catalysis. The human Rep1 gene has
been identified by positional cloning as that responsible
for choroideremia, which is an X-linked form of retinal
degeneration (131, 132, 187). Loss of Rep1 activity causes
the reduced prenylation of Ram/Rab27, which is expressed at high levels in the retinal cell layers, and the
degeneration of this protein in the progression of this
disease (647). Palmitoyltransferase that palmitoylates HaRas has been purified (406), but it is not yet known
whether this enzyme is the one that functions in vivo.
Methyltransferases that transfer the methyl moieties to
small G proteins having Cys-A-A-X, Cys-A-A-Leu, and CysX-Cys structures have not been well characterized. A
protease, Rce1, that removes the A-A-X and A-A-Leu portions of Ha-Ras, N-Ras, Ki-Ras, and Rap1B, has recently
been identified (345, 557).
B. A Role as Molecular Switches
According to the structures of small G proteins, they
have two interconvertible forms: GDP-bound inactive and
GTP-bound active forms (60, 250, 701) (Fig. 4). An upstream signal stimulates the dissociation of GDP from the
GDP-bound form, which is followed by the binding of
GTP, eventually leading to the conformational change of
the downstream effector-binding region so that this region interacts with the downstream effector(s). This interaction causes the change of the functions of the downstream effector(s). The GTP-bound form is converted by
the action of the intrinsic GTPase activity to the GDPbound form, which then releases the bound downstream
effector(s). In this way, one cycle of activation and inactivation is achieved, and small G proteins serve as molecular switches that transduce an upstream signal to a
downstream effector(s).
Thus the rate-limiting step of the GDP/GTP exchange
reaction is the dissociation of GDP from the GDP-bound
form. This reaction is extremely slow and therefore stimulated by a regulator, named GEP (also called GEF or
guanine nucleotide releasing factor), of which activity is
often regulated by an upstream signal. GEP first interacts
with the GDP-bound form and releases bound GDP to
form a binary complex of a small G protein and GEP.
Then, GEP in this complex is replaced by GTP to form the
GTP-bound form. Most GEPs, such as Son of Sevenless
(SOS), a Ras GEP, and Rab3 GEP, are specific for each
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FIG. 3. The COOH-terminal structures and posttranslational modifications of small G proteins. The COOH-terminal regions of small G
proteins are classified into at least four groups (1– 4). The Cys-A-A-X
structure is furthermore subclassified into two groups (1a and 1b). A,
aliphatic acid; X, any amino acid; P, palmitoyl; F, farnesyl; GG, geranylgeranyl.
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4. Regulation of small G protein activity.
member or subfamily of small G proteins (56, 75, 762), but
some GEPs, such as Dbl, a GEP active on Rho/Rac/Cdc42
proteins, show wider substrate specificity (259, 788). The
GDP/GTP exchange reactions of Rho/Rac/Cdc42 and Rab
proteins are furthermore regulated by another type of
regulator, named Rho GDI and Rab GDI, respectively (28,
206, 453, 629, 732). This type of regulator inhibits both the
basal and GEP-stimulated dissociation of GDP from the
GDP-bound form and keeps the small G protein in the
GDP-bound form. Rho GDI and Rab GDI show wider
substrate specificity than GEPs and GTPase-activating
proteins (GAPs) and are active on all Rho/Rac/Cdc42 and
Rab proteins, respectively (18, 275, 391, 627, 629, 732,
737). Thus the activation of Rho/Rac/Cdc42 and Rab proteins is regulated by positive and negative regulators.
Recently, Ran GDI, p10/NTF2, has also been reported
(125, 485, 563, 789), but GDIs have not been identified for
other small G proteins. The GTPase activity of each small
G protein is variable but relatively very slow and is stimulated by GAPs. Most GAPs, such as Ras GAP and Rab3
GAP, are specific for each member or subfamily of small
G proteins (56, 205, 727), but some GAPs, such as p190, a
GAP active on Rho/Rac/Cdc42 proteins, show wider substrate specificity (656).
pathway, such as neurons, neuroendocrine cells, and exocrine cells (140, 183, 476, 477, 625). Rab17 is detected in
epithelial cells (413). Most small G proteins are localized
either in the cytosol or on membranes. Ran is localized
either in the cytosol or in the nucleus. Each small G
protein is localized to a specific membrane. Ras proteins
are localized at the cytoplasmic face of the plasma membrane. This localization is mediated by the posttranslational modifications with lipid. The farnesyl moiety of
Ha-Ras and Ki-Ras alone is not sufficient for their binding
to the membrane (256). In the case of Ha-Ras, both the
farnesyl and palmitoyl moieties are necessary, whereas in
the case of Ki-Ras, both the farnesyl moiety and the
neighboring clustered polybasic amino acids are necessary. The farnesyl and palmitoyl moieties may interact
with the acyl moieties of the phospholipids, whereas the
polybasic amino acids may interact with the polar head
groups of the acidic phospholipids. The methyl moiety
also contributes markedly to efficient membrane association (255). Rap1 is geranylgeranylated and has clustered
polybasic amino acids. Most Rab proteins have either a
Cys-X-Cys or Cys-Cys structure of which Cys residues are
both geranylgeranylated. These small G proteins are localized at the cytoplasmic faces of distinct membrane
compartments. It has not been experimentally clarified
how Rap1 and Rab proteins exactly interact with the
membranes, but it is likely that both the prenyl moiety
and the polybasic region or two prenyl moieties are necessary. In contrast, Arf proteins have one myristoyl moiety and Sar1 has no lipid moiety, but they interact with the
cytoplasmic faces of membranes. Arf proteins interact
with membrane lipids by its myristoylated and amphipathic NH2-terminal helix (21, 47). In the case of Sar1, it
may interact with the phospholipid through only peptide
region. Small G proteins, such as Rho/Rac/Cdc42 and Rab
proteins, located on the plasma membrane and the cytosol are translocated between these two sites. Ran is also
translocated between the cytosol and the nucleus through
the nuclear pore complexes (NPCs).
III. RAS PROTEINS AS REGULATORS
OF GENE EXPRESSION
C. Localization
A. Outline
Small G proteins as well as heterotrimeric G proteins
are present only in eukaryotes from yeast to human,
although G proteins involved in protein synthesis such as
elongation factors exist in both prokaryotes and eukaryotes. Most small G proteins are widely distributed in
mammalian cells, and most cells have the Ras, Rho, Rab,
Sar1/Arf, and Ran families, although expression levels of
their members may vary from one type to another. A few
members show tissue-specific expression; for instance,
Rab3A is expressed in cells having a regulated secretion
Three Ras proteins are now known, Ha-Ras, Ki-Ras,
and N-Ras, which are capable of transforming mammalian
cells when activated by point mutations (71, 84, 182, 681).
In the yeast S. cerevisiae, there are two members of Ras
proteins, Ras1 and Ras2, that are essential for cell viability, and these yeast genes are functionally replaceable by
mammalian genes (141, 577). The downstream effector of
Ras proteins was first identified to be adenylate cyclase in
the yeast S. cerevisiae (66, 721). Mammalian adenylate
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FIG.
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SMALL G PROTEINS
B. Ras Protein Cycle: Activation/Inactivation
Ras protein activity is regulated by GEPs and GAPs,
and activation is induced by a large variety of extracellular signals, most notably signals that activate receptors
with intrinsic or associated tyrosine kinase activity (168,
207, 399, 549, 617) (Fig. 5). Phosphotyrosines serve as
docking sites for the adaptor proteins, such as GRB2 and
SHC/GRB2 complex, which then recruit SOS, the most
characterized Ras GEP, from the cytosol to produce a
receptor-adaptor-GEP complex. SOS recruited to the
plasma membrane then stimulates a Ras protein located
at the cytoplasmic face of the plasma membrane and
converts it from the the GDP-bound form to the GTPbound form. It is believed that GRB2 recruits SOS from
the cytosol to the plasma membrane without affecting its
GEP activity, but the possibility has not been totally excluded that GRB2 both recruits and activates SOS. Receptors not directly associated with tyrosine kinases, such as
T-cell receptors, may activate Ras proteins indirectly
through Src-like tyrosine kinases or ligand-independent
activation of receptor tyrosine kinases (243, 690, 774).
Moreover, heterotrimeric G protein-coupled receptors,
such as ␣-adrenergic receptors, muscarinic acetylcholine
receptors, and lysophosphatidic acid, have also been
shown to activate Ras proteins (266, 285, 294). In addition,
an increase of cytoplasmic Ca2⫹ induced by activation of
these receptors in neurons also induces activation of
another type of GEPs, p140 Ras GRF, that contains an IQ
motif regulated by calcium-bound calmodulin (178).
After the GTP-bound forms of Ras proteins accomplish their effects on downstream effector(s), they are
converted to the GDP-bound form by the action of Ras
GAPs. However, how termination of Ras protein signaling
is achieved is not fully understood. Phosphorylation of
SOS by the Raf/MAP kinase pathway (see below) may
induce the dissociation of SOS from GRB2 (281, 356), and
another possible mechanism is that Ras GAPs are activated by their binding to tyrosine-phosphorylated growth
factor receptors such as the platelet-derived growth factor (PDGF) receptor (17, 332).
Three GEPs of Ras (SOS, Cdc25, and Ras GRF) have
FIG. 5. Mode of action of Ras proteins in gene expression. Ras, Ras proteins.
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cyclase is directly regulated by heterotrimeric G proteins,
but not by Ras proteins. Subsequently, genetic, cell biological, and biochemical studies in Caenorhabditis
elegans, Drosophila, and mammalian cells established the
mode of action of Ras proteins; they directly bind to and
activate Raf protein kinase (82, 151a, 254a, 743, 759, 767,
815), which then induces gene expression through the
mitogen-activated protein (MAP) kinase cascade in response to various extracellular signaling molecules (145,
299, 416). Other studies have clarified that Ras proteins
regulate not only cell proliferation but also differentiation
(41, 249, 523), morphology (41a, 182, 779), and apoptosis
(334). Ras proteins regulate these functions mainly
through gene expression, but it has not been established
whether the Ras protein-mediated morphological changes
are a direct effect or an indirect effect through gene
expression. Another characteristic feature of Ras proteins
is that the mutations of their genes and their regulator
genes cause human cancers (9, 42, 59, 445, 607, 756, 786).
Thus Ras proteins are crucially important molecules not
only for biology but also for human health.
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GAP. One is p62 Dok, a docking protein that contains
multiple tyrosine phosphorylation sites for its binding to
the SH2 domains of p120 Ras GAP and Nck (87, 796). The
other is p190 Rho GAP; however, the precise role of the
interaction between p190 Rho GAP and p120 Ras GAP is
not fully understood (486, 656). p120 Ras GAP-deficient
mice die during embryonic development, and the ability
of endothelial cells to organize a vascular network is
severely impaired in addition to extensive neuronal cell
death in these animals (268). Another Ras GAP named
GAP1m, which shows a high degree of similarity to the
Drosophila Gap1 gene, has been also identified (426). In
addition to the GAP catalytic domain, GAP1m has two
domains with sequences closely related to those of the
phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine
kinase.
S. cerevisiae contains two Ras GAPs, Ira1 and Ira2
(711, 712), which contain domains homologous to the
COOH terminus of p120 Ras GAP. In wild-type cells, Ras
proteins are generally inactive, but in the absence of
either the IRA gene product, they accumulate in their
GTP-bound state, becoming hyperactive and leading to
overproduction of cAMP. In yeast, at least, Ras GAPs are
therefore not the effectors of Ras proteins; rather, they
serve as negative regulators.
Neurofibromin (NF1), the human protein defective in
von Recklinghausen neurofibromatosis (a benign tumor),
contains a domain homologous to the catalytic domains
of p120 Ras GAP, Ira1, and Ira2 (445, 786). Like p120 Ras
GAP itself, neurofibromin possesses GAP activity in vitro
and can complement the loss of Ira function in the yeast
S. cerevisiae, indicating that it may be the mammalian
homolog of Ira1 and Ira2 (34). These proteins share regions of significant similarity outside the GAP-related domain and presumably perform similar functions.
Small G protein GDP dissociation stimulator (Smg
GDS) is a regulator that is entirely distinct from GEPs,
GDIs, and GAPs (328, 795). Smg GDS has two biochemical
activities on a group of small G proteins including Ki-Ras
and the Rho/Rac/Cdc42/Rap1 proteins: one is to stimulate
their GDP/GTP exchange reactions and the other is to
inhibit their binding to membranes (18, 275, 479, 480, 788).
A detailed kinetic study of Smg GDS with Ki-Ras as a
substrate has revealed that it interacts with not only
GDP-Ki-Ras and the guanine nucleotide-free Ki-Ras but
also GTP-Ki-Ras, under the conditions where a Ras GEP,
mCdc25, does not form a ternary complex with GTP-KiRas (506). This property of Smg GDS suggests that it
stimulates the GDP/GTP exchange reaction only once.
The physiological role of Smg GDS remains to be established, although it shows mitogenic and transforming activities in cooperation with Ki-Ras in fibroblasts (198).
Studies on Smg GDS-deficient mice have revealed that
mice die of heart failure shortly after birth (708). En-
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been discovered to date. The first GEP for Ras, Cdc25, has
been identified genetically in S. cerevisiae (67, 81, 605).
Cdc25 has a GEP domain that is required for its catalytic
activity, located in its COOH-terminal region. In addition,
Cdc25 has an SH3 domain in the NH2-terminal region. In
higher eukaryotes, in addition to mammalian Cdc25
(mCdc25), two different types of proteins with homology
to Cdc25 have been found. The first group includes SOS.
SOS was first identified in Drosophila. Genetic studies
have shown that SOS is downstream of Sevenless, a receptor tyrosine kinase, which is homologous to the epidermal growth factor (EGF) receptor (609, 669). One
human and two murine homologs of SOS have been
cloned. SOS has one pleckstrin homology (PH) domain
that may interact with a membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to determine its
localization (62, 98). In addition, SOS has a GEP domain
that is required for its catalytic activity in the middle
portion and a GRB2-binding site in its COOH-terminal
region. The second GEP group includes p140 Ras GRF
that is primarily expressed in neural tissues (667). p140
Ras GRF has also a PH domain and a GEP domain. In
addition, p140 Ras GRF contains an IQ motif that is
regulated by calcium-bound calmodulin (178). Ras GRP, a
GEP also predominantly expressed in brain, binds Ca2⫹
directly through a structure similar to the calcium binding
EF hand; moreover, Ras GRP is regulated by direct binding of diacylglycerol (166). SOS is active on Ha-Ras and
Ki-Ras but not on R-Ras, whereas Ras GRF is active on
Ha-Ras and N-Ras but not on RalA or Cdc42 (56, 75,
507, 667).
p120 Ras GAP is the first GAP to be characterized at
a molecular level (221, 727, 728, 755). p120 Ras GAP is
active on Ha-Ras, Ki-Ras, N-Ras, and R-Ras, but not on
Rho/Rac/Rab proteins (56, 221, 727). It contains a hydrophobic NH2 terminus, two Src homology-2 (SH2) domains, one Src homology-3 (SH3) domain, one PH domain, and a region similar to the calcium-dependent lipid
binding region of phospholipase A2. Although it is clear
that p120 Ras GAP acts as a negative regulator of Ras
proteins, it has long been speculated that it also has other
functions. One possible role of p120 Ras GAP is that it is
a downstream effector of Ras proteins (463, 804). However, it now seems unlikely that p120 Ras GAP plays a
major downstream role of Ras proteins. A number of
tyrosine protein kinases have been shown to stimulate
tyrosine phosphorylation of p120 Ras GAP in a variety of
cells (169, 481), although the stoichiometry of these phosphorylations is generally low, and no alteration in the
activity or localization of p120 Ras GAP has been demonstrated upon tyrosine phosphorylation. Protein tyrosine
kinases may control the activity of p120 Ras GAP by
forming a complex with it. In fact, p120 Ras GAP forms a
complex with activated PDGF receptors (332, 339). Two
other proteins have been found to associate with p120 Ras
Volume 81
January 2001
SMALL G PROTEINS
hanced apoptosis is observed in at least heart, thymus,
and neuron in Smg GDS-deficient mice. This phenotype is
apparently similar to those of Ki-Ras-deficient mice (321,
362), providing another line of evidence that Smg GDS
plays a role in Ki-Ras-mediated signaling pathway in vivo.
C. Raf Protein Kinase Activation by Ras Proteins
for both its localization and biological activity. Furthermore, lipid-modified Ras proteins have been shown to
more efficiently activate adenylate cyclase in the yeast
system (288, 376). Posttranslational modifications of KiRas are also required for MAP kinase activation in a
cell-free system (309). In addition, posttranslational modifications of Ha-Ras are required for activation of, but not
for association with, Raf protein kinase (392, 547).
The intracellular signals that couple growth factors
to MAP kinase may determine the different effects of
growth factors; for example, transient activation of MAP
kinase by EGF stimulates proliferation of PC12 cells,
whereas sustained activation of MAP kinase by nerve
growth factor (NGF) induces differentiation of PC12 cells.
Activation of MAP kinase by NGF involves two distinct
pathways: the initial activation of MAP kinase requires
Ras proteins, but its activation is sustained by Rap1 (806)
(see below). Rap1 is activated by C3G, a GEP for Rap1,
and forms a stable complex with B-Raf (806). Activation
of B-Raf by Rap1 represents a common mechanism to
induce sustained activation of the MAP kinase cascade.
D. Modifiers of the Ras Protein-Induced Raf
Protein Kinase Activation
In addition to Ras proteins, a protein named 14 –3-3
seems to also interact with Raf-1 and activate it (176, 197).
14 –3-3 is a specific phosphoserine-binding protein (500).
Raf-1 itself contains two phosphorylation sites that may
interact with 14 –3-3. 14 –3-3 may have two different roles:
first, 14 –3-3 may be required for maintaining Raf-1 in an
inactive conformation, as Raf-1 that is unable to stably
interact with 14 –3-3 is activated (467). In response to
signaling events and Ras protein activation, 14 –3-3 may
subsequently play a second role in facilitating activation
of Raf-1 and stabilizing activated Raf-1.
The observation that Raf-1 becomes hyperphosphorylated in response to many signaling events (492) has
long suggested that phosphorylation plays a role in regulating Raf-1 activity. Mechanisms by which phosphorylation could regulate Raf-1 function include direct alteration of the intrinsic activity of Raf-1 and alteration of
critical protein interactions, such as with 14 –3-3. The
rapid and transient nature of Raf-1 activation further complicates the issue, making it difficult to distinguish between activating and inactivating modifications. Nevertheless, by the use of overexpression systems and
mutational analysis, the phosphorylation of tyrosine residues 340 and 341 has been shown to enhance the catalytic activity of Raf-1 (441). The tyrosine kinases implicated in phosphorylating Raf-1, and thereby enhancing its
activity, include members of the Src kinase family (441,
562, 576).
