Physiol Rev 92: 237–272, 2012
doi:10.1152/physrev.00045.2010
INHIBITION AND TERMINATION OF
PHYSIOLOGICAL RESPONSES BY GTPASE
ACTIVATING PROTEINS
Erzsébet Ligeti, Stefan Welti, and Klaus Scheffzek
Department of Physiology, Semmelweis University, Budapest, Hungary; Structural and Computational Biology
Unit, European Molecular Biology Laboratory, Heidelberg, Germany; and Division of Biological Chemistry,
Biocenter, Innsbruck Medical University, Innsbruck, Austria
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
INTRODUCTION
GUANINE NUCLEOTIDE-BINDING...
ROLE OF GAPs IN THE NERVOUS SYSTEM
ROLE OF GAPs IN THE ACTION OF...
ROLE OF GAPs IN BLOOD CELLS
ROLE OF GAPs IN HOST-PATHOGEN...
ROLE OF GAPs IN TUMORIGENESIS
CONCLUDING REMARKS
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254
255
258
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I. INTRODUCTION
Intercellular communication occurs mostly via interaction
of soluble or surface-bound ligands with specific receptor
proteins localized in the plasma membrane or at intracellular sites. The receptor-ligand interaction may induce various biochemical reactions such as changes of enzyme activity, protein-protein interactions, or transmembrane ion
movements, resulting in cellular responses. To transmit information, signals have to be dynamic, i.e., be terminated
and reelicited with definite time courses. Studies on cellular
signaling concentrate mostly on activation processes,
whereas counteracting mechanisms resulting in signal termination or downregulation are less frequently investigated. However, examples of human pathology alongside
with phenotypes of genetically modified animals clearly indicate the fundamental physiological importance of signal
terminating processes. This review focuses on a family of
proteins that can be regarded as professional cellular signal
downregulators or terminators.
Guanine nucleotide-binding proteins (GNBPs or G proteins) function as regulated time switches, the general functional scheme of which is commonly summarized in the
so-called GTPase cycle (FIGURE 1). In the resting state, they are
usually bound to GDP. They are activated with the help of
guanine nucleotide exchange factors (GEFs) that promote
dissociation of GDP and rebinding of intracellularly abundant GTP. In the GTP-bound, active form they interact with
specific target proteins, thereby switching on enzyme activity or induce the formation of protein complexes. Intrinsic
hydrolytic activity of G proteins transforms the bound GTP
into GDP, and in this inactive form, the processes regulated
by the given G protein become switched off. The generally
low rate of GTP hydrolysis (GTPase activity) is encoded in
the primary structure of the individual G proteins and varies
in a broad range. However, it is not a constant property but
is dynamically modulated by regulatory proteins. GTPase
activating proteins (GAPs) enhance the intrinsic GTP hydrolyzing activity of monomeric G proteins by up to five
orders of magnitude (44, 365) and thereby accelerate the
switch-off of the signal. Regulators of G protein signaling
(RGSs) are GAPs for G␣ subunits of heterotrimeric G proteins carrying out similar functions in G protein signaling.
In the present review, we direct the attention on the important physiological role played by GAPs. Our paper is by far
not meant to give a comprehensive list of GAPs. In this
respect, we refer the readers to excellent recent reviews (32,
39, 163, 169, 308, 351) and apologize to authors whose
237
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Ligeti E, Welti S, Scheffzek K. Inhibition and Termination of Physiological Responses by GTPase Activating Proteins. Physiol Rev 92: 237–272, 2012;
doi:10.1152/physrev.00045.2010.—Physiological processes are strictly organized in space and time. However, in cell physiology research, more attention is given
to the question of space rather than to time. To function as a signal, environmental
changes must be restricted in time; they need not only be initiated but also terminated. In this
review, we concentrate on the role of one specific protein family involved in biological signal
termination. GTPase activating proteins (GAPs) accelerate the endogenously low GTP hydrolysis
rate of monomeric guanine nucleotide-binding proteins (GNBPs), limiting thereby their prevalence
in the active, GTP-bound form. We discuss cases where defective or excessive GAP activity of
specific proteins causes significant alteration in the function of the nervous, endocrine, and
hemopoietic systems, or contributes to development of infections and tumors. Biochemical and
genetic data as well as observations from human pathology support the notion that GAPs represent
vital elements in the spatiotemporal fine tuning of physiological processes.
GAPs IN SIGNAL TERMINATION
work we could not cite due to space limitations. We concentrate on examples where long-term and intensive research accumulated clear evidence to support the vital necessity for turning off G proteins by GAPs in the right place
and at the right time. We chose only examples where a
coherent picture has emerged up to now, and typical phenotypes are observable either in human pathology or in
genetically modified animals. Accordingly, we grouped our
examples with respect to physiological functions rather
than to groups or families of molecules.
II. GUANINE NUCLEOTIDE-BINDING
PROTEINS
Different types of GNBPs function at different locations in
the cell. Heterotrimeric GNBPs (also known as heterotrimeric G proteins) consist of three different subunits (␣, ␤,
and ␥) with the ␣-subunit containing the GTP/GDP binding
238
site. They are localized to the plasma membrane where they
interact with G protein-coupled receptors (GPCR). The human genome contains 17 G␣, 5 G␤, and 14 G␥ genes (384).
Ligand binding to a GPCR initiates the activation of the interacting G protein that results in exchange of GDP for GTP on
the ␣ subunit and dissociation of the active G␣GTP from the
complex of ␤␥. Both G␣GTP and ␤␥ subunits have important
physiological functions such as regulation of enzyme activity
(adenylyl cyclase, phospholipase C, phosphodiesterase, GRK)
or modulating channel function (K⫹, Ca2⫹). G␣ subunits possess high GTPase activity that allows relatively fast decay of
the biological signal and reassociation of G␣GDP with ␤␥.