A novel protein kinase that functions downstream of
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Ras proteins mediate their effects on cell proliferation mainly by activation of a cascade of protein kinases:
Raf protein kinase (c-Raf-1, A-Raf, and B-Raf), MEK (MAP
kinase kinases 1 and 2), and MAP kinase. Ras proteins
activate this protein kinase cascade by directly binding to
Raf proteins (743, 759, 767, 815). Raf proteins then phosophorylate and activate MEK (145, 299, 378), which then
phosphorylates and activates MAP kinase (134, 450). The
activated MAP kinase translocates to the nucleus, where
it phosphorylates and stimulates the activity of various
transcription factors, including Elk-1 (442). The recent
observation that Ras proteins interact with two distinct
NH2-terminal regions of Raf-1 suggests that Ras proteins
promote more than just membrane translocation of Raf-1
and instead may also facilitate the subsequent events that
lead to Raf-1 activation (495, 513, 759).
The initial step of the Raf-1/MEK/MAP kinase cascade is the activation of Raf-1 by direct interaction with a
GTP-Ras protein (GTP-Ras) (743, 759, 767, 815). The interaction with GTP-Ras localizes Raf-1 to the plasma
membrane (389, 687). The first described and best-characterized Ras-binding domain (RBD) is contained within
residues 51–131 of Raf-1 (121, 513, 759). The association
of the RBD with the Ras effector domain is a high-affinity
interaction that is mediated primarily by residues Gln-66,
Lys-84, and Arg-89 of Raf-1 (55). The interaction between
the RBD and GTP-Ras appears to then allow for a second
RBD of Raf-1 to contact GTP-Ras (72, 159). This second
RBD (residues 139 –184 of Raf-1) encompasses the conserved Cys finger motif within the Raf-1 NH2 terminus and
is referred to as the Cys-rich domain (CRD) (495). In
terms of the Ras-Raf-1 interaction, the CRD associates
with different residues of GTP-Ras than does the RBD
(159), and posttranslational modifications of Ras proteins
may be important for CRD binding (297, 376). Thus, in the
full-length molecule, the CRD is inaccessible for GTP-Ras
binding, but either mutational events or RBD binding can
unmask the CRD and allow it to interact with GTP-Ras.
Thus, for the Ras-Raf-1 interaction to result in Raf-1 activation, binding to both the RBD and the CRD appears to
be required.
Ha-Ras is localized at the cytoplasmic surface of the
plasma membrane, while mutant forms of Ha-Ras, which
lack posttranslational lipid modification, are cytosolic and
lack biological activity. These findings suggest that posttranslational lipid modifications of Ha-Ras are required
161
162
TAKAI, SASAKI, AND MATOZAKI
E. Other Effectors of Ras Proteins
A variety of candidate Ras protein effectors have
been reported in addition to Raf proteins. These include
Ral GDS (279, 342, 676), RIN1 (254), and phosphatidylino-
sitol (PI) 3-kinase (608). AF6/Canoe is also suggested to
be a binding partner of Ras proteins (373, 746), but this
result has been called into question (434). It has recently
been shown that Rap1 shows a much higher affinity to
AF6 than Ras proteins do (404). p120 Ras GAP may participate in Ras protein-mediated gene expression, although it is still unclear whether p120 Ras GAP is a
regulator, an effector, or both for Ras proteins. In contrast, activation of PI 3-kinase by Ras proteins may promote cell survival (334, 608). However, it has not been
established whether these effector molecules other than
Raf proteins really play a role in the downstream pathway
of Ras proteins.
F. Transport of Newly Synthesized Ras Proteins
From the Endoplasmic Reticulum to the
Plasma Membrane
The plasma membrane localization of Ras proteins is
crucial for their functions. The mechanism by which Ras
proteins get to the plasma membrane has not fully been
understood. It has recently been shown that Ras proteins
do not directly travel to the plasma membrane from the
cytosol, but interact with intracellular membranes (25,
114). Ras proteins first associate with the endoplasmic
reticulum and then with the Golgi apparatus. The initial
association of Ras proteins with the endoplasmic reticulum requires only the COOH-terminal Cys-A-A-X structure
and farnesylation. N-Ras and Ha-Ras seem to be transported by exocytic vesicles following association with the
endoplasmic reticulum and the Golgi apparatus. Ki-Ras
takes a faster route that may not involve the Golgi apparatus. The hypervariable domains of the three Ras proteins are necessary and sufficient to account for their
differential localizations. Inhibition of vesicle transport
with brefeldin A (BFA), an inhibitor of Arf protein GEP
(see below), blocks the transit of N-Ras to the plasma
membrane, demonstrating the importance of vesicle
transport for N-Ras function. Carboxylmethylation and
A-A-X proteolysis are also necessary for proper association with the plasma membrane (255).
G. Ras Proteins and Cancer
Mutated versions of the three human Ras genes have
been detected in ⬃30% of all human cancers, implying an
important role for aberrant Ras protein function in carcinogenesis. For example, Ras gene mutations are highly
prevalent in pancreatic (90%) (9), lung (30%) (607), and
colorectal (50%) (59, 756) carcinomas. Because Ras proteins regulate diverse extracellular signaling pathways for
cell growth, differentiation, and apoptosis, the deregulated function of other cellular components can cause
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Ras proteins, kinase suppressor of Ras (KSR), has recently been identified by genetic screening as a suppressor of phenotypes caused by an activated Ras protein in
both Drosophila and C. elegans (366, 694, 717). Epistasis
analysis in Drosophila suggests that KSR functions downstream of Drosophila Ras-1 but upstream or in parallel to
Raf protein (717). Characterization of a mouse KSR homolog suggests that KSR facilitates signal transmission
between Raf proteins, MEK, and MAP kinase (718). In
addition, upon Ras protein activation, KSR translocates to
the plasma membrane, where it forms a stable complex
with Raf proteins (718). Moreover, KSR, via its kinase
domain, forms a stable complex with MEK in the cytosol
of quiescent cells (144). Therefore, in response to an
activated Ras protein, KSR might shuttle MEK from the
cytosol to activated Raf proteins at the membrane.
With the use of a screen for eye development defects in Drosophila, the connector enhancer of KSR
(CNK) protein has been identified as an enhancer of a
KSR dominant negative mutant phenotype (719). Mutation of CNK suppresses the phenotype of activated Ras
proteins or Sevenless but not Raf proteins, suggesting
that it acts upstream of Raf proteins. CNK has several
protein interaction domains. These domains include a
sterile alpha motif (SAM) domain, a PSD-95/Dlg-A/ZO-1
(PDZ) domain, two proline-rich (potential SH3-binding)
domains, and a PH domain; such domains are found in
many proteins involved in signaling and suggest further
interactions of CNK with other proteins and small molecules. In two-hybrid assays in S. cerevisiae, a COOHterminal portion of CNK that contains the PH domain
interacts with the Raf kinase domain (719). Thus the
SAM, PDZ, and novel domains might be available for
other interactions, although it is not known whether
CNK also binds other proteins in the Ras-Raf signaling
pathway. CNK has a molecular structure similar to that
of recently identified rat neuronal proteins, named
MAGUINs, that interact with the PDZ domains of PSD95/SAP90 and S-SCAM (802). MAGUINs interact with
c-Raf-1 but do not affect its enzymatic activity (803).
PSD-95/SAP90 and S-SCAM are neuronal membraneassociated guanylate kinases, and these proteins function as synaptic scaffolding proteins (264). In fact,
PSD-95/SAP90 further interacts with synGAP, which
regulates the activity of Ras proteins (346). Therefore,
MAGUINs may also bind Raf proteins and link it to
PSD-95/SAP90 and S-SCAM in synaptic junctions.
Volume 81
January 2001
SMALL G PROTEINS
aberrant Ras protein function in the absence of mutations
in the Ras genes themselves. Overexpression of ErbB2 or
EGF receptor tyrosine kinase is common in breast cancers, and their transforming actions are dependent on
signaling through the loss of negative Ras protein regulators (155). Similarly, the loss of function of negative Ras
protein regulators, such as neurofibromin defective in
type 1 neurofibromatosis-associated tumors, can cause
aberrant upregulation of Ras protein function (42). Therefore, the importance of aberrant Ras protein function in
human cancers may be greater than expected and may
extend to tumors that do not harbor mutated Ras alleles.
The Rap subfamily consists of Rap1A, Rap1B, and
Rap2. Rap1 proteins have been independently isolated by
three laboratories by different methods: they have been
isolated as homologs of Ras proteins by hybridization
(573), they have been purified as small G proteins (smg
p21) by column chromatography (337, 534), and they have
been identified as K-Rev1 in a screen for cDNAs that
revert the morphology of Ki-Ras-transformed cells (353).
Interestingly, Rap1 proteins have an effector domain virtually identical to that of Ras proteins, suggesting that
both proteins theoretically interact with similar effectors
and show similar or antagonistic effects. The antagonistic
function of K-Rev1 on Ras-transforming activity was the
first studied (353). Rap1A binds to the two Ras-binding
regions of Raf-1 (RBD and CRD), and this binding of
Rap1A to CRD is competitive with Ras proteins (296, 297).
Rap1 does not induce Raf-1 activation in intact cells but
inhibits the Ha-Ras-induced Raf-1 activation in intact cells
when Rap1 is overexpressed (124). However, most extracellular signals that induce Raf-1 activation, such as
PDGF and EGF, activate rather than inhibit Rap1 (828).
Furthermore, a phorbol ester induces Rap1 activation in
Rat1 cells, but does not inhibit the PDGF- and EGFinduced activation of MAP kinase (828). These results
suggest that the suppression of Ras protein function by
Rap1 is simply due to the artificially competitive inhibition of the Ras protein binding to RBD or CRD.
In contrast to the role of Rap1 antagonistic to that of
Ras proteins, evidence is accumulating that Rap1 functions independently of Ras protein signaling, utilizing effectors similar or identical to those of Ras proteins, like
Raf proteins. Rap1, as well as Ki-Ras, induces DNA synthesis in Swiss 3T3 cells (11, 807). Rap1, as well as Ki-Ras,
binds and activates B-Raf in vitro (541). In intact PC12
cells in response to cAMP and NGF, Rap1 is activated and
induces B-Raf activation, causing sustained activation of
the MAP kinase cascade that is necessary for neuronal
differentiation (761, 806). Most recently, CD31, an impor-
tant integrin adhesion amplifier, has been shown to selectively activate Rap1, but not Ha-Ras, R-Ras, or Rap2 (587).
An activated mutant of Rap1 stimulates T lymphocyte
adhesion to intercellular adhesion molecule and vascular
cell adhesion molecule, as does C3G. Thus Rap1 regulates
ligand-induced cell adhesion, and it may play a more
general role in coordinating adhesion-dependent signals.
In contrast to Rap1, little is known about Rap2 (573).
Several distinct second messenger pathways, including those for calcium (194), diacylglycerol (462), phospholipase C-␥ (462), and cAMP (10), and perhaps others
(497a), are able to induce Rap1 activation. Clearly, Rap1
activation is a common event, which suggests a function
that is central in signal transduction processes. C3G is a
Rap1-specific GEP containing a proline-rich domain that
interacts with the SH3 domain of members of the Crk
adaptor proteins, Crk I, Crk II, and Crk L (239, 355). In
general, this association is constitutive, but tyrosine phosphorylation of Crk may disrupt the interaction (546). The
SH2 domain of Crk binds directly to various activated
receptor tyrosine kinases and phosphotyrosine-containing adaptor proteins (355). This association of Crk-C3G
with these complexes may enhance GEP activity of C3G
(301), suggesting that complex formation and dissociation
of C3G regulate Rap1 activation by tyrosine kinases. However, in a human Jurkat T cell leukemia line, T-cell receptor-dependent induction of a Cbl-Crk L-C3G signaling
complex does not activate Rap1 (586). Therefore, more
work will be required to clarify how C3G complex formation is coupled to Rap1 regulation. Recently, two novel
GEPs specific for Rap1, named Epac/cAMP-GEFI and
nRap GEP/PDZ-GEF1/Hs-RA-GEF, have been identified
(147, 148, 335, 402, 540). Epac/cAMP-GEFI has cAMPbinding and Ras GEP domains; thus this GEP activity is
dependent on cAMP (148, 335). nRap GEP has been isolated as a binding partner of S-SCAM, that interacts with
N-methyl-D-aspartate (NMDA) receptors and neuroligin
through PSD-95/Dlg-A/ZO-1 (PDZ) domains at synaptic
junctions (540). In contrast to Epac/cAMP-GEFI, nRap
GEP/PDZ-GEF1/Hs-RA-GEF has one PDZ, one Ras association, and one Ras GEP domains as well as one COOHterminal consensus motif for binding to PDZ domains.
However, nRap GEP/PDZ-GEF1/Hs-RA-GEF has an incomplete cAMP-binding domain and its GEP activity is
independent of cAMP. SPA-1, a Rap1 GAP, has been
shown to interfere with Rap1 activation by membranetargeted C3G (729). Overexpression of SPA-1 in HeLa
cells suppresses Rap1 activation upon plating on dishes
coated with fibronectin and results in the reduced adhesion. In addition, overexpression of SPA-1 in promyelocytic 32D cells also inhibited both activation of Rap1 and
induction of cell adhesion by granulocyte colony-stimulating factor, suggesting that Rap1 is required for the cell
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H. Rap Proteins
163
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TAKAI, SASAKI, AND MATOZAKI
adhesion induced by both extracellular matrix and soluble ligands (729).
I. Ral Proteins
J. Other Ras Family Members
R-Ras has been shown to be involved in multiple
biological functions: the ability to transform NIH 3T3
cells, the promotion of cell adhesion, and the regulation of
apoptotic response in hematopoietic cells (128, 621, 695,
816). Unlike other Ras family members, R-Ras does not
activate Raf proteins or MAP kinases in cells, whereas it
stimulates PKB/Akt effectively through PI 3-kinase (444).
TC21 has a highly oncogenic potential and is found mutated in some human tumors and overexpressed in breast
cancer (37, 94, 300). As to the activation of Raf proteins by
TC21, it is controversial (242, 611). Recently, new members of the Ras family, Rit and Rin, have been identified by
an expression cloning screen (385). Rit is ubiquitously
expressed, whereas Rin is expressed only in neural tissue.
A unique feature of their structures is that they lack a
known recognition signal for COOH-terminal prenylation.
Nonetheless, both proteins localize on the plasma membrane, probably through a COOH-terminal cluster of basic
amino acids. Rin binds calmodulin through a COOH-terminal motif, suggesting that Rin may be involved in calcium-mediated signaling in neurons (385). Rad is another
member of Ras-like proteins that has originally been isolated as a gene overexpressed in the skeletal muscle of
humans with type II diabetes (594). Kir/Gem has also been
cloned as a gene that is overexpressed in cells transformed by abl tyrosine kinase (123) or cloned from mitogen-induced human peripheral blood T cells (429). Kir/
Gem and Rad constitute a new family of Ras-related
proteins. The distinct structural features of this family
include the G3 GTP-binding motif, extensive NH2- and
COOH-terminal extensions beyond the Ras-related domain, and a motif that determines membrane association
(429). Rheb, another Ras protein-related molecule, has
been isolated by differential cloning techniques to identify
genes that are rapidly induced in brain neurons by synaptic activity (790). Expression of Rheb is rapidly and
transiently induced in hippocampal granule cells by seizures and by NMDA-dependent synaptic activity (790).
The amino acid sequence of Rheb is most closely homologous to yeast Ras1 and human Rap2. In the developing
brain, Rheb mRNA is expressed at relatively high levels.
Its close homology with Ras proteins and its rapid inducibility by receptor-dependent synaptic activity suggest
that Rheb may play an important role in long-term activity-dependent neuronal responses (790). More recently,
Ras protein-like proteins, named ␬B-Ras1 and ␬B-Ras2,
have been identified (181). These proteins interact with
I␬B␣ and I␬B␤, which are inhibitors for the nuclear transcription factor kappa B, NF-␬B, and decrease the rate of
degradation of I␬Bs. In cells, ␬B-Ras proteins are associated only with NF-␬B:I␬B␤ complexes and therefore may
provide an explanation for the slower rate of degradation
of I␬B␤ compared with I␬B␣ (181).
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The Ral subfamily consists of RalA and RalB (100,
101). Ral GDS, a Ral GEP, has been found to be a Ras
protein effector (279, 342, 676). Moreover, insulin and
EGF induce activation of Ral proteins, and this activation
is inhibited by a dominant negative mutant of Ras proteins, suggesting that RalA is downstream of the Ras
protein signaling pathway (783). In NIH 3T3 cells, both a
dominant active mutant of Ha-Ras and Ral GDS synergize
with Raf-1 in the induction of cell transformation and the
activation of c-fos promoter (548, 741), and a dominant
negative mutant of RalA inhibits the Ha-Ras- and Raf-1induced transformation (741). These observations suggest
that the Ral GDS-Ral protein pathway contributes to cell
transformation and gene expression. However, a dominant active mutant of RalA alone cannot efficiently induce
the oncogenic transformation or the c-fos induction compared with a dominant active mutant of Ha-Ras and Ral
GDS (548, 741), suggesting that the transformation and
the gene expression induced by Ral GDS may require
other factors in addition to Ral proteins.
Three effectors for Ral proteins are known: RalBP1,
phospholipase D, and filamin. RalBP1 contains a Rho GAP
homology domain that exhibits the GAP activity for Rac/
Cdc42 proteins, but not for Rho proteins (179). Although
Rac/Cdc42 proteins contribute to the Ha-Ras-induced oncogenic transformation (582) (see below), it is unclear
whether the association of Ral proteins with RalBP1 regulates the activity of these Rho/Rac/Cdc42 proteins.
RalBP1 has been found to interact with POB1 and Reps1
(302, 791), which have proline-rich sequences responsible
for interaction with Grb2 and Crk, and an Eps15 homology domain. Ral proteins are involved in endocytosis of
the growth factor receptors probably through RalBP1,
POB1, Eps15, and Epsin (511). Another Ral protein effector, phospholipase D, is also implicated in vesicle trafficking (179, 316). The activity of phospholipase D is induced
by Src and Ras proteins. A dominant negative mutant of
RalA inhibits both v-Src- and v-Ras-induced phospholipase D activity (316). The third effector protein of Ral is
filamin (537). Either a dominant negative mutant of RalA
or the RalA-binding domain of filamin blocks Cdc42-induced filopodium formation. A dominant active mutant of
RalA elicits actin-rich filopodia, but it does not generate
filopodia in filamin-deficient cells. Thus the Ral signaling
appears to regulate vesicle trafficking, cytoskeletal organization, gene expression, and cell transformation. The
GAP proteins for RalA were characterized and partially
purified (48, 170); however, the molecular cloning of these
proteins has not yet been achieved.
Volume 81
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SMALL G PROTEINS
IV. RHO/RAC/CDC42 PROTEINS AS
REGULATORS OF BOTH CYTOSKELETAL
REORGANIZATION AND GENE EXPRESSION
A. Outline
B. Reorganization of the Actin Cytoskeleton
Reorganization of the actin cytoskeleton plays crucial roles in many cellular functions such as cell shape
change, cell motility, cell adhesion, and cytokinesis. The
actin cytoskeleton is composed of actin filaments and
many specialized actin-binding proteins (671, 688, 826).
Filamentous actin is generally organized into a number of
discrete structures (Fig. 6): 1) actin stress fibers: bundles
of actin filaments that traverse the cell and are linked to
the extracellular matrix through focal adhesions; 2) lamellipodia: thin protrusive actin sheets that dominate the
edges of cultured fibroblasts and many migrating cells;
membrane ruffles observed at the leading edge of the cell
result from lamellipodia that lift up off the substratum and
fold backward; and 3) filopodia: fingerlike protrusions
that contain a tight bundle of long actin filaments in the
direction of the protrusion. They are found primarily in
motile cells and neuronal growth cones. It is important,
therefore, that the polymerization and depolymerization
of cortical actin be tightly regulated. For the most part,
this regulation of actin polymerization is orchestrated by
Rho/Rac/Cdc42 proteins. Rho proteins regulate stress fiber formation (475, 600), while Rac proteins regulate
ruffling and lamellipodia formation (602), and Cdc42 regulates filopodium formation (368, 522).
C. Rho/Rac/Cdc42 Protein Cycle: Cyclical
Activation/Inactivation
The activation and inactivation of Rho/Rac/Cdc42
proteins are regulated by essentially the same mechanism
as Ras proteins by GEPs and GAPs, respectively. However, they are further regulated by another class of regulator, GDIs (206, 275, 280, 704, 732). In the cytosol, Rho/
Rac/Cdc42 proteins are complexed with the GDI and
maintained in the GDP-bound inactive form. The GDPbound form is first released from a GDI by a still unknown
mechanism and is converted to the GTP-bound form by
the action of a GEP. The GTP-bound form then interacts
with the downstream effector(s). Thereafter, the GTPbound form is converted to the GDP-bound form by the
action of a GAP. The GDP-bound form then forms a
complex with the GDI and returns to the cytosol.
Rho/Rac/Cdc42 proteins are posttranslationally modified with lipid as described above and therefore they have
to be in complex with GDIs to remain soluble in the
cytosol. However, it is unknown whether all the GDPbound form of Rho/Rac/Cdc42 proteins are complexed
with GDIs and remain in the cytosol. Some amount of the
GDP-bound form may be associated with membranes, and
it may be converted to the GTP-bound form and exert its
function on the membrane. In this case, GDIs would not
be essential for their cyclical activation and inactivation.
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The mammalian Rho family consists of at least 14
distinct members as shown in Table 1 and Figure 1. The
function of the Rho family was first demonstrated in yeast
(5, 45, 320). Phenotypes of the mutants that carry mutations in these genes indicated that Rho/Cdc42 proteins are
involved in the budding process, presumably through reorganization of the actin cytoskeleton (320, 798). In mammals, the function of Rac proteins was the first to be
clarified. GTP-Rac1, in addition to two other cytosolic
proteins, p47phox and p67phox, were shown to be required for the activation of NADPH oxidase of phagocytic
cells (1, 2, 18, 358, 479, 649). Then, the function of mammalian Rho proteins was elucidated by use of an exoenzyme of Clostridium botulinum, named C3, that specifically ADP-ribosylates Rho proteins (6, 343, 512). C3 ADPribosylates an amino acid (Asn-41) in the effector region
of RhoA and inhibits its function by preventing interaction
with downstream effectors (651). By the use of C3, Rho
proteins were first suggested to be involved in cytoskeletal control (97, 564). Rho proteins were subsequently
shown to regulate formation of stress fibers and focal
adhesions in fibroblasts by use of its dominant active
mutant and Rho GDI (475, 600, 601) and to regulate Ca2⫹
sensitivity of smooth muscle contraction (276). In contrast, Rac and Cdc42 proteins regulate formation of lamellipodia and filopodia, respectively (368, 522, 602). It has
now been established that at least Rho/Rac/Cdc42 proteins regulate primarily cytoskeletal reorganization in response to extracellular signals in mammalian cells. Evidence has also accumulated that they may play additional
roles in gene expression (126, 272, 473, 567, 692, 776).
Furthermore, involvement of Rho/Rac/Cdc42 proteins in
diverse cellular events, such as cell growth (341, 420, 552,
581–583, 794), membrane trafficking (4, 69, 86, 365, 380),
development (227), and axon guidance (412) and extension (277, 314, 369), have been reported. In these cellular
events, it is not known whether Rho/Rac/Cdc42 proteins
directly regulate them or indirectly regulate them through
cytoskeletal reorganization and gene expression. Many
upstream regulators and downstream effectors have been
identified for Rho/Rac/Cdc42 proteins, and although their
modes of activation and action have gradually been elucidated, our understanding remains incomplete. Posttranslational modifications of Rho proteins are also crucial for their various functions including cell shape
change, cell motility, cytoplasmic division of Xenopus
embryo, and regulation of 1,3-␤-glucan synthase of S.
cerevisiae (305, 352, 475, 705).
165
166
TAKAI, SASAKI, AND MATOZAKI
Volume 81
Many GEPs for Rho/Rac/Cdc42 proteins have been
isolated and characterized as shown in Table 2 and numbers are still increasing. Most GEPs have been isolated as
oncogenes. The GEPs thus far identified share a common
motif, designated the Dbl-homology (DH) domain, for
which the Dbl oncogene product is the prototype (93).
Biochemical analysis has confirmed that DH domains of
GEPs indeed show GEP activity on Rho/Rac/Cdc42 proteins in a cell-free assay system (259, 262, 468). In addition
to the DH domain, GEPs share a PH domain, which may
be involved in proper cellular localization presumably
through interaction with PIP2 (469, 824). Some members
of GEPs, such as Dbl and Vav1, have been shown to
exhibit exchange activity in vitro for a broad range including Rho/Rac/Cdc42 proteins, whereas others appear to be
more specific. Lbc, for example, and more recently discovered oncoproteins Lfc and Lsc are specific for Rho
proteins (226, 823), whereas FGD1 and frabin are specific
for Cdc42 (532, 738, 822). Although Vav1 is a GEP for Rac
proteins (133, 245), Vav2, a GEP closely related to Vav1,
functions preferably as a GEP for Rho proteins (642, 643).
Some GEPs, such as Dbl, prefer the lipid-modified form of
the substrate small G proteins to the lipid-unmodified
ones (788).
In addition to the PH and DH domains, many GEPs
have other domains that are commonly found in signaling
molecules, such as the SH2 domain for Vav or the SH3
domain for Vav and Dbs, suggesting that they may have
additional functions (93, 133, 642, 778). A GEP for Rho
proteins, named p115 Rho GEF, that contains the regulator of G protein (RGS) domain has recently been identified (262, 367). RGS stimulates the intrinsic GTPase activity of the ␣-subunit of G12 and G13. p115 Rho GEF acts
as an intermediary in the regulation of Rho proteins by
G␣12 and G␣13 (260, 367). In addition, another Rho GEP
(named PDZ-Rho GEF) that contains RGS and PDZ domains has been reported (204). These findings have provided a new model for a signaling pathway for Rho proteins from membrane receptors. Recently, SHP-2, a
protein tyrosine phosphatase containing SH2 domains,
has been demonstrated to suppress the activity of Vav2
and consequently to reduce the Rho’s ability to form
stress fibers and focal adhesions (360). SHP-2 thereby
positively regulates the hepatocyte growth factor (HGF)/
scatter factor (SF)-induced cell scattering. However, detailed information regarding signaling cascades coupling
the extracellular stimuli to activation of GEPs for Rac/
Cdc42 proteins is still limited. Some GEPs like Tiam1
(248) and Ras GRF (667) carry a second PH domain. For
Tiam1 and Ras GEF, this second, NH2-terminal PH domain mediates localization to cell membranes (74, 469). In
addition to the PH domain, frabin has an actin-binding
domain at its NH2-terminal region (532). The actin-binding
domain in addition to the DH and first PH domains is
essential for the filopodium formation mediated by frabin
through Cdc42 (532, 738). Frabin furthermore induces
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FIG. 6. Mode of action of Rho/Rac/Cdc42 proteins in cytoskeletal reorganization. A: mode of action of Rho proteins.
B: mode of action of Rac proteins. C: mode of action of Cdc42. Rho, Rho proteins; Rac, Rac proteins.
SMALL G PROTEINS
January 2001
TABLE
2. Regulators of Rho/Rac/Cdc42 proteins
Substrate
GEP
Dbl
Vav1
Dbs
Lbc
Lfc
Lsc
Vav2
p115 Rho GEF
PDZ-Rho GEF
Tiam-1
FGD1
Frabin
GAP
p50 Rho GAP
p190 Rho GAP
Graf
myr5
Bcr
n-Chimaerin
3BP-1
Abr
GDI
Rho GDI
D4/Ly-GDI
Rho GDI-3
Reference No.
Rho, Rac, Cdc42
Rho, Rac, Cdc42
Rho, Cdc42
Rho
Rho
Rho
Rho
Rho
Rho
Rac
Cdc42
Cdc42
259, 680
245
778
823
226
226
269
262
204
248
822
532, 738
Rho, Rac, Cdc42
Rho, Rac, Cdc42
Rho, Cdc42
Rho, Cdc42
Rac, Cdc42
Rac, Cdc42
Rac
Rac
36, 381
657
271
589
152
152
118
709
Rho, Rac, Cdc42
Rho
RhoB (not RhoA, RhoC)
206, 732
390, 635
809
GAP, resulting in increased accessibility of the effector
binding surface of its SH3 domain (298). A role for p190
Rho GAP in regulating Rho protein function in cells undergoing cytoskeletal rearrangements has been suggested
(95, 603), but it is not known whether this effect is induced by p190 Rho GAP as a downstream effector of Rho
proteins.
Recent studies have shown that a cycle of inactivation and activation of Rho/Rac/Cdc42 proteins is necessary for dynamic cell functions such as growth factorinduced cell scattering. Expression of dominant active
mutants of Rho/Rac/Cdc42 proteins inhibits HGF/SF-induced cell scattering (303, 331, 599), whereas C3 or Rho
GDI blocks HGF/SF-induced cell scattering (706). The
mode of action of Rho proteins in cell scattering remains
to be clarified, but the Rho protein-regulated assembly
and disassembly of stress fibers and focal adhesions have
been suggested to be, at least in part, involved in this
process (303, 331, 599, 706). It is not known how inactivation by GAPs is induced. In one case, integrin-induced
formation of stress fibers inhibits Rho protein activation
as part of a feedback inhibition system (593).
Rho GDI was originally isolated as a cytosolic protein
that preferentially associated with GDP-RhoA and GDPRhoB and thereby inhibited the dissociation of GDP (206,
732). Rho GDI requires the posttranslational lipid modifications of RhoA for its activity (286). Rho GDI prefers
GDP-RhoA and GDP-RhoB to the corresponding GTPbound forms and forms a ternary complex with the GDPbound form (628, 732). Rho GDI is also capable of inhibiting GTP hydrolysis by Rho proteins (116, 261, 628),
blocking both intrinsic and GAP-catalyzed GTPase activity. Rho GDI dissociates the GDP-bound form of prenylated RhoB from the membrane (308). Based on these
properties of Rho GDI, it has been proposed that Rho GDI
is involved not only in the regulation of the activation of
Rho proteins but also in their translocation between the
cytosol and the membrane (630, 702, 704). The GDPbound forms of Rho proteins are complexed with Rho
GDI and remain in the cytosol. When the GDP-bound form
is released from Rho GDI, it is converted to the GTPbound form by the action of Rho GEPs. The GTP-bound
form then activates its specific downstream effector(s)
until the GTP-bound form is converted to the GDP-bound
form by Rho GAPs. Once the GDP-bound form is produced on the membrane, it is captured by Rho GDI and
the complex returns to the cytosol.
Rho GDI has also been shown to be active not only
on Rho proteins but also on Rac/Cdc42 proteins (18, 275,
391). In addition to Rho GDI, at least two other isoforms,
named D4/Ly-GDI and Rho GDI-3, have been identified
(390, 635, 809). Now, the originally identified Rho GDI is
referred to as Rho GDI␣ or Rho GDI1; D4/Ly-GDI is
named Rho GDI␤ or Rho GDI2; and Rho GDI3 is named
Rho GDI␥ or Rho GDI3. Recent NMR studies have shown
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lamellipoidum formation through indirect activation of
Rac (553). The COOH-terminal FYVE and second PH domains, which associate with an unidentified membrane
structure, in addition to the DH and first PH domains are
necessary for this action (553).
The first GAP protein specific for Rho proteins was
biochemically purified from human spleen and bovine
adrenal gland (211, 212, 488). This protein, designated p50
Rho GAP, has GAP activity toward Rho/Rac/Cdc42 proteins in vitro (381). A number of proteins that exhibit GAP
activity for Rho/Rac/Cdc42 proteins have subsequently
been identified in mammalian cells (Table 2). These proteins all share a related GAP domain that spans ⬃140
amino acids of the protein but bears no significant resemblance to Ras GAP. The substrate specificity of Rho GAPs
toward Rho/Rac/Cdc42 proteins varies with each GAP
protein. Although some of these proteins exhibit GAP
activity for several small G proteins in cell-free assay
systems, their substrate specificities in vivo appear to be
more restricted. For instance, the substrate spectrum of
p50 Rho GAP in vitro encompasses Rho/Rac/Cdc42 proteins; however, in vivo, it appears to be restricted to Rho
proteins only (603). Although first identified as a tyrosinephosphorylated p120 Ras GAP-associated protein in Srctransformed cells and in growth factor-treated cells (169,
657), p190 Rho GAP was later shown to possess GAP
activity for Rho proteins (656). Although the biological
function of p190 Rho GAP is not well understood, the
interaction of p190 Rho GAP with p120 Ras GAP has been
suggested to induce a conformational change in p120 Ras
167
168
TAKAI, SASAKI, AND MATOZAKI
poietic differentiation but subtle defects in superoxide
production by macrophages derived from in vitro embryonal stem cell differentiation (244). Thus these two lines
of evidence suggest that Rho GDIs play physiologically
important roles.
Yeast Rom7/Bem4 is a novel type of Rho1- and
Cdc42-related molecule (273, 419). This protein appears
to be distinct from GEPs, GDIs, and GAPs, in the sense
that it interacts genetically with the Rho1 pathway but
does not show GEP, GDI, or GAP activity, and it is not a
downstream effector.
D. Mode of Action of Rho Proteins
in Cytoskeletal Reorganization
Mammalian Rho proteins are required for many actin
cytoskeleton-dependent cellular processes, such as platelet aggregation, lymphocyte and fibroblast adhesion, cell
motility, contraction, and cytokinesis (251, 704). In the
yeast S. cerevisiae, Rho proteins, including Rho1, Rho2,
Rho3, and Rho4, regulate budding and cytokinesis
through reorganization of the actin cytoskeleton (80, 119,
200, 713). The mode of action of Rho proteins was first
clarified for Rho1 in the yeast budding process. Pkc1, a
yeast homolog of mammalian protein kinase C, was first
shown to be a downstream effector of Rho1 (330, 525).
Pkc1 regulates gene expression through the MAP kinase
cascade, consisting of Bck1 (MAP kinase kinase kinase),
Mkk1/Mkk2 (MAP kinase kinase), and Mpk1 (MAP kinase) (173, 396). This MAP kinase cascade regulates expression of the genes necessary for cell wall integrity.
Bni1 is another effector of Rho1 (363). Bni1 also interacts
with GTP-Rho3, GTP-Rho4 (T. Kamei and Y. Takai, unpublished observations), and GTP-Cdc42 (175). Bni1 has
two domains, named formin homology (FH)1 and FH2
domains, which are found in a variety of proteins involved
in cytoskeletal rearrangement needed to achieve cell polarity and cytokinesis (196). Bni1 binds the actin monomer-binding protein profilin via its FH1 domain to regulate reorganization of the actin cytoskeleton (175, 363).
Furthermore, Bni1 binds Aip3 (Bud6), another actin-binding protein (15), and elongation factor 2, which is known
to stimulate actin polymerization (739), suggesting that
Bni1 is the downstream effector of Rho1 that directly
regulates reorganization of the actin cytoskeleton. More
recently, Bni1 has also been shown to participate in microtubule function, since disruption of BNI1 causes defects in spindle orientation (386), Kar9 localization (472),
and growth defect together with mutation either PAC1
and NIP100 (199), whose gene products are implicated in
microtubule function (215).
1,3-␤-Glucan synthase is the third effector of Rho1
(157, 456, 580). This enzyme synthesizes 1,3-␤-glucan, a
major component of the cell wall. This series of experi-
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that Rho GDI␣ has a pocket that masks the lipid moieties
of Rho proteins (238), consistent with biochemical analyses (286). More recently, the X-ray crystallographic
structure of the Cdc42/Rho GDI complex has revealed
two major sites of interaction between Rho GDI and
Cdc42 (280). The NH2-terminal regulatory region of Rho
GDI binds to the switch I and II domains of Cdc42, leading
to inhibition of both GDP dissociation and GTP hydrolysis. In addition, the geranylgeranyl moiety of Cdc42 inserts into a hydrophobic pocket within the immunoglobulin-like domain of the GDI molecule, keeping GDP-Cdc42
in the cytosol and permitting the dissociation of GDPCdc42 from membranes.