Physiological role and regulation of heterotrimeric G proteins
have been summarized in several excellent reviews (123, 146,
239, 250, 253, 384, 385).
Monomeric (or small) GNBPs (also known as small G proteins or small GTPases) consist of one single polypeptide
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FIGURE 1. The GTPase cycle of monomeric G proteins. Monomeric G proteins can adopt an active GTP- and
an inactive GDP-bound state, which is controlled by the relative activity of corresponding GEF and GAP proteins.
While GEFs support the release of GDP and thereby rebinding of intracellularly abundant GTP, GAPs increase
the slow intrinsic GTPase rate of G proteins leading to rapid GTP hydrolysis and phosphate release. Beyond
controlling the balance of this equilibrium, GEFs and GAPs also cause a high flux between the active and inactive
state of the G protein, making the system highly dynamic and responsive. GAP, GTPase activating protein; GEF,
G-nucleotide exchange factor.
LIGETI ET AL.
chain of ⬃20 kDa (348). They are localized in various compartments of the cell, both in the plasma membrane and in
the membrane of different vesicles but also in the cytosol
and the nucleus. Most small GNBPs contain hydrophobic
(COOH-terminal farnesyl or geranyl-geranyl or NH2-terminal myristoyl) chains that are important both for local-
ization and in interaction with certain regulatory proteins
(13, 152, 212, 222, 240, 279, 310). COOH-terminal modifications require the so-called CAAX-box motif (cysteinealiphatic-aliphatic-any amino acid) and differ in the detailed nature of the lipid and attachment site (FIGURE 2)
(273, 348, 390). Small GNBPs are activated by specialized
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FIGURE 2. Structure of the G-domain core and variations found in different monomeric G proteins. The
guanine nucleotide binding site of the G-domain is formed by the flexible, catalytically important switch I/II
regions, the phosphate binding P-loop, and the NKxD motif, which mediates nucleotide specificity. Mg2⫹
interacts as well with the phosphate groups of the nucleotide and is positioned by residues from switch I/II,
P-loop, and the DxxG motif. Binding of the guanine ring is further enhanced by direct and indirect interactions
with the SAK (G5) region. The hypervariable region (HVR) found at the COOH terminus is variably lipidated
providing the functionally important membrane anchor. Optional helices found in Rho, Arf, and Ran proteins are
indicated in green (1, 46, 365).
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GAPs IN SIGNAL TERMINATION
regulatory proteins, the GEFs (FIGURE 1, and below). Their
inherent GTPase activity is two or three orders of magnitude lower than that of G␣ subunis of heterotrimeric G
proteins; thus the spontaneous decay of the active conformation is very slow. GTP hydrolysis by small G proteins is
dramatically accelerated by GAPs. The human genome
codes for over 150 monomeric G proteins (“Ras superfamily”) (383), which participate in the regulation of almost all
cellular functions.
A. Structure of the G Domain
B. Families and Function of Monomeric
GNBPs
Traditionally, monomeric (small) GNBPs are grouped into
five families defined by Ras, Rho/Cdc42/Rac, Rab, Arf, and
Ran as representatives (TABLE 1), although some proteins
cannot be assigned to any of these families (348). The individual families have distinct and typical functions that are
summarized below. However, recent reports describe more
and more cross-interaction between the traditional functions of the individual families, and the boundaries become
less defined (100, 244, 281, 333).
At the heart of the biochemical activity of GNBPs lies the
catalytic machinery implemented in the globular core module termed G-domain (FIGURE 2). It is made up by a central
␤-sheet surrounded by ␣-helices (365). The nucleotide
(GTP or GDP) is bound in a shallow pocket on the surface
of the protein that is lined by the characteristic sequence
motifs G1-G5 (FIGURE 2) defining the determinants for nucleotide binding specificity and hydrolysis (46). These include the typical P-loop motif (G1) mediating most of the
commonly high nucleotide affinity, the guanine base binding motif (G4, G5), and the switch regions (G2, G3) that
sense the presence of the ␥-phosphate and are key to the
conformational differences defining GTP-bound ON and
GDP-bound OFF states, respectively (FIGURE 1) (45, 46,
365). This structural arrangement has historically been described with Ras (267, 355) and is found in the vast majority of GNBPs (387). In keeping with specific physiological
requirements, various structural variations are found in the
various subfamilies. These include NH2- or COOH-terminal peptide or helical extensions as well as the insertion of
structural elements or whole domains in loops connecting
the core structural elements (365).
Ras is activated by growth factors, cytokines, and hormones via different plasma membrane receptors. First discoveries reported activation of Ras by growth factors such
as EGF or PDGF via receptor tyrosine kinase (RTK) pathways involving the adaptor protein Grb2 and the exchange
factor SOS (56) (FIGURE 3). Subsequently, other pathways
of SOS activation have been characterized as well as other
RasGEFs have been discovered, which relay Ras activation
to production of the second messenger cAMP or to Ca2⫹
signaling (57, 231).