Although the mechanism by which Rho proteins are
released from Rho GDI has largely been unknown, it has
been shown that ERM, which consists of ezrin, radaxin,
and moesin, has the capacity to displace the GDP-bound
form of Rho proteins from Rho GDI␣ (700). ERM has two
functional domains; the NH2-terminal plasma membranebinding and COOH-terminal F-actin-interacting domains
(30, 730). ERM is translocated to the plasma membrane
probably through the interaction with the cytoplasmic
domain of integral plasma membrane proteins such as
CD44, providing the F-actin binding sites. Rho GDI␣ interacts with the NH2-terminal fragments of ERM, and this
interaction reduces Rho GDI␣ activity (700). The unfolding of ERM may induce Rho GDI␣ interaction with ERM
NH2-terminal regions, causing release of the Rho-GDP
and making it sensitive to the action of each Rho GEP
(699, 700). The interaction of full-length ERM with the
cytoplasmic fragment of CD44 does not induce the association of ERM with Rho GDI␣ (700). However, CD44 and
Rho GDI␣ are coimmunoprecipitated with moesin (274).
It is likely that full-length ERM unfold to permit interactions with Rho GDI␣ and CD44 via ERM NH2-terminal
regions and F-actin via ERM COOH-terminal regions; a
signal from CD44 may trigger this process. These findings
suggest that the activation of Rho proteins is regulated in
a temporally and spatially dynamic manner and is distinct
from that of Ras proteins, which appears to be regulated
in a unidirectional manner.
Little is known about the physiological function of
Rho GDIs in vivo, but microinjection studies have shown
that Rho GDI␣ inhibits several downstream functions of
Rho proteins (303, 352, 475, 521, 630, 705, 706). Rho
GDI␣-deficient mice have revealed several abnormal phenotypes (722). These mice are initially viable, but they
develop massive proteinuria mimicking nephrotic syndrome in humans, leading to death due to renal failure
within a year. Histologically, degeneration of tubular epithelial cells and dilatation of distal and collecting tubules
are readily detected in the kidneys. Rho GDI␣-deficient
mice are also infertile and show impaired spermatogenesis with vacuolar degeneration of seminiferous tubules. In
contrast, Rho GDI␤-deficient mice show normal hemato-
Volume 81
This action of GTP␥S is inhibited by C3 or an exoenzyme
of Staphylococcus aureus, named EDIN, which also inhibits the function of Rho proteins, and it is mimicked by
GTP␥S-RhoA, but not by GDP-RhoA, indicating that Rho
proteins are involved in GTP␥S-induced Ca2⫹ sensitization of smooth muscle contraction (276). Subsequently,
myosin light-chain phosphatase has been shown to be a
downstream effector of Rho proteins (232, 524). ROCK/
Rho kinase has finally been shown to phosphorylate and
to inhibit myosin light-chain phosphatase, causing the
sustained contraction of smooth muscle contraction even
after decrease in Ca2⫹ concentrations (347). In this mode
of action, Rho proteins do not induce smooth muscle
contraction without Ca2⫹ triggering; rather, they modify
Ca2⫹ sensitivity of smooth muscle contraction. In this
sense, the physiological relevance of the ROCK/Rho kinase-induced direct phosphorylation of the myosin light
chain and the subsequent activation of the myosin ATPase
could not be considered (13). The physiological relevance
of the phosphorylation of other substrate proteins, including moesin, is also controversial. ROCK/Rho kinase phosphorylates moesin, which induces microvilli formation
(202), but another report indicates that ROCK/Rho kinase
is not involved in this phosphorylation (452). p140mDia, a
mammalian homolog of Bni1 and Bnr1 in the yeast S.
cerevisiae (304, 363) and that of Drosophila diaphanous
(92), has also been implicated as a downstream effector
of Rho proteins (770). mDia has FH1 and FH2 domains.
mDia as well as Bni1 and Bnr1 bind profilin via its FH1
domain to regulate reorganization of the actin cytoskeleton. Overexpression of mDia induces weak formation of
stress fibers without affecting the formation of focal adhesions (510, 769). ROCK and mDia cooperatively regu-
3. Effectors of Rho/Rac/Cdc42 proteins
Rho Proteins
Effectors
Mammal
Yeast
PI 3-kinase
Phospholipase D
PI 4,5-kinase
Citron
ROK/ROCK/
Rho kinase
MBS
PKN
Rhophilin
Kinectin
Rhotekin
p140mDia
DGK␨
Pkc1
Fks1, 2,
(glucan synthase)
Bni1
Bnr1
PI, phosphatidylinositol.
Rac Proteins
Cdc42
Reference no.
Effectors
Reference no.
Effectors
Reference no.
812
431
112, 592
423
307, 393, 451
NADPH oxidase
PAK
PI 3-kinase
PI 4,5-kinase
IQGAP
POR1
S6-kinase
MLK2, 3
Sra-1
POSH
DGK
2, 358, 479
439
57, 724
263, 724
65, 374, 458
745
113
502
359
716
725
ACK
PI 3-kinase
PAK
WASP
S6-kinase
IQGAP
MLK2, 3
N-WASP
MRCK
MSE5
Borg
438
820
439
31, 698
113
65, 374, 458
502
470
394
76
318
Ste20
Cla4
Bni1
Skm1
Gic1, 2
819
137
175, 363
446, 652
106
347
14, 768
768
292
588
770
293
330, 525
580, 456
363
304
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ments has established that Rho proteins regulate complicated cell functions through multiple effectors in a cooperative manner. Another protein having FH domains has
also been found in yeast and named Bnr1 (304). This
protein serves as an effector of Rho4 and binds both
profilin and Aip3 (Bud6), indicating that Rho4 regulates
the actin cytoskeleton at least though Bnr1. Rho2 has 53%
identity to Rho1, and a RHO2 null mutant does not show
any growth defect (422). Hence, it has been assumed that
Rho2 has redundant functions with Rho1, although several lines of evidence suggest Rho2-specific functions
(149, 436). Rho3 and Rho4 are implicated in polarized
growth presumably through regulating the actin cytoskeleton via their interactions with Bni1 and Bnr1 (175, 331a).
Moreover, it has been shown that Rho3 interacts with
Myo2 (type V myosin) and Exo70 (a component of the
exocyst, a multiprotein complex which is involved in
exocytosis) (606), suggesting the involvement of Rho3 in
the polarized secretion.
Numerous downstream effectors of mammalian Rho
proteins have been identified (Table 3). p160ROCK, also
named ROK␣/Rho kinase, is a recently identified, serine/
threonine protein kinase (307, 395, 451) and a downstream effector of Rho proteins. The activity of this protein kinase is stimulated by GTP-Rho proteins. Many
ROCK/Rho kinase substrates have been identified; these
include the myosin binding subunit of myosin light-chain
phosphatase (347), myosin light chain (13), ERM (202),
and cofilin (425). Of these substrates, myosin light-chain
phosphatase appears to be a physiological substrate
(347). Guanosine 5⬘-O-(3-thiotriphosphate) (GTP␥S), a
poorly hydrolyzable GTP analog, increases the sensitivity
of smooth muscle contraction to Ca2⫹ (354, 487, 785).
TABLE
169
SMALL G PROTEINS
January 2001
170
TAKAI, SASAKI, AND MATOZAKI
E. Mode of Action of Rac/Cdc42 Proteins
in Cytoskeletal Reorganization
The function of Rac/Cdc42 proteins in cytoskeletal
reorganization was first demonstrated in the yeast. In the
yeast S. cerevisiae, Cdc42 participates in recognition of
the landmark that defines the incipient bud site (5, 320,
827). In fibroblasts, Rac proteins regulate formation of
lamellipodia and membrane ruffles and subsequent stress
fiber formation (602). In contrast, Cdc42 plays a key role
in the formation of filopodia at the cell periphery followed
by the formation of lamellipodia and membrane ruffles
(368, 522). Both Rac/Cdc42 proteins induce the assembly
of multimolecular focal complexes at the plasma membrane of fibroblasts (522). In addition, Rac/Cdc42 proteins
regulate the cadherin-based cell-cell adhesion and hence
control the HGF- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell scattering of Madin-Darby canine
kidney cells (284, 324, 331, 361, 624, 686, 707). Cdc42 may
play a role in the polymerization of both actin and microtubules toward antigen-presenting cells (689). Furthermore, the involvement of Cdc42 in the control of cytokinesis in HeLa cells and Xenopus embryos has been
reported (156, 164). In Drosophila, Rac/Cdc42 proteins
are implicated in axonal guidance, wing hair formation,
actomyosin-driven furrow canal formation, and nuclear
positioning (130, 165, 214).
To date, several potential effectors of Cdc42 and Rac
proteins have been identified (Table 3). Several of these
proteins are common effectors for both Rac and Cdc42
proteins. Among them, the family of serine/threonine protein kinases known as PAKs are especially interesting.
Homologs of the mammalian PAKs have been identified in
S. cerevisiae (Ste20 and Cla4) (137, 384), S. pombe (Pak1,
also called Shk1) (443, 556), Drosophila (PAK1) (258),
and C. elegans (Ste20) (108). In the yeast S. cerevisiae,
Cdc42 interacts with Ste20, which in turn associates with
Ste5 and Bem1, both of which interact with actin (387,
821). Drosophila PAK1 also plays a role in dorsal closure
(258). Thus it is likely that PAK proteins are involved in
mediating the effect of Cdc42/Rac proteins on the cytoskeleton. Most recently, Drosophila Trio, a GEP for Rac
proteins, has been shown to play an important role in
axon guidance (32, 43, 403, 515). One of the two Trio GEP
domains is critical in photoreceptor axon guidance (515).
This GEP domain activates Rac, which in turn activates
PAK. Trio interacts genetically with Rac proteins, PAK,
and DOCK, an SH2-SH3 docking protein. Thus Trio regulates Rac proteins, which subsequently activates PAK,
linking guidance receptors to the growth cone cytoskeleton (515). However, in mammalian cells, the role of PAKs
remains unclear (Fig. 6B). Expression of a mutant form of
Rac or Cdc42 protein that is unable to bind and activate
PAKs can still induce the formation of membrane ruffles
and lamellipodia (323, 379), indicating that PAKs are not
essential for the Rac protein-elicited membrane ruffling
and lamellipodium formation or for the Cdc42-triggered
filopodium formation. This does not exclude the possibility, however, that PAKs themselves may play a role in
cytoskeletal rearrangement by inducing actin reorganization independently of Rac/Cdc42 proteins. Alternatively,
PAKs may mediate effects on the cytoskeleton induced by
Rac/Cdc42 proteins, which are different from the immediate actin reorganization (437, 653).
All PAKs identified to date share a similar 18-amino
acid CRIB (Cdc42/Rac interactive binding) motif that mediates the interaction with Rac/Cdc42 proteins. Most recently, N-WASP, a ubiquitously expressed Cdc42-interacting protein (470), and the Arp2/3 complex have been
shown to participate in the downstream cascade of
Cdc42-induced actin polymerization (49, 417, 418, 610,
697, 775, 782) (Fig. 6C). Wiskott-Aldrich syndrome protein
(WASP), which is only expressed in hematopoietic cells,
was originally identified as a protein mutated in patients
with Wiskott-Aldrich syndrome (31, 698). WASP and NWASP (470) possess a PH domain that binds PIP2 and a
CRIB domain. The binding of both GTP-Cdc42 and PIP2 to
N-WASP activates N-WASP by stabilizing the active conformation of this molecule. The COOH terminus of NWASP binds the Arp2/3 complex and consequently stimulates its ability to nucleate actin polymerization in vitro
(49, 415, 417, 418, 610, 697, 775, 782). Therefore, the
interaction of N-WASP with the Arp2/3 complex is a core
mechanism that directly connects the Cdc42-mediated
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late the Rho protein-induced reorganization of the actin
cytoskeleton (510, 769) (Fig. 6A). ROCK is part of another
downstream cascade of Rho proteins, a Rho-ROCK-LIM
kinase pathway (425). ROCK directly phosphorylates LIM
kinase, which in turn is activated to phosphorylate cofilin.
Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIM kinase (425,
693). Overexpression of LIM kinase induces the formation
of actin stress fibers in a ROCK-dependent manner. Thus
phosphorylation of LIM kinase by ROCK and subsequent
increased phosphorylation of cofilin by LIM kinase contribute to the Rho protein-induced reorganization of the
actin cytoskeleton.
In addition to ROCK, a variety of Rho effector proteins have recently been discovered (Table 3). These include serine/threonine protein kinase PKN (14, 768),
citron (424), rhotekin (588), and rhophilin (768). Citron
contains a protein kinase domain that is related to ROCK.
Citron kinase localizes to the cleavage furrow and midbody of cultured cells (423). Overexpression of citron
mutants results in the production of multinucleate cells,
and a kinase-active mutant causes abnormal contraction
during cytokinesis, suggesting that citron kinase is involved in the Rho protein-regulated cytokinesis (423).
Volume 81
January 2001
SMALL G PROTEINS
F. Mode of Action of Rho/Rac/Cdc42 Proteins
in Gene Expression
Whereas Ras proteins control the activation of the
p42/44 MAPK cascade, in certain cell types, Rac/Cdc42
proteins regulate the activation of Jun NH2-terminal kinase (JNK) and p38 MAP kinase (126, 473). Expression of
a constitutively active mutant of Rac or Cdc42 protein in
HeLa, NIH 3T3, and COS cells results in a stimulation of
JNK and p38 activities (126, 473). Furthermore, the same
effects have been observed with oncogenic GEPs for
Rho/Rac/Cdc42 proteins, such as Dbl (473). The exact role
of Rho/Rac/Cdc42 proteins in MAP kinase activation is,
however, unclear, and it is still largely unknown whether
physiological activation of the JNK pathway is induced by
endogenous Rac/Cdc42 protein activity. Genetic analysis
of dorsal closure in Drosophila also supports a role for
Rac proteins in JNK regulation (227).
Rho/Rac/Cdc42 proteins have each been reported to
activate serum response factor (SRF) (272, 776). They
have also been shown to activate the transcription factor
NF␬B (567, 692). In addition, generation of reactive oxy-
gen species by Rac proteins might be the trigger for NF␬B
activation. This observation may be related to the fact
that Rac proteins regulate an NADPH oxidase enzyme
complex in phagocytes to produce superoxide as described earlier (1, 2, 18, 358, 479, 649). Rho/Rac/Cdc42
proteins are required for G1 cell cycle progression (420,
552, 794), but it is unclear whether this is due to their
effects on the actin cytoskeleton and integrin adhesion
complexes or, instead, to more direct effects on gene
transcription.
G. Other Functions of Rho/Rac/Cdc42 Proteins
Various studies have suggested the involvement of
Rho/Rac/Cdc42 proteins in membrane-trafficking processes. Cytoskeletal rearrangements are closely coupled
to the onset of phagocytosis. Rho/Rac/cdc42 proteins are
implicated in one or more steps of the phagocytic response (86, 129, 449). Two distinct mechanisms for the
phagocytic response have recently been defined (8, 514).
In type I phagocytosis, plasma membrane protrusions
extend to engulf the particle and drag it into the cell; this
is mediated by coordinated actions of Rac/Cdc42 proteins
(86). In type II phagocytosis, particles sink into actin-lined
investigations in the plasma membrane; here, internalization is dependent on RhoA (86). There are morphological
similarities between these processes and the invasion of
mammalian cells by certain pathogenic bacteria. Shigella
invasion starts with actin nucleation and Rho proteininduced actin polymerization (4, 496). This is followed by
continued actin polymerization around membrane-bound
protrusions that fold over the bacterium and coalesce to
engulf it. The complete inhibition of Shigella-induced
membrane folding by C3 suggests that actin polymerization is essential for the generation of the surface extensions (4, 496). Interestingly, Cdc42 has been shown to
play a direct role in Salmonella internalization (107) and
in Shigella flexneri motility (696).
Rho/Rac proteins have also been implicated in the
regulation of endocytosis. In mammalian cells, expression
of dominant active mutants of Rho/Rac proteins reduces
the efficiency of endocytosis of the transferrin receptor
(380), and a dominant active mutant of RhoA also blocks
internalization of muscarinic acetylcholine receptors
(757). Expression of a dominant active mutant of RhoA
causes an increase in clathrin-independent endocytosis in
Xenopus oocytes (639). In Swiss 3T3 cells, expression of
a dominant active mutant of Rac proteins stimulates pinocytosis (602), and the pinocytic vesicles are coated
with the Rac signaling partner PAK1 (150). However,
expression of a dominant active Rac protein has no effect
on pinocytosis in baby hamster kidney cells (398); thus
the role of Rac proteins in pinocytosis is still unclear.
Rho/Rac proteins have also been implicated in the
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signal transduction pathway to the stimulation of actin
polymerization.
In addition to the CRIB-containing proteins, a 34-kDa
protein, POR1 (partner of Rac), interacts specifically with
GTP-Rac proteins and is implicated in mediating Rac
protein-dependent membrane ruffling (745). A dominant
active mutant of Rac1 (Rac1V12L37) that fails to bind
POR1 also fails to induce membrane ruffling (323). Arf6 is
also implicated downstream of Rac proteins and mediates
membrane ruffling through PIP2 kinase ␣ (283). Arf6 and
POR1 function on distinct signaling pathways to mediate
cytoskeletal reorganization induced by Rac proteins (Fig.
6B). Another protein with a potential role in cytoskeletal
organization is IQGAP (65, 374, 458). IQGAP interacts
with both GTP-Rac1 and GTP-Cdc42 and localizes to
membrane ruffles. IQGAP has been shown to be localized
to cell-cell adhesion sites (375). It has been suggested that
activated Cdc42 blocks the ability of IQGAP to inhibit
asse bly of a cadherin-catenin complex and thereby promotes formation of adherens junctions (375), but this
model has not yet been substantiated. In addition, no
apparent abnormality is observed in the formation of adherens junctions in the IQGAP1-deficient mice (400). Although IQGAP contains some interesting motifs found in
signaling molecules such as a WW domain, an SH3-binding domain, a calmodulin-binding domain, and, somewhat
surprisingly, a Ras GAP-like motif, its function remains
unknown. Although a number of potential Rho family
effectors have been identified, a major goal for the future
will be to determine the physiological roles of these proteins in Rho family-mediated cytoskeletal rearrangement.
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TAKAI, SASAKI, AND MATOZAKI
teins regulate intracellular vesicle trafficking was first
obtained genetically in the yeast S. cerevisiae. Many
genes essential for secretion were isolated and named the
SEC genes (527). One of the SEC genes, the SEC4 gene,
was shown to encode a small G protein that is required for
vesicle trafficking from the Golgi apparatus to the plasma
membrane (623). The yeast YPT1 gene was first discovered as a gene located between the actin and tubulin
genes in 1983 (208) and was later identified to encode a
small G protein (641). Ypt1 was shown to be later involved in vesicle trafficking from the endoplasmic reticulum to the Golgi apparatus (33, 650). Subsequently, a large
body of evidence has accumulated in support of a role for
Rab proteins in vesicle trafficking from yeast to human
(447, 528, 529, 550, 636).