The GTPase reaction is generally slow, ranging from 1–2
min⫺1 with G␣ subunits (123) to 0.039 min⫺1 for RhoA
(316) and to virtually undetectable GTP hydrolysis for Arf
proteins (170). The mechanism of GTP hydrolysis has been
studied in detail in many laboratories, with Ras being the
probably best-studied example. The spatial constraints imposed by the P-loop as well as the interactions of the switch
regions with the ␤,␥-phosphate moiety are key components
of the catalytic machinery and are major biochemical determinants of the pathogenicity of oncogenic Ras mutants that
are unable to hydrolyze GTP at a rate sufficient to terminate
the downstream signal (4, 356). In the currently accepted
mechanism, Ras employs substrate-assisted catalysis with
GTP itself acting as a general base of G protein-mediated
nucleotide hydrolysis (312), with the nucleophilic water
molecule stabilized by the conserved glutamine of switch II.
After the revelation of the first GAP mechanisms (290, 309),
GTPase mechanistic research with other GNBPs concentrated on the GTP hydrolysis as catalyzed by the GAP components, thus considering the catalytic machinery as a heterodimeric complex made of the GNBP and its cognate
GAP (308).
GTP-bound Ras couples to multiple distinct downstream effectors (FIGURE 3). Interaction is usually mediated by the socalled Ras binding domain (RBD), a ubiquitin structure like
protein module that senses the nucleotide bound state of Ras
mediated by the switch regions (148, 386). The first discovered
and best characterized Ras effectors are the Raf kinases that
control, via activation of the MAP kinase cascade, gene expression, cell proliferation, differentiation, and survival (231,
383). Another well-characterized direct effector of Ras is represented by the phosphatidylinositol 3-kinases (PI3K) that, via
enrichment of phosphatidylinositol 3,4,5-trisphosphate
[PI(3,4,5)P3] in the membrane, regulate several kinases, the
most versatile being perhaps Akt (also called protein kinase B).
In addition to protein and lipid kinases, active Ras also interacts with regulatory proteins of other small GTPases mediating cross-talk to other superfamily members, specifically Rac
and Ral (36). The most important signaling pathways initiated
by active Ras proteins are summarized in FIGURE 3. On a
pathophysiological level, prominent features of Ras-related
signaling processes have been described in the context of tumorigenesis (22, 42, 172, 214) and in brain function (175,
220, 377).
Ras genes have originally been discovered as the cellular
counterpart of a rat sarcoma virus-derived retroviral gene
that is associated with oncogenic transformation (94) encoding H-Ras and K-Ras. Together with the later discovered N-Ras, these proteins constitute the founding members
of the canonical Ras proteins (22). Additional members of
the Ras family include R-Ras, M-Ras, Ral, Rap1/2, and
Rheb and a few less explored examples (348).
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1. Ras family
LIGETI ET AL.
Table 1. Families of the Ras superfamily small GTPases
Ras
Rac/Rho
Rab
Arf
Ran
Number of smg
Representative members
33
N-Ras
K-Ras
H-Ras
Rap1,2
Rheb
15
P120RasGAP
Neurofibromin
RASAL
DAP2IP
SynGAP
22
Rac 1,2,3
Rho A,B,C
Cdcd42
60
Rab5
Rab8A
Rab10
27
Arf1
Arf6
1
⬃70
Abr
Bcr
Chimerins
DLC-1
Oligophrenin
P50GAP
P190GAP
38
TBC1D4
TBC1D1
31
ARAP1
ASAP
1
⬃80
Orientation
53
Intracellular vesicular
transport
18
Assembly of molecular
coat around budding
vesicles
1
Nucleocytoplasmic
transport
Number of GAPs
Representative GAPs
No. of GEFs
Major function
RapGAP
Tuberin
29
Proliferation
Survival
Apoptosis
Migration
Actin cytoskeleton
Myosin
contraction
NADPH oxidase
Only those members of the GTPases and their GAPs are listed which are mentioned in the text. For complete
data, readers are referred to References 32, 163, 348, and 351.
Rap proteins (Rap1A/B, Rap2A/B, Rheb) are close Ras homologs (311) and as such are counted in the same subfamily.
They have originally been found antagonizing Ras-induced
transformation but can oppose other actions of Ras including
regulation of cell growth and differentiation, integrin-dependent responses, and synaptic plasticity (343, 344, 407). Although closely related to Ras, they are functionally different
(410). Rap proteins control primarily cell adhesion, cell junction formation, cell secretion, and cell survival (38, 41, 183).
Rheb turned out to be an important element relaying growth
factor signaling to translation and protein synthesis on ribosomes (215).
2. Rho family
The most prominent members of the Ras homologous
(Rho) family of small GTPases are RhoA, Rac1, and Cdc42
(TABLE 1), which are ubiquitously expressed, whereas expression of Rac2 is restricted only to hematopoietic cells
(328).
The major function of Rho family small GNBPs is regulation of cell morphology, cell polarity, cell motility, cell adhesion, as well as endocytosis and exocytosis. They are
involved in vital biological processes such as embryonic
development, tissue renewal from stem cells (e.g., skin, intestinal epithel, hematopoiesis), wound healing, immune
surveillance, or influence of tumor metastasis (107, 160,
241, 359). Many of these functions are achieved via regulation of the actin cytoskeleton and the actin-binding motor
protein myosin (135, 284, 285).
The first fundamental observations have been made on fibroblasts microinjected with a constitutively active form of
Rho family GNBPs. Expression of constitutively active Rho
induced the formation of stress fibers, i.e., parallel actin
bundles within the cell body (286). Constitutively active
Rac initiated formation of lamellipodia which are broad,
flat cellular extensions (287). Finally, constitutively active
Cdc42 induced the formation of long, thin protrusions,
called filopodia (261). These phenotypical changes could be
reproduced by stimulating the cells with growth factors
such as PDGF, EGF, or insulin (lamellipodia) or lysophosphatidic acid (LPA; stress fibers) (135, 261).