B. Vesicle Trafficking
Transmembrane proteins and secreted, soluble proteins are transported from one membrane compartment
to another by vesicles (465, 579, 616) (Fig. 7). Newly
synthesized secretory proteins are translocated into the
endoplasmic reticulum and are then transported to the
plasma membrane via the Golgi apparatus by vesicles;
some proteins are delivered to prelysosomes. In parallel,
macromolecules that are taken up from the plasma membrane are transported inward to endosomes and lysosomes by vesicles. Some cell-surface proteins including
receptors for extracellular ligands transit through a recycling endosome and are recycled back to the plasma
membrane. Thus exocytosis, endocytosis, and recycling
are performed by intracellular vesicle trafficking. There
are two exocytotic pathways: regulated and constitutive
pathways. In the regulated pathway, secretory proteins
are stored at very high concentrations in the core of
V. RAB PROTEINS AS REGULATORS
OF VESICLE TRAFFICKING
A. Outline
Rab proteins exist in all eukaryotic cells and form the
largest branch of the small G protein superfamily (103,
447, 528, 529, 550, 636). The yeast S. cerevisiae genome
sequence encodes 11 Rab proteins (383). In mammalian
cells, over 50 Rab proteins (including isoforms) are
known (447, 528, 529, 550, 636). Evidence that Rab pro-
FIG. 7. Principal mechanism of intracellular vesicle trafficking. Rab,
Rab proteins; Sar1/Arf, Sar1/Arf proteins.
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regulation of secretory vesicle transport (69, 555a). In
mast cells, Rho/Rac proteins stimulate the exocytosis of
secretory granules, whereas C3 and dominant-negative
mutants of Rho/Rac proteins inhibit GTP␥S-induced secretion (526). In PC12 cells, RhoA, Rac1, and Rho GDI are
also involved in Ca2⫹-dependent exocytosis at least partly
through the reorganization of actin cytoskeleton, and the
activation of RhoA or Rac1 alone is not sufficient for this
reaction (365). RhoD and RhoB are localized to endocytic
vesicles, suggesting that they might perform roles in the
regulation of intracellular trafficking (209, 498).
Rho/Rac/Cdc42 proteins have been implicated in
morphogenesis by regulation of cytoskeletal rearrangements. For instance, the Drosophila homologs of Rac1,
Rac2, and Cdc42, namely, DRac1, DRac2, and DCdc42,
respectively, are highly expressed in the nervous system
and in the mesoderm during development, respectively
(411, 412). Particularly, the inhibition of axonal outgrowth
induced by a dominant active or dominant negative mutant of DRac1 is due to different cytoskeletal defects in
developing neurons, suggesting that cyclical activation
and inactivation of DRac1 is crucial for axonal development (214, 412). In addition, expression of a dominant
active mutant of DCdc42, DCdc42V12, in the neurons of
fly embryos results in a similar but qualitatively different effect on neuronal development, compared with
DRac1V12 (412). In mammalian N1E-115 neuroblastoma
cells, Rho proteins appear to inhibit neurite outgrowth,
whereas Rac/Cdc42 proteins promote neurite outgrowth,
presumably by regulating the formation of growth cone
filopodia and lamellipodia (277, 369). In contrast, in chick
dorsal root ganglion neurons, Rho proteins induce neurite
outgrowth, while an activated mutant of Rac proteins
increases the proportion of collapsed growth cones (317).
DRac1 and DCdc42 have also been implicated in muscle
differentiation (172, 412). Rac/Cdc42 proteins also regulate muscle development, probably via their effects on
membrane fusion and the actin cytoskeleton. Finally, little is known at present about the role of Rho proteins in
neuronal and muscle development. In summary, Rho/Rac/
Cdc42 proteins have multiple essential functions during
morphogenesis.
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SMALL G PROTEINS
regulate the budding process, which is mainly regulated
by Sar1/Arf proteins (see sect. VI).
There are two types of Rab proteins: one type is
involved in regulated secretion, and the other type is
involved in other vesicle transport steps and is essential
for this function (447, 528, 529, 550, 636). Of the first type,
the mode of action of Rab3A has most extensively been
investigated (703). Rab3A plays a key regulatory role in
Ca2⫹-dependent exocytosis, particularly in neurotransmitter release from nerve terminals. Studies on Rab3Adeficient mice have revealed that Rab3A is not essential
for basal neurotransmission but modulates it, thereby
contributing to synaptic plasticity (91, 216, 217, 218, 408).
In contrast, mutations of Rab proteins mediating other
vesicle transport steps can have more dramatic consequences. In the yeast S. cerevisiae (383), Rab mutants are
often characterized by a massive accumulation of the
vesicles in the respective secretory pathways (623). Moreover, some of them are essential for cell viability.
FIG. 8. Subcellular localization of Rab proteins. Plain,
localization of each Rab protein; italic, functioning site of
each Rab protein.
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secretory granules that can be mobilized by an exocytosis
pathway in response to neural or hormonal stimuli. In
contrast, constitutive exocytosis occurs independently of
extracellular stimuli and proceeds at a steady rate. Constitutive exocytosis is responsible for the insertion of
plasma membrane proteins and secretion of extracellular
proteins, such as matrix components and plasma proteins. Vesicle trafficking is needed for various other cell
functions, such as establishment of cell polarity, cytokinesis, and cell motility.
There are at least four principal events in a given step
of intracellular vesicle transport: 1) budding of a vesicle
from the donor membrane; 2) targeting of the vesicle to
the acceptor membrane; 3) docking of the vesicle to the
acceptor membrane; and 4) fusion of the vesicle with the
acceptor membrane (Fig. 7). Rab proteins regulate these
processes (447, 528, 529, 550, 636). Substantial evidence
has accumulated that most Rab proteins regulate the
targeting/docking/fusion processes and that some of them
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TAKAI, SASAKI, AND MATOZAKI
C. Localization of Rab Proteins
D. Rab Protein Cycle: Cyclical
Activation/Inactivation and Translocation
Rab proteins cycle between the GDP-bound inactive
and GTP-bound active forms and between the cytosol and
the membranes, and these cyclical activation, inactivation, and translocation are regulated by at least three
types of regulators: GEPs, GDIs, and GAPs (447, 528, 703)
(Fig. 9). In the cytosol, a Rab protein is maintained in the
GDP-bound inactive form by a GDI. The GDP-bound form
is first released from the GDI by a still unknown mechanism that is coupled to the delivery of the Rab protein to
a specific membrane compartment (673, 735); the Rab
protein is subsequently converted to the GTP-bound form
by the action of a GEP. The GTP-bound form then interacts with a downstream effector(s). Thereafter, the GTPbound form is converted to the GDP-bound form by the
action of a GAP. The GDP-bound form produced on the
membrane then complexes with the GDI and returns to
the cytosol as a Rab protein-Rab GDI complex. Thus a
characteristic of Rab proteins is that a cycle of membrane
association/dissociation is superimposed onto their GDP/
GTP cycle.
Yeast Dss4 and its mammalian counterpart, Mss4,
were first isolated and characterized as Rab GEPs (79,
497). Dss4 is active on both Sec4 and Ypt1 and Mss4 is
active on a subset of Rab proteins. Moreover, the exchange activities of both GEPs are very low compared
with the values of other GEPs for Ras and Rho/Rac/Cdc42
proteins. These proteins are unlikely to serve as physiologically relevant GEPs, and their real roles are currently
unknown. Only three physiological Rab GEPs have been
isolated and characterized: Rab3 GEP, specific for the
Rab3 subfamily members (762); Rabex-5, specific for the
Rab5 subfamily members (290); and Sec2 for Sec4 (763).
Rab3 GEP is ubiquitously expressed but abundant in
brain (762). It is active at least on Rab3A, Rab3C, and
Rab3D and is inactive on other Rab proteins, including
FIG. 9. Cyclical activation/inactivation of Rab proteins
and their translocation. Rab, Rab proteins.
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Rab proteins contain unique, hypervariable COOHterminal domains with two Cys residues, both of which
require geranylgeranyl moieties by geranylgeranyltransferase type II as described above. This lipid modification
is essential for the membrane association of Rab proteins
(27, 322, 340, 349, 568, 765). The COOH-terminal hypervariable region is important for the correct targeting of
Rab proteins to their target membranes (64, 102), but how
it directs localization remains to be clarified.
At steady state, 10 –50% of a given Rab protein is
detected in the cytosol. This pool is maintained in the
GDP-bound form through interaction with Rab GDIs.
Each Rab protein is localized at organelles and is involved
in the vesicle transport of the organelle (447, 528, 529, 550,
636) (Fig. 8). For example, Rab1, Rab2, and Rab6 are
localized at the endoplasmic reticulum and the Golgi apparatus and regulate vesicle transport along the biosynthetic/secretory pathway (104, 240, 265, 448, 575, 720);
Rab3 is localized on secretory granules including synaptic
vesicles and is involved in Ca2⫹-dependent exocytosis
(91, 140, 183–185, 216, 217, 282, 319, 408, 476, 477, 625);
Rab4 and Rab5 are present on early endosomes and regulate early steps of the endocytic process, mediating endosome-endosome fusion and receptor recycling, respectively (73, 104, 237, 747, 748). However, the sites of action
of the majority of Rab proteins have not yet been elucidated. In addition, it seems likely that some functional
specialization of Rab proteins takes place, both in different cell types and in different organisms. One example is
Rab11A and Rab11B and its two yeast homologs Ypt31p
and Ypt32p. A role for Rab11 has been documented in late
recycling of transferrin receptors (590, 736) and in transGolgi network-to-plasma membrane transport (109). On
the other hand, in yeast, Ypt31p and Ypt32p are involved
in intra-Golgi transport or protein export from the transGolgi (46).
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SMALL G PROTEINS
membrane fusion. In contrast, for Rab3A, the GTP-bound
form might interact with a prefusion complex, thus preventing fusion. The GTPase-deficient mutant of Rab3A
inhibits Ca2⫹-dependent exocytosis from PC12 cells and
chromaffin cells (282, 319) (see below). In this case, Rab3
GAP plays a crucial role in the function of Rab3 proteins.
Three isoforms of Rab GDI have been isolated: Rab
GDI␣ (Rab GDI-1), Rab GDI␤, and Rab GDI␥ (Rab GDI2)(315, 453, 520, 629, 666). Southern blot analysis of
genomic DNA indicates that both mouse and rat contain
at least five Rab GDI genes (315). In contrast, there is only
one Rab GDI gene (GDI1) in the yeast S. cerevisiae, and
it is essential for cell viability (213). The properties of Rab
GDI␣ and Rab GDI␤ have most extensively been studied
(26, 27, 28, 315, 520, 629, 665, 666, 800). Rab GDI␤ is
ubiquitously expressed, whereas Rab GDI␣ is abundantly
expressed in neuronal cells (520). Despite the different
tissue distribution, both isoforms show similar biochemical properties (26, 28, 520, 629, 800). 1) Both proteins
bind the lipid-modified, GDP-bound form, but not any
other forms such as the lipid-modified, GTP-bound form,
the lipid-unmodified GDP-bound form, and the lipid-unmodified GTP-bound form. 2) Both proteins inhibit the
basal and GEP-stimulated dissociation of GDP from the
GDP-bound form. 3) Both proteins inhibit the binding of
the GDP-bound form to the membrane and keep it in the
cytosol. 4) When the GDP-bound form is produced from
the GTP-bound form on the membrane, both proteins
bind the GDP-bound form and retrieve it from the membrane. In addition to these activities, Rab GDI plays an
important role in specific delivery of Rab proteins to their
target membranes (572, 673, 703, 735). For instance, Rab5Rab GDI and Rab9-Rab GDI complexes mediate in vitro
binding of Rab5 and Rab9 to organelles where these proteins normally reside (673, 735). The GDP/GTP exchange
reaction does not seem to be a prerequisite for the Rab
protein binding to the membranes, because the GDPbound form can be transiently detected in the membranes
after delivery of Rab protein-Rab GDI complexes onto
membranes is likely to be followed by GDP/GTP exchange and the release of the GDI. A GDI dissociation
factor (GDF) has been identified that is active on Rab5
and Rab9 and devoid of GEP activity but is not yet fully
characterized (154).
Rab GDI␣ and Rab GDI␤ are significantly related to
the mammalian Choroideremia gene product CHM,
which delivers Rab proteins to the catalytic subunit of
geranylgeranyltransferase II as described above (645).
Choroideremia is an X-linked form of retinal degeneration (267). Recently, the crystal structure of Rab GDI␣ has
been determined, and the sequence-conserved regions are
clustered on one face of the molecule (633). The two most
sequence-conserved regions, which form a compact structure at the apex of Rab GDI␣, play a crucial role in the
binding of Rab proteins. Mutations of this region in the
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Rab2, Rab5A, Rab10, and Rab11. It prefers the lipid-modified form to the lipid-unmodified form as a substrate. A C.
elegans counterpart gene of this mammalian GEP, Aex-3,
has been identified genetically; mutation of this gene
causes a defect in synaptic transmission (310). Rabex-5
forms a tight complex with Rabaptin-5, a Rab5 effector,
and activation of Rab5 by Rabex-5 is likely coupled to
effector recruitment (290). Rabex-5 is homologous to
Vps9, a yeast protein involved in endocytosis (77). In the
yeast, Sec2, a GEP for Sec4, is genetically essential for the
Golgi-to-plasma membrane transport (763). Sec2 shares
sequence homology to Rabin-3 that specifically interacts
with Rab3A and Rab3D (68). However, Rabin-3 has no
detectable GEP activity. These data suggest that each
member or each subfamily of the Rab family could interact with a specific GEP. It is not known whether Rabex-5
and Sec2 require lipid modifications of their small G protein substrates.
Only two GAPs for Rab proteins have been isolated
and characterized in mammalian cells: Rab3 GAP, specific for the Rab3 subfamily members (205, 501) and
GAPCenA, specific for Rab6 (135). Rab3 GAP is ubiquitously expressed and enriched in the soluble synaptic
fraction in brain (205, 501, 543). Rab3 GAP consists of two
subunits, a catalytic and a noncatalytic subunit (205, 501).
It is active at least on Rab3A, Rab3B, Rab3C, and Rab3D
and is inactive on other Rab proteins, including Rab2,
Rab5A, and Rab11 (205). It prefers the lipid-modified form
to the lipid-unmodified form as a substrate. The role of the
noncatalytic subunit of Rab3 GAP is unknown, but Sar1
GAP also consists of catalytic (Sec23) and noncatalytic
(Sec24) subunits (270, 808) (see sect. VI). GAPCenA is
ubiquitously expressed (135). It is mainly cytosolic, but a
minor pool is associated with the centrosome. It forms a
complex with ␥-tubulin and may play a role in microtubule nucleation. In the yeast S. cerevisiae, five GAPs have
been isolated and characterized: Gyp1 (GAP for Sec4 and
Ypt1), Gyp6 (GAP for Ypt6), Gyp7 (GAP for Ypt7), Mdr1p/
Gyp2p (GAP for Ypt6 and Sec4), and Msb3p/Gyp3p (GAP
for Sec4, Ypt6, Ypt51, Ypt31/ Ypt32, and Ypt1) (7, 163, 691,
760). Interestingly, Gyp proteins and GAPCenA (but not
Rab3 GAP) share significant homology to yeast cell cycle
checkpoint proteins, Bub2 and Cdc16, raising a possibility
that these Rab GAPs serve to coordinate membrane trafficking with other events taking place during mitosis, such
as the control of microtubule nucleation.
GAPs terminate the function of Rab proteins, but it is
not yet clear whether GTP hydrolysis is important for Rab
proteins to accomplish their functions. The cycle of GTP
hydrolysis of Rab5 has been shown to be uncoupled to the
Rab5-regulated endosome-endosome fusion (619). Similarly, the GTP hydrolysis of Ypt1 seems dispensable for
the Ypt1-mediated fusion events in the yeast (598). In
these cases, the GTP-bound form seems to be requisite or
even stimulatory for the progression of a vesicle toward
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TAKAI, SASAKI, AND MATOZAKI
human Rab GDI␣ gene are responsible for X-linked nonspecific mental retardation (see sect. VG) (138).
E. SNAREs and Tethering Proteins in Vesicle
Targeting/Docking/Fusion
endosome fusion process (115). EEA1 has recently been
found to be an effector of Rab5 (115) (see below). It is
likely that the events that precede stable, SNARE-dependent docking of vesicles or membranes are the result of a
network of interactions between many proteins including
tethering proteins. Although the direct interaction between Rab proteins and the tethering proteins has only
been documented for Rab5/EEA1, Sec4, and the exocyst,
Rab proteins are likely candidates for orchestrating vesicle targeting through tethering proteins as described below.
F. Mode of Action of Rab Proteins in Vesicle
Targeting/Docking/Fusion
Several Rab protein effectors have been isolated and
characterized. The first effector to be identified is Rabphilin-3 for the Rab3 subfamily members (662, 663). Rabphilin-3 is expressed in neurons and interacts preferentially
with GTP-Rab3A. Rabphilin-3 is present on the surface of
synaptic vesicles (478). It has not been established how
Rabphilin-3 localizes to synaptic vesicles: one model is
that it is recruited to the vesicles by Rab3A, analogous to
the Ras protein-Raf protein kinase system (397, 683);
another model is that it is targeted to synaptic vesicles
independently of Rab3 (460, 654, 664). The NH2-terminal
region constitutes the Rab3-binding region; the COOHterminal region contains two C2 domains that bind Ca2⫹
and phospholipid (566, 662, 792). Expression or microinjection of either the NH2-terminal or the COOH-terminal
region of Rabphilin-3 blocks Ca2⫹-dependent exocytosis
in several different systems (78, 117, 364). Recently, it was
shown that the abnormalities of synaptic transmission
and synaptic plasticity that are observed in Rab3A-deficient mice are not observed in Rabphilin-3-deficient mice
(638). This observation suggests that another Rab3 effector is present and compensates for loss of function of
Rabphilin-3 in these mice. Rim is another effector of Rab3
proteins (766). Rim has an NH2-terminal Rab3A-binding
domain that is homologous to that of Rabphilin-3, a cen-
FIG. 10. Possible modes of action of Rab
proteins, tethering proteins, and SNARE proteins in vesicle targeting/docking/fusion processes. Rab, Rab proteins.