Cell migration is directed by environmental cues such as
extracellular matrix proteins, attractive or repellent chemical stimuli via integrins or plasma membrane receptors. To
start directed movement, the cell has to polarize, i.e., an
asymmetric alteration occurs in the distribution of mem-
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Family
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FIGURE 3. Overview of major Ras-mediated signaling pathways. Ras can either be activated by RTKs directly
(86, 268) or is transactivated by GPCRs via RTKs. This involves the shedding of growth factors, resulting in the
autocrine stimulation of RTKs (173, 197, 263, 300). Main downstream targets of Ras include PI3K (112),
PLC-⑀ (28, 138), MAPK (62, 177, 221), Ral (36, 113, 136, 230), Tiam1 (44, 80), and RASSF1A (326,
362). ADAM, a disintegrin and metalloprotease; Akt (PKB), murine thymoma viral oncogene; APC, anaphase
promoting complex; BAD, Bcl2 antagonist of cell death; CDC42, cell division cycle 42; cFos, FBJ (Finkel-BiskisJinkins) osteosarcoma; cJun, ju-nana (jap. for 17), v-Jun avian sarcoma virus 17 oncogene homolog; cMyc,
v-Myc avian myelocytomatosis viral oncogene homolog; DAG, diacylglycerol; Erk, extracellular signal-regulated
kinase; ETS, erythroblastosis, v-ETS erythroblastosis virus E26 oncogene homolog (avian); FOXO, forkhead box
O1; GPCR, G protein-coupled receptor; GRB2, growth factor receptor-bound 2; HB-EGF, heparin-binding EGF
(epidermal growth factor)-like growth factor; IP3, inositol trisphosphate; KSR, kinase suppressor of Ras; Mek,
mitogen-activated protein kinase kinase 1; NF␬B, nuclear factor ␬B; PDK1, pyruvate dehydrogenase kinase,
isozyme 1; PI3K, phosphatidylinositol-3-kinase; PI345P3, phosphatidylinositol-3,4,5-trisphosphate; PI45P2,
phosphatidylinositol-4,5-bisphosphate; PKB, protein kinase B; PKC⑀, protein kinase C⑀; PLC⑀, phospholipase
C⑀; p120GAP, p120 GTPase activating protein; Rac, Ras-related C3 botulinum toxin substrate 1; Raf,
replication-defective acutely transforming; RalA, Ras like A; RalB, Ras like B; RalGDS, Ral guanine-nucleotide
dissociation stimulator; Ras, rat sarcoma viral oncogene homolog; RASSF1A, Ras association domain family
1 isoform A; RLIP, Ral interacting protein; RTK, receptor tyrosine kinase; Shc, Src homology 2 domain
containing; SOS, son of sevenless; SRC, v-Src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene; TBK1,
TANK binding kinase 1; Tiam1, T-cell lymphoma invasion and metastasis protein-1.
LIGETI ET AL.
brane components. Cdc42 is the key regulator of cell polarization, and it becomes activated toward the front of the
migrating cell. Cdc42 initiates the formation of filopodia
that can be regarded as fine sensors of the environment.
Filopodia contain long parallel bundles of actin filaments.
Extension of nonbranching actin filaments is organized by
the Cdc42-target proteins Diaphanous-related formins Dia
1–3 (285) (FIGURE 4). Polarization also involves typical distribution of phosphoinositides and their synthesizing and
hydrolyzing enzymes: PI3K that is required for synthesis of
phosphoinositides phosphorylated in position 3= is enriched
at the front, whereas the phosphatase PTEN is accumulated
in the tail (223, 288, 303). Accordingly, at the leading edge
an enrichment of PI(3,4,5)P3 and phosphatidylinositol 3,4bisphosphate [PI(3,4)P2] can be typically observed.
Finally, retraction of the tail involves contraction by the actomyosin complex, where a kinase regulated by active Rho
(ROCK) participates in activation of myosin. Elaborate fluorescent microscopic techniques involving energy transfer between interacting proteins have indicated a fine spatial and
temporal sequence in activation of the different members of
Rho family GTPases in migrating cells (121, 188). In the spatio-temporal orchestration of the cellular processes, several
positive-feedback loops and cross-talk between the members
of Rho family GTPases have also been revealed (288).
Beyond their central role in cytoskeletal organization, Rho
family members also function as direct regulators of important
enzymes. Rac1 and Rac2 are key components in the enzyme
complex responsible for superoxide formation in phagocytes
(NADPH oxidase, NOX2) (2) and in many other tissues by its
homologs NOX1 and NOX3 (30). Rho is a critical regulator
of smooth muscle contraction via its effector Rho kinase
(ROCK), mainly via inhibition of myosin light-chain phosphatase (289) and phosphorylation of myosin light chain (234). In
addition to protein kinases, several kinases regulating phospholipid metabolism (e.g., PI4P-5-kinase, PI3K, diacylglycerol
kinase, phospholipase D, or phospholipase C isoforms) have
been shown to be regulated by one or several members of the
Rac/Rho family (160).
Last but not least, members of the Rho subfamily of small
GTPases also play a fundamental role in the control of cell
3. Rab and Arf family
Small GNBPs of these two families participate in the organization of intracellular vesicular traffic including endo-,
exo-, and transcytosis as well as anterograde and retrograde
traffic between ER and Golgi (25, 85, 340, 403) (FIGURE 5).
In mammals, the Rab family consists of ⬃60 different proteins
that are localized in the membrane of the various subcellular
organelles and function in specifying membrane identity (64).
Most intracellular vesicles contain more than one type of Rab
protein; however, these may be segregated in different vesicular domains (336). The large number of Rab proteins and their
regulatory proteins in mammalian cells serves probably the
fine organization of trafficking of many different types of
cargo molecules (118).
In their active, GTP-bound form, Rab proteins bind to specific
effector proteins and initiate the formation of large protein
complexes stabilized both by protein-protein and protein-lipid
interactions. Perhaps the best studied function of Rab proteins
is in tethering, i.e., specific identification of membrane compartments, that precedes vesicle docking and fusion (FIGURE 6).
Tethering factors [such as the early endosome antigen 1
(EEA1) localized to early endosomes] contain coiled-coil
regions and multiple binding sites for the same or different
Rab proteins. In this way, they provide a first connection
between two identical or different membrane compartments, a prerequisite for activation of the fusion machinery,
including the SNARE complexes (340).
Whereas Rabs are present on almost every type of intracellular
organelle, members of the Arf family have a more restricted
function. Arf1 is localized to the Golgi membrane where it is
involved in regulation of the assembly of coat complexes
around budding vesicles. The other well-investigated member
of the Arf family, Arf6, is localized in the plasma membrane,
where it regulates the endocytic pathway via organization of
both clathrin-dependent and clathrin-independent processes
(85). Cooperation between Rab and Arf proteins is represented by an increasing number of reports on the existence of
golgin proteins with multiple binding sites for different members of the Rab and Arf family of GTPases (58, 111).
Both Rab and Arf proteins were reported to regulate several
enzymes of the phospholipid metabolism (85, 340). The
resulting local changes in the phospholipid concentrations
(mainly of phosphoinositides) seem to provide significant
contribution to the assembly of the membrane-associated
complexes.
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Activation of Rac at the leading edge, via RacGEF(s) localized
by receptor-initiated protein and/or lipid interactions, results
in development of lamellipodia that contain a branched network of actin filaments. The branching proteins Arp2/3 and
the adaptor proteins WASP/WAVE, which are direct effectors
of active Cdc42 and Rac, respectively, play the central role
(FIGURE 4). Lamellipodia form focal adhesions to extracellular
matrix proteins or neighboring cells, which provide traction
sites for forward movement. Inhibition of Rac activation prevents migration, whereas inhibition of Cdc42 activation transforms directed migration into random movements (11).
cycle progression. Both Rho, Rac, and Cdc42 are able to
activate the JNK and p38 MAP kinase pathways (120, 266,
352, 353), and Rac and Cdc42 were shown to stimulate
cyclin D1 transcription (224, 381). This way Rho family
proteins play an essential role in G1/S transition (73).
GAPs IN SIGNAL TERMINATION
4. Ran family
The Ras-like nuclear protein (Ran) is a key regulator of
nucleocytoplasmic transport (190, 380). The active, GTPbound form of Ran is significantly enriched in the nucleus,
due to the exclusive localization of the two regulatory proteins. The Ran-GEF RCC1 (for regulator of chromosome
condensation) is associated with the chromatin, whereas
RanGAP prevails in the cytoplasm. RanGTP binds to the
nuclear transport proteins named importins and exportins;
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LIGETI ET AL.
however, this binding alters the affinity to the transported
cargo in opposite directions. RanGTP enhances the dissociation of the cargo molecule from importin, but it promotes
the association of cargo molecules with exportins.
The function of Ran is not restricted to the interphase, but
plays important roles also in other phases of the cell cycle (71,
201). In the metaphase, RanGTP enriched around the chromatin stimulates microtubule polymerization and the assembly of the mitotic spindle, whereas a decrease in the local
concentration of RanGTP impairs the alignment of chromo-
somes (190). In the telophase RanGTP is required for reconstruction of the nuclear membrane (71).
C. Regulatory Proteins Acting on
Monomeric GNBPs
Two types of regulatory proteins act on each family of small
GNBPs (39, 44) (FIGURE 1). GEFs catalyze the exchange of the
bound nucleotide, and in the cellular environment, where the
GTP concentration largely exceeds the GDP concentration,
FIGURE 4. Selected signal transduction pathways involved in Rho/Rac/CDC42 signaling. Rho, Rac, and CDC42 are main regulators of the
actin cytoskeleton, utilizing various overlapping signaling cascades to address key components of the F-actin nucleation, polymerization, and
branching machinery (dark green/yellow). The GNBPs themselves are regulated by a network of GEF (green) and GAP (red) proteins downstream
of both GPCRs and RTKs (10, 136, 141, 143, 180, 218, 265, 271, 334). ARP2/3, actin related protein 2/3; CDC42, cell division cycle 42;
Ena/VASP, enabled/vasodilator-stimulated phosphoprotein; Fak, focal adhesion kinase; GNBP, G-nucleotide binding protein; GPCR, G proteincoupled receptor; GRIN1, G protein-regulated inducer of neurite outgrowth; IRSp53, insulin receptor substrate p53; IQGAP, IQ motif-containing
GAP 1; LIMK, LIM (lin-11, Isl-1, mec-3) kinase; LMW-PTP, low-molecular-weight protein tyrosine phosphatase; mDIA, mammalian diaphanous;
mDIA2, mammalian diaphanous 2; MLC, myosin light chain; MYPT, myosin phosphatase targeting; PAK, p21 activated kinase; PI3K,
phosphatidylinositol-3-kinase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; Rac, Ras-related C3 botulinum toxin substrate 1; Rho, Ras
homologous protein; ROCK, Rho-associated kinase; RTK,receptor tyrosine kinase; Src, v-Src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene; WASP, Wiskott-Aldrich syndrome protein; WAVE, WASP-family verloprolin-homologous protein.