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According to the SNARE hypothesis, there are vesicle and target membrane SNAREs (v- and t-SNAREs) and
these two types of SNAREs interact with each other,
resulting in the docking of vesicles with their target membranes (615) (Fig. 10). Recent results suggest that SNARE
pairing cannot be solely responsible for the appropriate
targeting and docking of transport vesicles. For example,
the docking of endoplasmic reticulum-derived vesicles
with Golgi membranes can occur in the absence of
SNARE components (83). Similarly, initial events leading
to the homotypic fusion of vacuoles are not dependent on
SNARE components (632, 740). Moreover, SNARE components can form promiscuous complexes in vitro (799).
These results suggest that SNAREs are not primarily involved in the specific targeting of vesicles.
Evidence is accumulating that targeting/docking
specificity is determined by the collaboration of several
factors. Tethering proteins play key roles in vesicle targeting/docking (571, 771). This group includes Uso1 (83),
TRAPP (620), p115 (505, 620), exocyst (246), and EEA1
(115), all of which bind membranes before the formation
of SNARE complexes. In S. cerevisiae, Uso1 and a large
protein complex named TRAPP are implicated in endoplasmic reticulum-to-Golgi transport (83, 620, 749); the
exocyst, a large protein complex consisting of Sec3, Sec5,
Sec6, Sec8, Sec10, Sec15, and Exo70, is involved in targeting of post-Golgi vesicles to the plasma membrane
(246). A mammalian version of the exocyst has been
identified in mammalian cells, where it may be involved in
targeting of vesicles (or docking with) the basolateral
membrane of epithelial cells (243a, 295). In mammalian
cells, a complex of p115, GM130, and giantin induces the
interaction of Golgi-derived vesicles with Golgi membranes (505, 675), and EEA1 is involved in the homotypic
Volume 81
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SMALL G PROTEINS
(787), although the function of this domain remains unknown. Rabkinesin-6 is an effector of Rab6, and a member
of the kinesin family (167). This protein displays a conventional kinesin structure with an NH2-terminal motor
domain, followed by a region predicted to form an ␣helical coiled-coil stalk and a tail domain. Rab6 may regulate microtubule-dependent retrograde transport from
the Golgi apparatus through Rabkinesin-6 (777).
Rab11BP/Rabphilin-11, an effector of Rab11, is also involved in microtubule-based vesicle transport (432, 668,
810), although it is not a motor protein. This protein
contains a proline-rich domain within its NH2-terminal
half and WD40 repeats, important for the protein-protein
interactions, within its COOH-terminal half.
Thus several effectors have been identified within the
past 5 years. In addition, the recent determination of the
X-ray crystal structure of Rab3A complexed to the Rab3binding domain of Rabphilin-3 has enabled the identification of complementarity-determining regions, which are
potentially involved in the interactions of Rab proteins
with their effectors (555). These regions exhibit a high
degree of sequence variability among Rab proteins and
might enable them to interact with a wide variety of
effectors (482).
Although it has not been fully elucidated how each
Rab protein regulates vesicle targeting/docking/fusion
processes through their specific effectors, one probable
mechanism is to regulate or facilitate the assembly of
SNARE complexes (Fig. 10). Rab proteins are not core
components of SNARE complexes. However, genetic
studies in yeast have shown that functions of Rab and
SNARE proteins are linked: the effects of a mutation in
the effector domain of Sec4 is suppressed by overexpression of Sec9, the SNAP-25-like protein (63), and Ypt1 is
involved in the priming of t-SNARE (401). In this process,
Rab proteins may function to recruit tethering proteins
onto membranes and coordinate loose membrane tethering to induce the SNARE complex-mediated, tighter and
stable docking process.
GTP-Sec4 interacts with Sec15, a component of the
exocyst (246). Then, a chain of protein-protein interactions leads to the assembly of the exocyst and its binding
to Sec3, which marks the specific site of exocytosis at the
plasma membrane. After Rab5 is activated by Rabex-5
complexed with Rabaptin-5, Rab5 recruits EEA1, which
interacts with PIP3 to early endosome (115, 670). PIP3
might serve to stabilize EEA1 on membranes through
interaction with its FYVE domain. Uso1 is needed to allow
the assembly of SNARE complex and Uso1-regulated tethering is helped by Ypt1 (83). Although it is not clear how
Rab proteins and tethering proteins induce the assembly
of SNARE complexes, they may induce the dissociation of
the Sec1/Munc18 family members which impair the association of t-SNARE with v-SNARE (Fig. 10). Consistent
with this possibility, Vac1, a mammalian homolog of
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tral PDZ domain, and two COOH-terminal C2 domains
that are separated by alternatively spliced sequences. In
contrast to Rab3A and Rabphilin-3, Rim is clearly absent
from synaptic vesicles but enriched on the presynaptic
plasma membrane, especially at the active zone. Although
the precise function of Rim is still obscure, overexpression of its NH2-terminal fragment suggests that Rim is
implicated in neurotransmitter release. The localization of
Rim to the presynaptic plasma membrane is intriguing in
terms of its interaction with vesicle-associated GTPRab3A and the postulated role of Rab proteins in vesicle
docking.
Rabaptin-5 was identified in a yeast two-hybrid
screen using the GTPase-deficient mutant of Rab5 as a
bait (685). Rabaptin-5 consists of two isoforms: Rabaptin-5␣ and Rabaptin-5␤ (241). Rabaptin-5 is recruited from
the cytosol to endosomal membranes by GTP-Rab5. In
addition, its overexpression induces the formation of
large endosomes, a phenotype that is also observed upon
overexpression of a GTPase-deficient mutant of Rab5.
Rabaptin-5 has an NH2-terminal coiled-coil region that
serves as a self-association determinant and a COOHterminal Rab5-binding domain. The NH2 terminus of
Rabaptin-5 has recently been found to also interact with
the GTPase-deficient mutant of Rab4, which is involved in
the receptor recycling process (753). Rabaptin-5 is part of
a large complex required for membrane docking/fusion
processes (457). EEA1, another effector of Rab5, is also
included in this complex (115, 457, 670). EEA1 may represent the core component of this complex and serve as a
tethering protein, because it alone could replace the requirement for cytosol in an early endosome fusion assay
(115). EEA1 contains two spatially separate Rab5-binding
domains and a phosphatidylinositol 3,4,5-trisphosphate
(PIP3) binding, “FYVE finger” at its COOH terminus (670).
Rabaptin-5 also interacts with the NH2-terminal region of
Rabphilin-3, and this interaction is inhibited by GTPRab3A (542). Because endocytosis is often coupled with
exocytosis, particularly at nerve terminals, this Rabphilin3-Rabaptin-5 interaction may contribute to the coupling of
these two events.
A Rab8-interacting protein (Rab8ip) is a serine/threonine protein kinase, closely related to the GC kinase
found in lymphoid germinal centers (591). Like Rab8, this
kinase localizes to the Golgi apparatus where it may
regulate vesicle transport to the cell surface in response
to Rab8. Rab8ip shares sequence homology to PAKs, protein kinases that are effectors of Rac/Cdc42 proteins
(439). An effector of Rab9, p40, is associated with endosomes and shows synergy with Rab9 in its ability to
stimulate transport of the mannose 6-phosphate receptor
from endosome to the trans-Golgi network in an in vitro
transport assay (151). p40 is comprised entirely of six
internally repeated sequences, named kelch repeats,
which were first identified in the Drosophila kelch protein
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TAKAI, SASAKI, AND MATOZAKI
EEA1, interacts not only with an effector of Vps21 (a
yeast Rab protein), but also with Vps45, a member of the
Sec1/Munc18 family (569). In addition, other docking
complexes regulated by Rab proteins have been characterized. For example, Rabphilin-3 and Rim, two effectors
of Rab3 proteins, may be part of a complex that links
synaptic vesicles to the presynaptic plasma membrane in
neurons.
G. Rab3A in Ca2ⴙ-Dependent Exocytosis
H. Rab Proteins and Cytoskeleton
Recently, a kinesin-like protein, termed Rabkinesin-6,
has been identified as a downstream effector of Rab6, for
the first time linking Rab proteins to the microtubule
cytoskeleton (167). Rab6 is associated with highly dynamic tubular structures that move along microtubules
from the Golgi apparatus to the cell periphery (777). Rab6
may facilitate the transport of these structures to their
acceptor compartment, probably the endoplasmic reticulum, either by recruiting Rabkinesin-6 onto membranes or
by changing the biochemical properties of the motor.
Similarly, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility
toward the minus ends of microtubules (516). Rab11 is
colocalized with its effector, Rab11BP/Rabphilin-11, along
microtubules (88, 432, 668, 810). Rab11 and its effector
protein are involved in cell migration, probably via regulation of the vesicle recycling (303, 432). Sec4 promotes
polarized transport of post-Golgi vesicles along the actin
cytoskeleton through an interaction with its GEP, Sec2
(763). Rab proteins may also serve to link vesicles and
target membranes to the cytoskeleton at docking sites.
For example, Rabphilin-3, an effector of Rab3 proteins,
interacts with the actin-bundling protein ␣-actinin (333).
This interaction may induce the local organization of the
actin cytoskeleton for the fusion of synaptic vesicles with
the plasma membrane (233).
I. Rab Proteins in Vesicle Budding
There are several lines of evidence that Rab proteins
may also play a role in the budding process (see below).
Depletion of Ypt31 and Ypt32 in the S. cerevisiae leads to
the accumulation of stacks of membranes that resemble
the typical Golgi cisternae of mammalian cells, suggesting
a role for these proteins in intra-Golgi transport and in the
formation of transport vesicles at the exit face of the
Golgi apparatus (46). Dominant negative mutants of Rab1
and Rab9 appear to block the budding of the vesicles from
the endoplasmic reticulum and late endosomes, respectively (530, 604). A complex of Rab5 and Rab GDI appears
to be required for coat invagination and receptor sequestration in an in vitro assay for clathrin-coated pit assembly
(461). Similarly, clathrin-coated vesicles can recruit Rab5
(287). However, it has not yet been demonstrated that Rab
proteins regulate the assembly of coat proteins as described for Sar1/Arf proteins (see below). Currently, the
possibility cannot be excluded that the effects of Rab
proteins in the budding process may be an unknown,
secondary effect in the targeting/docking process, because after or during the bud formation, coat proteins are
disassembled to produce uncoated vesicles. Before, during, or after this uncoating process Rab proteins may
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Transient overexpression or microinjection of a dominant active mutant of Rab3A inhibits Ca2⫹-dependent
secretion from bovine chromaffin cells and PC12 cells as
described above (282, 319). In contrast, Rab3B stimulates
secretion: in antisense RNA experiments performed in
anterior pituitary cells, reduction in Rab3B mRNA inhibited Ca2⫹-dependent secretion from these cells (407), and
when a dominant active mutant of Rab3B was stably
expressed in PC12 cells, where endogenous Rab3B is
absent, Ca2⫹-dependent norepinephrine secretion was
markedly stimulated (772). The explanation for these apparently opposite effects of Rab3A and Rab3B on Ca2⫹dependent exocytosis is currently unknown.
Studies on Rab3A-deficient mice have revealed an
important insight into Rab3A function. Synaptic depletion
is much faster in the hippocampal CA1 region of these
mice, although two forms of short-term synaptic plasticity, paired-pulse facilitation and posttetanic potentiation,
are unaffected (216). These findings suggest that Rab3A
plays a role in the recruitment of synaptic vesicles for
exocytosis. In Rab3A-deficient, cultured hippocampal
cells, Rab3A is involved in a late step in vesicle fusion
(217). Moreover, Rab3A-deficient mice show reduced formation of postsynaptic long-term potentiation in the CA3
region (91). Thus Rab3A is not essential for basal synaptic
transmission; rather, it modulates synaptic vesicle trafficking, thereby contributing to synaptic plasticity.
Recent genetic analysis of X-linked, nonspecific,
mental retardation has revealed that mutations or deletions of the Rab GDI␣ gene can cause this disease (138).
Notably, Rab GDI␣ is localized to the distal part of chromosome Xq28. Mutations in this region have recently
been described to cause a syndromic form of X-linked
nonspecific mental retardation that comprises epileptic
seizures (29). Consistent with these findings, analysis of
Rab GDI␣-deficient mice has shown that synaptic potentials remain elevated and show reduced depression during repetitive stimulation in the hippocampal CA1 region
(306). In addition, the mice have increased propensity for
seizures. These observations suggest that Rab GDI␣ is
essentially important for vesicle trafficking in neural cells,
and its function may be needed to suppress hyperexcitability, likely through Rab3 proteins.
Volume 81
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SMALL G PROTEINS
be associated with the vesicles (Fig. 7). Consistently,
Rab11BP/Rabphilin-11 (432, 668, 810), a downstream effector of Rab11 mainly implicated in vesicle recycling
(590, 736), directly interacts with mammalian Sec13 (433).
In yeast, Sec13 counterpart is involved in Sar1-induced
vesicle coat assembly (714) (see below). This result provides a physical connection between a Rab protein and a
structural component necessary for vesicle budding.
VI. SAR1/ARF PROTEINS AS REGULATORS
OF VESICLE BUDDING
A. Outline
B. Coat Proteins and Vesicle Budding
Vesicle budding requires the assembly of specific
proteins coating the cytoplasmic face of a donor mem-
brane (634). The assembly of coat proteins is thought to
serve at least two functions: it provides the mechanical
force to pull the membrane into a bud, and it helps to
capture specific membrane receptors and their bound
cargo molecules. Three classes of coated vesicles have
been well-characterized to date: clathrin-, COP (coat protein) I-, and COPII-coated vesicles (634, 640). Clathrin
coats contain clathrin and heterotetrameric adaptor protein (AP) complexes, AP-1, AP-2, AP-3, or AP-4 (278, 350).
Two different clathrin-coated vesicles containing AP-1
and AP-2 carry selected proteins from the trans-Golgi
network to endosomes and lysosomes and from the cell
surface to endosomes, respectively. Another AP complex,
AP-3, is involved in the biogenesis of specialized organelles such as pigmented granules and synaptic vesicles
and in an alternative Golgi-to-vacuolar pathway in yeast.
AP-4 has recently been identified as a novel complex
related to other AP complexes through searches in EST
data, but its function remains to be determined. COPIIcoated vesicles have been shown to be involved exclusively in the export of cargo molecules from the endoplasmic reticulum (35, 38, 634). COPI-coated vesicles
seem to be involved in membrane trafficking between the
endoplasmic reticulum and the Golgi apparatus, in intraGolgi transport, and possibly in endosomes. However, it is
currently a matter of debate as to how many transport
steps COPI is involved in, and whether it functions in
transport in the anterograde or retrograde direction or
both (35, 405, 410). Sar1 and Arf proteins play crucial
roles in the membrane recruitment of the COPII and COPI
components, respectively (38, 405). Arf proteins are also
involved in the recruitment of AP-1 and AP-3 components
to the membranes (278). In contrast, neither of these
small G proteins is involved in the clathrin/AP-2-coated
vesicle formation from the cell surface.
C. Arf Protein Cycle: Cyclical
Activation/Inactivation and Translocation
Arf proteins cycle between GDP-bound, inactive and
GTP-bound, active forms, and the cycling is regulated by
specific GEPs and GAPs (312, 494) (Fig. 11). This cycling
is not regulated by GDIs. The GDP-bound form of Arf
proteins is present in the cytosol and is converted to the
GTP-bound form by the action of a GEP, inducing the
conformational change that allows the myristoylated NH2terminal amphipathic helix of the Arf proteins to interact
with phospholipid bilayers. Nucleotide exchange also induces a conformational change in the switch I and II
regions, allowing Arf proteins to interact with downstream effector (110, 229, 613).
Certain recent studies indicate that the myristate is
exposed and can interact with the phospholipid bilayer
when Arf proteins are still in the GDP-bound form (21).
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The SAR1 gene was originally isolated as a multiple
copy suppressor of a ts mutant of the SEC12 gene (Sar1
GEP gene) that had genetically been identified to be required for transport from the endoplasmic reticulum to
the Golgi apparatus in the yeast S. cerevisiae (508). Subsequently, the SAR1 gene was also shown to be involved
in transport from the endoplasmic reticulum (509, 545).
There is only one SAR1 gene in yeast. Two Sar1 proteins
(Sar1a and Sar1b) have been found in mammals (372).
Arf proteins are homologous to Sar1 with ⬃35% identity. Six Arf proteins and some Arf-like proteins (ARLs
and ARD) have thus far been identified in mammals, and
three members exist in S. cerevisiae (493). Arf proteins
were originally purified from rabbit liver and bovine brain
membranes based on their ability to stimulate in vitro the
cholera toxin-catalyzed ADP-ribosylation of Gs (326, 327).
The cDNAs of bovine and yeast Arf1 were then isolated
(658). It was shown that Arf1 is localized to the Golgi
apparatus in some cell lines and that Arf1 mutations cause
secretion defects in S. cerevisiae (684). Subsequently,
small G proteins involved in vesicle formation from the
Golgi apparatus were identified to be Arf1 and another Arf
protein (655). It is now established that Sar1 and Arf
proteins are involved in the budding of vesicles from yeast
to human (103, 493, 494, 634).
Mammalian Arf proteins are structurally grouped
into three classes: class I including Arf1, Arf2, and Arf3;
class II including Arf4 and Arf5; and class III including
Arf6 (103, 494). Of these Arf proteins, Arf1 has been the
most extensively characterized and is established to be
involved in the budding of vesicles from the Golgi. Earlier
studies implicated Arf6 in the formation of vesicles from
the plasma membrane (12, 161, 162, 585, 814). However,
recent studies indicate that it is also involved in remodeling of the actin cytoskeleton and cell motility downstream of Rac1 (584).
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TAKAI, SASAKI, AND MATOZAKI
Volume 81
FIG. 11. Cyclical activation/inactivation of Arf proteins and their translocation. Arf, Arf proteins.
cludes mammalian ARNO, cytohesin-1, GRP1, and cytohesin-4 (312, 614). No homologs exist in yeast. These
proteins consist of ⬃400 amino acids and have in common an NH2-terminal coiled-coil region, a central Sec7
domain, and a COOH-terminal PH domain. Furthermore,
all are insensitive to BFA. EFA6, a GEP specific for Arf6
(191), can also be grouped into this subfamily because it
is BFA insensitive and has all the structural features,
despite a slightly larger size. Although earlier studies
reported that ARNO, cytohesin-1, and GRP1 showed preferential GEP activity on class I and II Arf proteins, recent
studies have revealed that these small GEPs are colocalized with Arf6 at the cell periphery and cause elevation of
GTP-Arf6 (192, 193, 382).