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FIGURE 5. Anterograde and retrograde vesicular traffic directed by Rab and Arf proteins. The large numbers
of different Rab and Arf proteins specifically mediate defined sets of endosomal transport routes. For details,
see selected reviews (49, 126, 340). ER, endoplasmatic reticulum; MVB, multivesicular body; TGN, transGolgi network.
GAPs IN SIGNAL TERMINATION
they bring about the activation of the small GNBPs. In contrast, GAPs accelerate the slow intrinsic hydrolysis of GTP
and thereby promote the deactivation of the small G protein.
The third type of regulatory proteins, guanine nucleotide dissociation inhibitors (GDIs), act only on small GTPases of the
Rho and Rab family.
1. GEFs
In the resting state, GNBPs are bound to GDP. To become
activated, the usually tightly bound GDP has to be exchanged
for GTP, requiring the activity of GEFs. GEFs play a similar
role as the ligand-receptor complex plays in activation of the
␣-subunit of heterotrimeric G proteins. These proteins essentially destabilize nucleotide binding, thereby allowing release
of GDP and binding of the intracellularly abundant GTP (44).
GEFs are generally family specific, and the GEF domain is
frequently embedded as a module of 20 –30 kDa in multidomain proteins. The best explored examples include the Rasspecific Sos, the Rho-family Dbl-homology and DOCK, and
the Arf-specific Sec7 domains. GEF mechanisms have been
explored intensively since the cloning of the first mammalian
GEF (329) more than 15 years ago, and structural information
is available for a number of examples (44, 66).
ments of the nucleotide binding residues, especially the switch
regions and the P-loop to break up the nucleotide binding pocket.
Expelling the catalytic cofactor Mg2⫹ also seems to represent a
common feature of GEF mechanisms (44).
Regulation of GEFs occurs via a variety of strategies as reflected in their multidomain modular architecture. They include protein-protein interactions, lipid binding, interaction
with second messenger molecules [cAMP, diacylglycerol
(DAG), Ca2⫹], and posttranslational modification such as
phosphorylation. Localization and local activation along with
phosphorylation appear to be predominant strategies how
GEFs act in the right place at the right time (44). Signaling
complexes consisting of a GEF, its GTPase substrate, and the
effector protein of the GTPase have been identified for the
RhoGEF p115RhoGEF (161) and the RacGEFs Tiam1 (76)
and Cool-2/␣-Pix (20), indicating the ability of multidomain
GEFs to carry out also scaffolding function. In the case of
p63RhoGEF, the crystal structure of the complex of the GEF,
its regulator G␣ and its target RhoA has been solved (211),
providing the molecular mechanism for activation of a monomeric G protein by a plasma membrane G protein-coupled
receptor (320, 392).
2. GAPs
Although the GEFs for the various families are unrelated in either
sequence or structure, the derived mechanisms in general appear
to follow similar strategies. Accordingly, GEFs induce rearrange-
246
In the human genome, ⬃150 genes code for proteins with
potential GAP activity. The sizes of these proteins range
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FIGURE 6. Rab5-mediated vesicle fusion. Rab5-mediated vesicle fusion is well investigated and could be
reconstructed in vitro: RabGDF recruits Rab5 to the membrane by displacing RabGDI. In a positive-feedback
loop, Rab5 recruits its own GEF Rabex-5, thereby stabilizing the population of active Rab5 at the membrane.
Via PI3K, the membrane microenvironment is enriched with PI345P3, resulting in the recruitment of tethering
factors and SNARE complexes. Membrane fusion is then caused by conformational changes in the SNARE
complexes, which are subsequently regenerated (74, 264). EEA1, early endosome antigen 1; NSF, Nethylmaleimide-sensitive factor; PI3K, phosphatidylinositol-3-kinase; PI345P3, phosphatidylinositol-3,4,5-trisphosphate; Rab, ras-related in brain; RabGDF, Rab GDI displacement factor; RabGDI, Rab guanine nucleotide
dissociation inhibitor; SNAP, soluble NFS attachment protein; SNARE, soluble N-ethylmaleimide-sensitive
factor attachment protein; VPS45, vacuolar protein sorting associated protein 45.
LIGETI ET AL.
from 50 to 250 kDa. Several GAPs are not monospecific;
rather, they recognize and interact with a few small GNBPs
of the same family. GAPs acting on Rac/Rho family GTPases are especially abundant: the human genome contains
⬃70 potential GAPs (32, 275), which are in almost threefold excess over Rac/Rho small GTPases. Inverse relation
holds for the Rab family: 38 GAPs act on 60 Rab proteins
(TABLE 1).
By acceleration of GTP hydrolysis, GAPs terminate the active state of the G protein and promote the formation and
prevalence of the GDP-bound, inactive form of the GTPase.
Whether this results in inhibition of the regulated biological
process or rather in termination of the signal depends on the
rate of activation of the G proteins by GEFs or GPCRs
(296). When the rate of activation largely exceeds the rate
of GTP hydrolysis, then signal termination will be dominant, whereas in the case of comparable activation and
hydrolysis rates, substantial inhibition of signaling will occur (45). The same GAP can inhibit signaling or enhance
termination (or influence both) in different processes of the
same cell (296).
The substrate specificity of different GAPs has been determined first under in vitro conditions, measuring the GTP
hydrolytic activity of bacterially expressed small GTPases
in the absence and presence of suspected GAPs (316, 356).