ARNO prefers myristoylated Arf proteins and its activity is enhanced by PIP2 (99). In contrast, GRP1 binds to
PIP3 but poorly to PIP2 through its PH domain (357). It is
therefore likely that these small GEPs use the phosphoinositide-PH domain for their recruitment to a specialized
membrane subdomain, where the GEPs activate myristoylated GDP-Arf proteins. Supporting this model are the
recent observations that translocation of these small
GEPs to the plasma membrane is stimulated by growth
factors and is blocked by inhibitors of PI 3-kinases (382,
503, 751).
The crystal structure of GDP-Arf1 has revealed a
patch of positively charged amino acids on its surface (16). Thus Arf1 may also interact with negatively
charged phospholipids on membranes via this patch.
Crystal structure analyses of 5⬘-guanylylimidodiphosphate [Gpp(NH)p]-bound Arf1 and of nucleotide-free Arf1
complexed with the Sec7 domain of Gea2 have provided
insight into the activation mechanism of Arf1 (229). Arf1
in its inactive conformation cannot fit into the recognition
surface of the Sec7 domain. Taken together with the
biochemical data that a low-affinity interaction of myristoylated GDP-Arf1 with the phospholipid membrane is
required for subsequent nucleotide exchange by GEPs
(21, 47, 190), it is likely that phospholipids act on Arf1 to
induce the conformational change that permits the binding of Arf1 to the Sec7 domain, followed by Arf1 activation.
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This weak but measurable membrane association is completely abolished if the myristate is removed (190). Arf
protein-membrane interaction permits the activation of
Arf proteins by a GEP. Membrane association is stabilized
in the GTP-bound form by a conformational change of the
NH2-terminal helix that exposes several hydrophobic
amino acid residues, including Leu-8 and Phe-9 (which are
buried inside the Arf-GDP), and permits their insertion
into the membrane (21, 47). A cytosolic GAP is recruited
to the membrane after vesicle budding to stimulate the
hydrolysis of bound GTP to GDP, resulting in the release
of the Arf protein and in turn the COPI coat.
Many GEPs for Arf proteins have been isolated and
characterized in yeast and mammals (312, 494, 613). Although they show different substrate specificities, they
have a conserved ⬃200-amino acid region, called as the
Sec7 domain, that shows strong similarity to yeast Sec7.
Sec7 was initially identified as a protein required for
transport at different stages of the yeast secretory pathway and is localized to the Golgi apparatus (195). The
Sec7 domain itself was subsequently shown to be a minimum GEP unit capable of binding to and activating Arf
proteins (99, 229, 440, 489, 570, 631).
Members of the Arf GEP family can be grouped into
two major subfamilies on the basis of their sequence
similarities and functional differences (312, 614). The
high-molecular-weight Arf GEP subfamily includes yeast
Sec7, Gea1, and Gea2, and mammalian BIG1/p200, BIG2,
and GBF1, which all consist of 1,400 –2,000 amino acid
residues. Plants also have related Arf GEPs. There is a
Sec7 domain in the middle of these polypeptides, as well
as some additional, homologous regions. It is a notable
feature of these Arf GEPs that all but one (GBF1) are
sensitive to a fungal metabolite, BFA (490, 570, 631, 723),
which inhibits their Arf GEP activity in an uncompetitive
manner (440, 570). BIG1/p200 has been shown to be also
a Golgi-localized protein in mammalian cells (440). GBF1
is colocalized with COPI to the Golgi apparatus, and,
when overexpressed in cells, confers BFA resistance, suggesting that it may be involved in the formation of COPIcoated vesicles through activation of Arf proteins (122).
The second subfamily (low-molecular-weight type) in-
January 2001
SMALL G PROTEINS
D. Arf Proteins in Vesicle Budding
Although it is currently unclear at which stage Arf
GEP is activated or how its activation is controlled, GTPArf proteins, once produced by the action of Arf GEP,
associate with the Golgi apparatus and recruit coatomer,
a seven subunit complex (410, 780), or a clathrin-adaptor
complex, AP-1 or AP-3 (278). In the case of COPI-coated
vesicle formation, GTP-Arf1 has been shown to interact
with ␤-COP and ␥-COP (780, 817, 818). However, it is
currently unknown which subunits of the AP complexes
interact with GTP-Arf proteins. Acidic phospholipids,
such as phosphatidic acid, enhance Arf1-induced binding
of coatomer (370, 614). Assembled COPI captures cargo
molecules through binding to cargo receptors, such as
KDEL receptor and the p24 family of proteins (410, 780).
In the latter case, COPI recognizes the phenylalanine or
dilysine motif (or both) within the cytoplasmic tail of the
p24 family members. Thus vesicle formation and cargo
selection are coupled through a bivalent interaction of
coatomer with Arf1 and a putative cargo receptor. It is
speculated that coat protein assembly induces local membrane deformation into a nascent vesicle bud.
The entire process of the formation of COPI-coated
and clathrin/AP-1-coated vesicles can be reproduced with
pure components in vitro (677, 825). The simplest systems
consist of GTP-Arf1 and either coatomer, or clathrin and
AP-1, added to liposomes of defined composition that
support the formation of small coated vesicles (40 –70 nm
diameter for COPI and 60 – 80 nm diameter for clathrin/
AP-1) (677, 825). The results indicate that Arf1 and
coatomer are sufficient to pinch off vesicles. Although it
has been proposed that production of phosphatidic acid
by Arf1-induced activation of phospholipase D triggers
binding of coat proteins to membranes and subsequent
vesicle formation, the physiological relevance of these
events is unclear (612). On the other hand, a recent study
has suggested that production of phosphatidic acid by
acylation of lysophosphatidic acid may play a role in
vesicle budding from the Golgi apparatus (773).
Either concomitant with or after vesicle budding,
GTP-Arf1 is converted to the GDP-bound form by the
action of a GAP. Recent studies have shown that
coatomer and a transmembrane cargo receptor control
membrane association of Arf1 GAP and its activity (23, 24,
230). KDEL receptor enhances the recruitment of Arf1
GAP to the membranes of the Golgi apparatus (23, 24).
Arf1 GAP activity may then be stimulated by coatomer on
the membranes (230). Consistent with this observation,
the crystal structure of GTP-Arf1 complexed with the
minimal GAP domain of Arf1 GAP indicates that, unlike
most G proteins, the GAP binding site on Arf1 does not
overlap with its effector binding site (230). Thus Arf1 may
simultaneously interact with Arf1 GAP and coatomer, and
both coatomer and cargo receptors may regulate the termination of the Arf activity.
E. Arf6 in Endocytic Recycling
and Cytoskeletal Reorganization
The subcellular localization of Arf6 is quite different
from that of class I and class II Arf proteins. Various
studies have shown that Arf6 is involved in recycling of
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Many Arf GAPs have been identified in yeast and
mammals (494, 614). These GAPs each contain a zinc
fingerlike Arf GAP domain, which has been shown to be a
minimum unit for the GAP activity. However, their substrate specificities have not yet been systematically characterized. An Arf GAP purified from rat spleen cytosol is
activated by PIP2 and shows broad substrate specificity,
being active on Arf1, Arf3, Arf5, and Arf6 (153). This
activity does not require Arf myristoylation. Another Arf
GAP (Arf1 GAP), the first GAP identified in mammals
(136), is localized to the Golgi apparatus and is stimulated
by diacylglycerol, which may be produced from phosphatidylcholine by the sequential actions of phospholipase D (a downstream effector of Arf proteins; see below), and phosphatidic acid phosphohydrolase (22).
Recruitment of cytosolic Arf1 GAP to the vesicle membranes is enhanced by cargo receptors such as the KDEL
receptor, which recognizes a Lys-Asp-Glu-Leu (KDEL)
COOH-terminal motif on certain soluble proteins destined
for Golgi-to-endoplasmic reticulum retrieval (23, 24). Centaurin-␣ and PIP3-BP, two closely related proteins, have
two PH domains that are involved in binding to PIP3 (253,
710). ASAP1/DEF-1 and PAP, also close relatives, have a
PH domain, three ankyrin repeats, a proline-rich region,
and an SH3 domain and have been shown to interact with
Src and Pyk2, respectively (19, 70, 348). Both enhance the
GTPase activity of Arf1 and Arf5 but act only weakly on
Arf6. PKL and GIT are closely related and contain three
ankyrin repeats and a PH domain (120, 528, 731). PKL was
identified as a protein that binds to the paxillin LD4 motif
and serves as a connector between paxillin and molecules
of the Rac1/Cdc42 pathway, PAK and PIX (731). GIT1 was
identified as a protein that interacts with a G proteincoupled receptor kinase; it regulates endocytosis of various G protein-coupled receptors (120, 578). To date, the
crystal structures of the GAP domain of PAP and that of
Arf1 GAP complexed with Arf1 have been determined
(230, 435). A unique chimera protein, ARD1, contains both
Arf protein and Arf GAP structures (474). The GAP domain is specific for the Arf domain and shows GDI activity
that slows the dissociation of GDP from the Arf domain
(754).
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TAKAI, SASAKI, AND MATOZAKI
F. Sar1 as a Regulator of Vesicle Budding
The function of Sar1 in vesicle budding has been
extensively characterized in the yeast S. cerevisiae but in
less detail in mammals (38, 634). Sar1 does not undergo
any known lipid modification, but it is associated with the
endoplasmic reticulum and is involved in the formation of
COPII-coated transport vesicles from the endoplasmic
reticulum (39, 518, 544). The Sar1 cycle is regulated by a
GEP (Sec12) and a GAP (Sec23) (40, 44, 371, 808). Sec12
is an integral endoplasmic reticulum membrane protein
involved in activation of Sar1 (40, 142, 143). However, its
mammalian homolog has not yet been identified. Sec23
accelerates the GTPase activity of Sar1 and is tightly
associated with Sec24 (the Sec23 complex) (808). Sec23
homologs are present in mammalian cells, and they localize to compartments between the endoplasmic reticulum
and the Golgi apparatus (554, 558). A Sec24 homolog is
also present in mammalian cells, and as expected, it interacts with mammalian Sec23 (559, 715). Thus the Sar1/
COPII system is well conserved between yeast and mammalian cells.
COPII assembly and disassembly are regulated by the
Sar1 cycle (44, 371, 679). Sec12-induced activation of Sar1
promotes association of the Sec23 complex to the budding site at the endoplasmic reticulum. Then, the Sec13
complex comprised of Sec13 and Sec31 is recruited onto
the Sec23 complex, and this binding and the subsequent
polymerization of the Sec13 complex are proposed to lead
to the concentration of cargo proteins and the deformation of the membrane into a coated bud. Thus vesicles
coated with GTP-Sar1, the Sec23 complex, and the Sec13
complex, are produced. Sec23-induced GTP hydrolysis of
Sar1 leads to dissociation of these proteins from the
vesicle, although hydrolysis appears not to be a prerequisite for the vesicle budding. Two other factors have
been implicated in COPII-vesicle budding. Sec16 interacts
directly with the Sec23 complex and may serve as a
scaffold for COPII coat assembly (174, 225). Sed4 is highly
homologous to Sec12 in its cytoplasmic domain, although
it does not seem to show GEP activity on Sar1. However, unlike Sec12, Sed4 interacts directly with Sec16
(224). It is thus possible that Sar1, along with Sed4 and
Sec16, may be involved in a distinct vesicle formation
process.
Proteins necessary for the targeting and docking processes, such as v-SNARE and Rab proteins, must be incorporated into transport vesicles. However, the mechanism for this is currently unknown. Two facts are
established. 1) The Sec23 complex interacts simultaneously with GTP-Sar1 and two v-SNAREs, Bet1 and
Bos1, in a cooperative manner during the formation of
endoplasmic reticulum-to-Golgi COPII transport vesicles
(678), and 2) that Rab11BP/Rabphilin-11 (432, 668, 810), a
downstream effector of Rab11 mainly implicated in vesicle recycling (590, 736), interacts directly with mammalian Sec13 (433).
VII. RAN FUNCTION IN NUCLEOCYTOPLASMIC
TRANSPORT AND MICROTUBULE
ORGANIZATION
A. Outline
There is only one Ran gene in many cell types (including human and Schizosaccharomyces pombe), although cells of other species (S. cerevisiae and tomato)
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endosomal vesicles and regulates receptor-mediated endocytosis (12, 161, 162, 585, 814). However, Arf6 is also
implicated in remodeling of the cytoskeleton underlying
the plasma membrane. Arf6 is localized to the plasma
membrane, especially to membrane ruffles in spreading
cells. Activation of Arf6 induces remodeling of the actin
cytoskeleton and cell spreading, and expression of a dominant negative mutant of Arf6 blocks cell spreading (160,
585, 674). Furthermore, ARNO and EFA6, two low-molecular-weight Arf GEPs, have been reported to modulate
growth factor- and protein kinase C-mediated cytoskeletal reorganization through activation of Arf6 (191, 193).
In addition, a dominant negative mutant of ARNO inhibits
both Arf6 translocation and cortical actin formation induced by growth factors (750). In this context, it is noteworthy that recent studies have suggested that there is
cross-talk between the Arf6 and Rac1 pathways in actin
remodeling. First, EFA6-induced cytoskeletal remodeling
is blocked by coexpression of a dominant negative mutant
of Arf6 or Rac1 (191). Second, a dominant negative mutant of Arf6 inhibits growth factor- and Rac1-mediated
membrane ruffling (584, 813). Third, a deletion mutant of
POR1/arfaptin-2, which interacts with both Rac and Arf
proteins, but not a dominant negative mutant of Rac1,
inhibits Arf6-mediated cytoskeletal rearrangements (160).
Taken together, these results indicate that Arf6 functions
either downstream of or in concert with Rac1. Further
evidence suggesting a connection between Arf6 and Rac1
pathways is the finding that PKL, an Arf GAP, serves as a
connector between paxillin and the Rac/Cdc42 pathway
components PAK and PIX (731). An effector of Arf6 that
is implicated in membrane ruffling is phosphatidylinositol
4-phophate 5-kinase (283). This kinase translocates to
ruffling membranes and produces PIP2 synergistically
with Arf6 and phosphatidic acid, the production of which
is catalyzed by phospholipase D. Because phospholipase
D itself is an effector of Arf proteins and PIP2 is an
activator of Arf proteins, it is tempting to speculate that
the local phospholipid metabolism that is regulated by
Arf6 may play a crucial role in membrane ruffle formation.
Volume 81
January 2001
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SMALL G PROTEINS
contain two or more closely related Ran genes (483). Ran
protein (Ras-related nuclear protein: Ran) was originally
cloned on the basis of its homology to Ras proteins (158).
The first evidence for the involvement of Ran in nuclear
transport was obtained in 1993 by showing that Ran is
essential for the nuclear import in permeabilized cells of
a reporter construct containing the nuclear localization
signal (NLS) of the simian virus 40 T antigen (PKKKRKV)
(464, 484). It is now clear that Ran plays a central role in
nucleocytoplasmic transport. Recent studies have uncovered another role for Ran in microtubule organization
during the M phase of the cell cycle (85, 325, 329, 351, 504,
533, 781).
B. Nucleocytoplamsic Transport
Macromolecules are transported back and forth between the cytoplasm and the nucleus through NPCs. The
NPCs contain more than 50 different proteins (454, 517,
536, 565). Movement of macromolecules through the
NPCs occurs by at least two distinct mechanisms: passive
diffusion and active transport (180). Small molecules diffuse quickly through the NPCs in either direction,
whereas those larger than 50 – 60 kDa, including proteins
and RNAs, are actively and selectively transported. The
transport of cargo proteins requires at least three types of
soluble factors: transport receptor molecules, adaptor
molecules, and Ran and its binding proteins (139, 234,
454). Transport receptors shuttle continuously between
the nucleus and the cytoplasm, interact with NPCs, bind
cargo molecules, and facilitate cargo translocation
through the NPCs. There are two types of transport receptors: nuclear import receptors, called importins, and
C. Ran Cycle: Cyclical Activation/Inactivation
and Translocation
Like other small G proteins, Ran is cyclically activated and inactivated, but the most characteristic feature
of the Ran cycle is that the GTP-bound form and the
GDP-bound form are asymmetrically distributed in the
nucleus and the cytoplasm, respectively. This asymmetric
distribution of the two forms are due to asymmetric distributions of the regulators, a GEP and a GAP (Fig. 12).
Only one Ran GEP, regulator of chromosome condensation (RCC1) (54, 538), and only one Ran GAP, Ran GAP1
(51, 52), have been identified in mammals. RCC1 was
originally identified as a regulator of chromatin condensation (538). RCC1 is localized exclusively in the nucleus
where it is associated with the chromatin (539) and converts the GDP-bound form to the GTP-bound form (54). In
contrast to RCC1, Ran GAP1 is localized exclusively in the
FIG. 12. Cyclical activation/inactivation of
Ran and its translocation.
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export receptors, called exportins (734). Importins and
exportins recognize NLS and nuclear export signal (NES)
of cargos, respectively, bind them, and transport bound
cargos through the NPCs (3, 483). In some cases, transport receptors do not directly bind cargos and indirectly
bind them through adaptors that can recognize their NLS.
For instance, importin-␣ and importin-␤ are well-known
adaptor and receptor molecules, respectively. Transport
receptors constitute a superfamily of proteins that share a
binding domain of GTP-Ran (189, 235). In the yeast S.
cerevisiae, 14 members of importin-␤ superfamily are
predicted from the genome sequence (3). Fewer receptors
have been identified in mammals, but more receptors will
be found in the future.
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TAKAI, SASAKI, AND MATOZAKI
aspect of this cycle is that GTP-Ran forms a gradient
across the nuclear envelope and that this gradient plays a
key role in controlling the directionality of nuclocytoplasmic transport (236, 311). Ran does not undergo any posttranslational lipid modification and probably, as a result,
does not bind to membranes inside the cell or require
lipids for its activity (618).
D. Mode of Action of Ran in Nucleocytoplasmic
Transport
As described above, one role of Ran is to export
cargos via exportins from the nucleus to the cytoplasm.