Later investigations carried out in cellular models indicated
that the substrate specificity determined under in vitro or in
vivo conditions may diverge (68, 69, 127, 235, 373). This
may be due to structures outside of the catalytic domain of
FIGURE 7. Mechanisms of GAP/RGS-assisted GTP hydrolysis by GNBPs. Key factors for the efficient
hydrolysis of GTP are 1) correct positioning of the attacking water molecule, 2) stabilization of the transition
state of the hydrolysis reaction by neutralization of developing negative charges at the nucleotide (Arg-Finger),
3) stabilization of the water-positioning residue, and 4) stabilization of the switch regions of the GNBPs (not
depicted, valid for all GAPs). The relative importance of these factors varies within the depicted GNBP/GAP
systems. GNBP: light blue, GAP/RGS: light red, sandwiched between GNBP and GAP are GTP (yellow) and
water (dark blue). Amino acids are noted in the three-letter code; dotted arrows indicate an enhancing effect
regarding GTP hydrolysis (44, 72, 158, 269, 308, 313, 314, 335, 337). Arf, ADP-ribosylation factor; GAP,
GTPase activating protein; GNBP, G-nucleotide binding protein; Rab, ras-related in brain; Ran, ras-related
nuclear protein; Rap, ras-related protein; Ras, rat sarcoma viral oncogene homolog; RGS, regulator of G
protein signaling.
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GAP activities are normally family specific, with the catalytic machinery implemented in modules with molecular
weights ranging from ⬃20 to ⬃50 kDa. Family specific
GAP domains differ in their structural topology (fold) and
catalytic mechanisms to complement the G protein active
site (FIGURE 7). While Ras- and RhoGAPs employ a catalytic arginine provided in trans (249, 290, 309), genuine
RapGAPs use a catalytic asparagine that is believed to play
the role of the catalytic glutamine in Ras to position the
water molecule or derived nucleophile for nucleophilic attack (89). In addition, mixtures of strategies have been reported for RabGAPs (269) and RanGAP (314). As a general
strategy, GAPs seem to stabilize the G protein switch regions in the majority of the examples and use a catalytic
residue to contribute to in trans transition stabilization
(308). RGSs represent a large group of GAPs acting on
heterotrimeric G protein ␣-subunits that appear to primar-
ily adjust the existing catalytic machinery but apparently do
not provide a catalytic residue (297).
GAPs IN SIGNAL TERMINATION
GAPs or to posttranslational modification of the GAPs or
the substrate GTPases.
In the case of several GAPs (p50RhoGAP, oligophrenin,
Abr, chimerins), autoinhibition due to protein folding has
been described (75, 102, 108, 162, 240) that limits the
activity of the relevant GAP. Various regulatory mechanisms were shown to be required to relieve this autoinhibition (75, 102, 108, 162, 240). Alternatively, the posttranslational addition of lipid tail to the substrate GNBP was
necessary to allow the interaction with the relevant GAP
(240).
Different cells express their own “GAP repertoire.” Early
studies have identified six different Rac/RhoGAPs in neural
tissue (216) and three Rac/RhoGAPs in neutrophilic granulocytes (122). Expression of multiple GAPs with similar
substrate specificity in the same cell raises the question of
specific or overlapping functions. The multidomain structure and the large differences in the domain composition of
individual GAPs suggest that these proteins may participate
in large protein complexes and function also as scaffolds. In
fact, IQGAPs, which contain a RasGAP-homology domain
and bind the GTP-bound active form of Rac or Cdc42, are
devoid of GAP activity and function rather as Rac- or
Cdc42 effector scaffolds (48). Likewise, the breakpoint
cluster region of the p85 ␣-subunit of PI3K is structurally
homologuous to the canonical RhoGAP module but has no
detectable GAP activity towards Rho family members
(243).
Examples of cross-talk between small GNBPs and GAPs
have been demonstrated, e.g., Rac was shown to interact
with p190B thereby influencing its RhoGAP activity (59) or
Arf1 was shown to recruit ARHGAP10 to the Golgi membrane (100) or the activity of RA-RhoGAP was shown to be
increased by Rap1 binding (395). A few GAPs have been
reported to have dual or even triple G protein specificity.
248
3. GDIs
GDIs bind to the GDP-bound form of the relevant small G
protein and protect them from the effects of GEFs. Binding
of Rho family GNBPs to RhoGDIs involves both proteinprotein interactions and binding of the prenyl tail of the
small GTPase into the lipid binding pocket of RhoGDI
(152, 166, 310). This way, RhoGDI is able to extract its
target from the plasma or intracellular membranes and sequester it in an inactive form into the cytosol. Similarly,
RabGDI is able to bind Rab proteins with two geranylgeranyl tails (13, 279) and shuttle them through the cytosol
to another vesicle where GDI displacement factor (GDF)
contributes to the liberation of GDI and association of Rab
with the appropriate vesicular membrane.
III. ROLE OF GAPs IN THE NERVOUS
SYSTEM
In the developing nervous system migration of cells (or entire cellular layers), formation and guidance of axons as
well as dendritic branching and spine formation depend
largely on organization of the actin cytoskeleton. The central role of various members of the Rho/Rac family of small
GTPases (see sect. IIB2) in all these processes has been
shown and reviewed previously (106, 128, 209, 254, 255,
280, 375).
Antagonism between Rho and Rac activation has been observed both in neurite outgrowth and dendrite formation.
Typically, activation of Rac induces lamellipodia and
growth cone formation, promoting neurite outgrowth,
whereas activation of Rho results in growth cone collapse
and neurite retraction (184). A similar antagonism has been
reported in axon pathfinding directed by the guidance molecule netrin-1 (202). However, the situation may not be
generalized, as growth cone collapse induced by other guidance molecules (semaphorin 3A and 3D) has been reported
to be mediated via active Rac (165, 363).