The most well-characterized exportin is CRM1 (also
called exportin 1), which recognizes a specific, leucinerich type of NES (188, 203, 682). In the presence of
GTP-Ran, both GTP-Ran and a cargo bind to CRM1 in a
cooperative manner to form a GTP-Ran-CRM1-cargo complex (Fig. 13A). This complex is exported to the cytoplasm, where GTP-Ran is converted to GDP-Ran, causing
the cargo to dissociate from CRM1. CRM1 is then reimported to the nucleus in the empty state. In this example,
CRM1 functions without an adaptor. In some cases, NEScontaining proteins bind RNAs that are destined for nuclear export. In these cases, the NES proteins serve as
adaptors and the cargo is the RNA.
Ran has another role in nuclear import (536). In the
absence of GTP-Ran, importin-␤ forms a complex with
FIG. 13. Mode of action of Ran in nucleocytoplasmic transport. A: mode of action of Ran in nuclear export. B: mode
of action of Ran in nuclear import.
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cytoplasm (455, 596) but some can be detected in the
nucleus (726). In vertebrate cells, a significant portion of
Ran GAP1 acquires a 121-amino acid ubiquitin-like addition (called SUMO-1) (430, 455). This modification does
not target this protein for degradation but instead targets
it specifically to the filaments at the cytoplasmic face of
the NPCs. Moreover, two GTP-Ran-binding proteins, Ranbinding protein 1 (RanBP1) and Ran-binding protein 2
(RanBP2/NUP380), have been isolated (53, 784, 805).
RanBP1 is localized in the cytoplasm and enhances the
Ran GAP1 activity (53, 127, 597, 637), whereas RanBP2 is
a component of the NPCs at the cytoplasmic face that
recruits SUMO-Ran GAP1 and enhances its GAP activity
(430, 455). Once GTP-Ran is produced in the nucleus, an
exportin binds both GTP-Ran and its cargo in a cooperative manner, and this ternary complex is exported to the
cytoplasm through the NPCs. At the cytoplasmic face of
the NPCs, GTP-Ran in this complex is attacked by SUMORan GAP1 and Ran BP2 (430, 455, 622), and in the cytoplasm it is attacked by Ran GAP1 and RanBP1, leading to
conversion to GDP-Ran (50, 186, 409). Inactivation of
GTP-Ran induces the dissociation of the complex in the
cytoplasm. GDP-Ran then forms a complex with another
regulator, named p10/NTF2, which transports GDP-Ran
back to the nucleus through the NPCs (595). p10/NTF2
has a GDI activity, keeping Ran in the GDP-bound form
(789). GDP-Ran thus returned to the nucleus and is then
reutilized for the next cycle of transport. Thus the Ran
cycle is regulated by many factors. The most important
Volume 81
January 2001
SMALL G PROTEINS
The precise mode of action of Ran and RanBPM in spindle
formation is not understood, but in the absence of a
nucleus, chromatin-bound RCC1 and cytoplasmic Ran
GAP1 produce a natural gradient of GTP-Ran that may be
most concentrated at the chromosome surface (325).
Consequently, the GTP-Ran-RanBPM complex would
stimulate microtubule assembly adjacent to the chromosomes. In this case again, GTP hydrolysis is not essential
for microtubule assembly but may have some secondary
role in the elongation of previously nucleated microtubules. Other cellular factors, including the chromosomeassociated, kinesin-like protein, XKLP1 (752, 764), and a
complex of cytoplasmic dynein, dynactin and NuMA (466,
801), are also involved in organizing these microtubules
into spindles. Thus the observation that Ran and RanBPM
stimulate microtubule polymerization offers a significant
insight into the process by which chromosomes drive
spindle assembly.
VIII. SMALL G PROTEIN CASCADES
AND CROSS-TALKS
A. Small G Protein Cascades
E. A Role for Ran in Microtubule Organization
During G1, S, and G2 phases of the cell cycle, nucleocytoplasmic transport is active, but during the M phase,
the nuclear envelope is broken down and nucleocytoplasmic transport stops. At the onset of mitosis in eukaryotes,
the nuclear envelope and interphase microtubule array
disassemble, and the duplicated centrosomes nucleate
microtubules that interact with chromosomes, forming a
bipolar spindle. Sister chromosomes balanced at the spindle equator segregate and move to opposite spindle poles
during anaphase. How chromosomes influence spindle
assembly in the absence of microtubule-nucleating organelles has been a long-standing mystery. Recent studies
have revealed that Ran and its novel effector, named
Ran-binding molecule (RanBPM) (504), regulate aster formation and spindle assembly.
An initial hint for Ran involvement in microtubule
regulation was derived from the observation that, in the
yeast S. cerevisiae, overexpression of Prp20, the yeast
RCC1 homolog, suppresses the toxic effect of certain
hyperstable ␣-tubulin mutants (351). More direct evidence has recently been obtained by the identification of
a novel mammalian GTP-Ran-binding protein, RanBPM
(504). RanBPM associates with centrosomes, the microtubule-nucleating centers of mammalian cells. Overexpression of RanBPM in cultured cells or addition of GTPRan or GTP␥S-Ran to a cell extract induces aster
formation. Conversely, inhibition of RanBPM or Ran activity prevents in vitro aster formation (85, 329, 533, 781).
Multiple small G proteins form a signal cascade and
thereby transduce their signals to downstream effectors.
For instance, in the yeast S. cerevisiae, Rsr1, a member of
the Ras family, is thought to be first activated by an
unknown cue that is produced by the previous budding
site (45, 96) (Fig. 14). In the next step, GTP-Rsr1 binds to
Cdc24, a GEP for Cdc42, which in turn binds GDP-Cdc42
to activate it (821). The activation of Cdc42 induces not
only reorganization of the actin cytoskeleton but also the
recruitment of multiple small G proteins, including Rho1
and Sec4, to the future budding site. Sec4 would supply
the bud with vesicles necessary for bud enlargement (527,
623), while Rho1 would induce synthesis of the new cell
wall component, 1,3-␤-glucan, by directly activating 1,3␤-glucan synthase and stimulating expression of the
genes necessary for this synthesis (80, 157, 456, 580).
Rho1 would also regulate reorganization of the actin cytoskeleton necessary for the budding processes (200, 304,
363, 739).
Another typical example of small G protein cascade
includes Rho/Rac/Cdc42 proteins in mammalian cells
(420, 744) (Fig. 15). Rho proteins regulate formation of
stress fibers and focal adhesions (564, 600, 601), Rac
proteins regulate formation of lamellipodia and membrane ruffles (521, 522, 602), and Cdc42 proteins regulate
formation of filopodia (368, 522). The sequential activation of these three small G proteins by extracellular agonists has been best characterized in quiescent Swiss 3T3
fibroblasts (522, 600, 602). Agonists like bradykinin activate Cdc42 proteins in these cells to produce filopodia or
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importin-␣ that serves as an adaptor, binding NLS-containing proteins (Fig. 13B). This complex is imported into
the nucleus through the NPCs, where it encounters GTPRan. GTP-Ran binds to importin-␤, causing dissociation of
the complex into each component. Empty importin-␤,
probably still bound to GTP-Ran, is then reexported to the
cytoplasm. Recycling of importin-␣ requires a specific
exportin, named CAS, that is analogous to CRM1 (377).
The binding of importin-␣ to CAS requires cooperative
GTP-Ran binding, and CAS prefers importin-␣, which is
not associated with a cargo. Thus empty importin-␣ is
exported again by CAS. After inactivation of GTP-Ran to
GDP-Ran in the cytoplasm, importin-␣ dissociates, and
CAS returns to the nucleus for reutilization.
During nuclear transport, the nucleotide-bound state
of Ran acts as a simple switch to delineate the direction of
movement, and the energy of GTP hydrolysis is not
strictly required. Moreover, the binding of GTP-Ran to
importins induces conformational changes so that they
dissociate cargos. GTP-Ran is not essential for the transportation of importins through the NPCs, because the
NPC-interacting region of importins alone can be transported through the NPCs in the absence of GTP-Ran (3).
185
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TAKAI, SASAKI, AND MATOZAKI
Volume 81
FIG. 14. Small G protein cascade in control of polarized
cell growth in the yeast S. cerevisiae.
B. Cross-talk Between Small G Proteins
In addition to the sequential cascade of multiple
small G proteins, distinct families of small G proteins
regulate various cellular functions in a cooperative manner. Although a dominant active mutant of Rho proteins
does not cause transformation of fibroblasts, dominant
negative mutants of Rho proteins inhibit Ras proteininduced transformation (341, 583). Similarly, a dominant
negative mutant of Rac/Cdc42 proteins inhibits Ras protein-induced transformation, while a dominant active mutant of these Rac/Cdc42 proteins enhances oncogenic Ras
protein-triggered, morphologic transformation (581, 582).
Thus Rho/Rac/Cdc42 proteins are involved in a cooperative manner in Ras protein-induced transformation.
It has recently been shown that Rho/Rab proteins
coordinately regulate cell adhesion and migration of cultured MDCK cells (303). TPA induces cell scattering of
MDCK cells and induces early disassembly of stress fibers
and focal adhesions followed by their reassembly in
MDCK cells. Expression of a dominant active mutant of
RhoA inhibits the TPA-induced disassembly of stress fibers and focal adhesions, while microinjection of C3
blocks their reassembly. In addition, microinjection of C3
or a dominant active mutant of RhoA inhibits the TPA-
FIG. 15. Small G protein cascade in control of cytoskeletal reorganization in Swiss 3T3 fibroblasts. Rho,
Rho proteins; Rac, Rac proteins; LPA, lysophosphatidic
acid.
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 4, 2017
microspikes. Activation of Cdc42 proteins leads to localized activation of Rac proteins; hence, filopodia are often
associated with lamellipodia, which are produced by Rac
proteins. In fact, it is hard to see filopodium formation
unless Rac activity is first inhibited. Quiescent Swiss 3T3
cells have no detectable stress fibers, and activation of
Rac proteins under these conditions leads to weak and
delayed activation of Rho proteins that produces stress
fibers. This typical cascade of Rho/Rac/Cdc42 proteins
observed in Swiss 3T3 fibroblasts has not always been
seen in other cell lines. For instance, it has been shown
that Rho proteins are inhibited by Rac/Cdc42 proteins in
cultured cells such as neuroblastoma cells, N1E-115 cells,
and NIH 3T3 cells (277, 388). In addition, Rho proteins
may in turn inhibit the activity of Rac/Cdc42 proteins in
N1E-115 cells (624).
January 2001
SMALL G PROTEINS
IX. CONCLUSIONS AND PERSPECTIVES
A. Roles in Two Types of Cell Regulation
The mechanism of Gs-induced activation of adenylate
cyclase is different from that of EF-Tu-induced elongation
of polypeptide. Once GTP-Gs binds to adenylate cyclase, it
continuously activates it until it is converted to the GDPbound form. In contrast, GTP-EF-Tu elongates a single
amino acid, and further elongation requires the inactivation and reactivation of EF-Tu (60) (Fig. 16). GTP␥S-Gs
continuously activates adenylate cyclase, whereas GTP␥SEF-Tu stimulates just one cycle of peptide elongation and
stops further elongation. In this sense, Ras-induced Raf
protein kinase activation and Rho/Rac/Cdc42 protein-in-
duced activation of their specific downstream effector
protein kinases are similar to the Gs-induced adenylate
cyclase activation, whereas reorganization of the actin
cytoskeleton regulated by Rho/Rac/Cdc42 proteins, vesicle trafficking regulated by Rab and Sar1/Arf proteins, and
nucleocytoplasmic transport regulated by Ran are similar
to the EF-Tu-induced elongation of polypeptide and require cyclical activation and inactivation of the small G
proteins. Consistent with this mode of action of Gs and
Ras proteins, the ADP-riboyslation of Gs by cholera toxin
continuously activates adenylate cyclase, thereby causing
diarrhea (733), and point-mutated activated forms of Ras
proteins frequently found in a variety of cancers continuously activate Raf protein kinases that cause stimulated
cell proliferation (82). In contrast, no point-mutated activated forms of other small G proteins have been identified, and this may be due to the necessity for the cyclical
activation and inactivation of these small G proteins for
their cell functions. Instead, many GEPs for Rho/Rac/
Cdc42 proteins have been identified as oncogenes (93,
251, 744). Oncogenic GEPs increase relative ratios of
small G protein GTP-bound forms to GDP-bound forms
and show oncogenic phenotypes. From this point of view,
we should carefully interpret data obtained using dominant active mutants of small G proteins.
B. A Role as Biotimers Rather Than as Molecular
Switches
All downstream effectors of small G proteins have at
least two functionally distinct domains: a small G protein-
FIG. 16. Two types of cell regulation by G proteins. A: mode of action of EF-Tu in protein sysnthesis. G, EF-Tu; aa
tRNA, aminoacyl-tRNA. B: mode of action of heterotrimeric G protein. G, heterotrimeric G protein.
Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on July 4, 2017
induced cell scattering. In contrast, expression of Rab
GDI or a dominant negative mutant of Rab5 attenuates
either the TPA-induced reassembly of stress fibers and
focal adhesions and subsequent cell scattering of MDCK
cells (303). Expression of a dominant active mutant of
Rac1 or Cdc42 inhibits the HGF/SF- or TPA-induced cell
scattering (331, 361). Therefore, during cell migration at
least, Rho/Rac/Cdc42 proteins may regulate the reorganization of the actin cytoskeleton, while Rab5 may control
the recycling of adhesion molecules such as integrins by
intracellular vesicle trafficking. The temporal and spatial
activation and inactivation of these two families of small
G proteins appear to be required for reorganization of the
actin cytoskeleton and cell migration.
187
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TAKAI, SASAKI, AND MATOZAKI
C. A Role as Spatial Determinants
In addition to temporal regulation of small G proteins, spatial regulation is also essential for them to regulate various cell functions, particularly very dynamic cell
functions, such as cell migration and adhesion. The sites
of the activation of some small G proteins, such as Ras,
Sar1, and Ran proteins, are determined by their GEPs.
Upon stimulation of membrane receptors, Ras GEPs, such
as SOS, are recruited to the cytoplasmic region of the
plasma membrane receptors through adaptors, such as
GRB2/SHC, where Ras proteins are activated (75, 399,
FIG. 17. A role of small G proteins as biotimers rather than as
molecular switches. G, small G protein.
549, 617). A Sar1 GEP, Sec12, is a transmembrane protein
of the endoplasmic reticulum and interacts with GDPSar1 and activates it at the cytoplasmic surface of the
endoplasmic reticulum (40, 142, 143). A Ran GEP, RCC1,
is associated with chromatin and activates it in the nucleus (54, 539). In these cases, however, it remains unknown how activities of these GEPs are regulated. Chromatin structure may regulate RCC1 activity and cargo
proteins may regulate Sec12 activity. There is another
possibility that they are always active without being regulated and attack their substrates when they are available.
An Arf1 GEP, ARNO, binds to PIP2 and/or PIP3 and is
associated with a specialized membrane subdomain,
where it interacts with GDP-Arf1 and converts it to the
GTP-bound form (99). Thus lipid metabolism is crucial for
the localization of Arf1.
In contrast to these small G proteins, Rab proteins
stay in the cytosol in the GDP-bound form complexed
with Rab GDIs and they are translocated to their specific
membrane compartments where they are activated (572,
701–703). GEPs may also be determinants for their localization, but Rab3 GEP is mostly found in the cytosol, and
there is no evidence that Rab3 GEP is first associated with
membranes and then recruits Rab3 proteins (543, 762).
Another possible mechanism is that the downstream effector(s) may serve as determinants. A downstream effector of Rab3 proteins, Rabphilin-3, is associated with synaptic vesicles in a Rab3A-independent manner, and upon
its activation, it is associated with the vesicles through
Rabphilin-3 (460, 654, 664). However, there is a contradictory result that the association of Rabphilin-3 with synaptic vesicles is dependent on GTP-Rab3A (397, 683). Rab5
proteins are also reported to be first associated with
endosomes, followed by recruitment of their downstream
effector, Rabaptin-5, and the COOH-terminal regions of
Rab5 proteins are essential for this specific localization
(685). These mechanisms are similar to the Ras protein
system in which the Ras proteins activated at the cytoplasmic surface of the plasma membrane recruit their
downstream effector, Raf protein kinases (389, 687).
However, it is unknown how these activated Rab proteins
determine their specific localizations. They could be physically associated with membrane phospholipid through
their COOH-terminal prenyl moieties (27, 322, 340, 349,
568, 765), but lipid-lipid interactions alone presumably do
not determine their specific localizations. It also remains
unknown how these GEP activities are regulated.
The situation is similar for Rho/Rac/Cdc42 proteins.
Most Rho/Rac/Cdc42 proteins stay in the cytosol in the
GDP-bound form complexed with Rho GDIs (702, 704).
Upon activation by their specific GEPs, they are translocated to their functioning sites. Some GEPs, such as
Tiam1 for Rac proteins and frabin for Cdc42, require
specific localizations through the membrane or F-actinbinding domains for their physiological functions (469,
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binding domain and a catalytic domain (in the case of
enzymes) or a protein-interacting domain (in the case of
nonenzymes). In the case of enzymes, the binding of the
small G protein induces conformational change, resulting
in its activation or inactivation. The activation (or inactivation) continues until the small G protein is converted to
the GDP-bound form by the action of GAPs, causing its
release from the enzyme. In the case of nonenzymes, the
binding of the small G protein induces conformational
change in the effector, resulting in the interaction with or
the dissociation from a further downstream effector. The
interaction or the dissociation continues until the small G
protein is converted to the GDP-bound form. In both
cases, the small G protein-binding region directly or indirectly masks the catalytic domain or the other proteinbinding domain, and the binding of the small G protein
induces unfolding of this functional domain. In this sense,
small G proteins work as more than molecular switches,
that activate or inactivate the downstream effectors, and
work as biological timers (biotimers), that induce activation or inactivation and determine the periods of the
functioning time (Fig. 17). It would be, therefore, of crucial importance to understand the mechanisms when and
how small G proteins are activated by GEPs and inactivated by GAPs.
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SMALL G PROTEINS
738). When dominant active mutants of Rac or Cdc42
proteins are overexpressed, they could be artificially distributed in the cell, and some of them are located at the
real functioning site and show similar effects there. Under
physiological conditions, small amounts of Rac or Cdc42
proteins activated by their GEPs at specific sites are
involved in their functions. In many literatures, overexpressed dominant active mutants of these proteins show
a variety of phenotypes, but we should be careful for their
interpretations. It is of crucial importance to understand
the mechanisms of the temporal and spatial activation
and inactivation of these small G proteins.
14.
15.
16.
17.
18.
19.
20.
21.
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We thank Drs. Yoshito Kajiro, Kenichi Kariya, Tohru
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