In addition to effects on neurite outgrowth, Rac and Rho
activity had also different effects on dendritic development
and synaptogenesis: activated Rac initiated whereas activated Rho reduced the formation of dendritic spines (248,
350). Genetic manipulation of the expression of Rac/Rho
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GAPs are complex proteins containing a wide variety of
different domains (FIGURE 8) that allow interactions with
proteins or lipids and represent targets of many regulatory
processes. In fact, a broad spectrum of regulatory processes
has been described such as phosphorylation/dephosphorylation, protein-protein interaction, lipid binding, and degradation/resynthesis (33) that are able to modify the physiological effect of various GAPs. These regulatory processes
may alter either the catalytic activity of GAPs, or their localization and consequently their access to the GNBP substrates. Interestingly, there are examples where phosphorylation alone or in combination with lipid binding alters the
substrate preference of a GAP (200, 206, 228). In a complex
system like the cell, several modifying interactions may take
place simultaneously, and in many cases, clarification of the
precise mechanism in comparative in vitro and in vivo studies is still missing.
These include the Ras/Rap specific SynGAP (187) and
GAPIP4BP/Rasal/CAPRI (82) proteins. Equipped with a canonical RasGAP module it turned out that RapGAP activity
required portions outside the catalytic GAP module (194,
276). Biochemical studies suggested that the dual specificity
is mediated by components of the Rap protein itself (337).
Another interesting example is provided by ARAP1 that
contains both an ArfGAP and a RhoGAP domain, but the
two activities are regulated separately (232) and the protein
couples vesicular traffic with cytoskeletal movements (244).
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FIGURE 8. Domain organization of typical GAP representatives regulating families of G proteins. In the right
column, the Uniprot (www.uniprot.org) accession numbers of the depicted proteins are given.
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GAPs IN SIGNAL TERMINATION
also an NH2-terminal SH2 domain that allows interaction
with phosphotyrosines (FIGURE 8).
It is not exclusively the Rho/Rac family of small GNBPs that
plays the central role in development and functioning of the
nervous system. Members of the Ras/Rap family controlling the activity of the MAPK pathway, gene expression,
and protein synthesis have also been shown to be key elements (397).
The four studies provide a comprehensive picture on the
essential and specific role of ␣2-chimerin in EphA4 signaling (31, 159, 327, 378). In biochemical experiments, binding of ␣2-chimerin to EphA4 has been demonstrated in a
yeast two-hybrid system (378), by affinity chromatography
(31), and by coimmunoprecipitation (159). The interaction
has been localized to the NH2-terminal SH2 domain of
␣2-chimerin and the juxtamembrane tyrosines of EphA4
(31). Most of the data suggest that the kinase activity of
EphA4 is required for the binding and results in tyrosine
phosphorylation of ␣2-chimerin (31). EphA4 signaling reduced the level of active Rac, and this effect depended both
on tyrosine phosphorylation and on the GAP activity of
␣2-chimerin (327).
Investigations deciphering the molecular pathways leading
to activation of small GTPases have mainly focused on exchange factors and have successfully identified several GEFs
that function in different molecular complexes (193, 260,
376). However, the activity level of any small GTPase depends on the fine balance between activating and inactivating factors, i.e., on the ratio of GEF to GAP activities. In the
following sections we describe examples where alteration of
the GAP activity results in disturbance of the spatiotemporal fine regulation of different small GTPases with consequent disturbance of the development and function of neural activities.
A. Axon Guidance
Development of the neural network requires precise guidance of outgrowing axons. Pathfinding is directed by attractive and repulsive cues provided by soluble or transmembrane guidance molecules such as netrins, Slits, semaphorins, or ephrins (96). Signaling through the appropriate
receptor molecules in the plasma membrane of developing
neurons regulates cytoskeletal dynamics resulting in elongation or retraction of the growing axon. Interestingly, the
same family of receptors may mediate both repulsive and
attractive signals. Small GTPases have been implicated in
the signaling pathway of several guidance receptors.
1. Eph receptor signaling
Axons of the neurons in layer 5 of the motor cortex descend
in the corticospinal tract and form synapses with spinal
motoneurons on the contralateral side. They are prevented
from crossing the midline at spinal level by repelling cues
provided by ephrinB3 gradient along the midline of the
spinal cord. EphrinB3 is a ligand for the tyrosine kinase
receptor EphA4, and signaling via the EphrinB3/EphA4
pathway was discovered to play a specific role in formation
of the motor system.
In 2007, four different teams published concordant results
on involvement of ␣2-chimerin in EphA4 signaling. Chimerins are the products of two related genes (␣- and ␤- chimerin), both occurring in two splice variants. They function as
GAPs for the small GNBP Rac. In addition to the typical
GAP domain, ␣1- and ␤1-chimerin possess a DAG binding
C1 domain. The longer variants (␣2- and ␤2-chimerin) have
250
In ␣2-chimerin knock-out animals, a remarkable change in
motoric coordination has been observed: the alternating
movement of the hindlimbs has been changed to synchronized movement, and the mice showed a rabbit-like hopping movement (FIGURE 9). In histological analysis, recrossing of the descending corticospinal axons has been shown in
the spinal grey matter. Electrophysiological data are consistent with the histological finding: in ␣2-chimerin⫺/⫺ animals unilateral stimulation of the motor cortex resulted in
bilateral movement and EMG activity in the hindlimbs (31).
Recrossing of the midline was not restricted to descending
corticospinal axons but could be observed also in the case of
axons of spinal interneurons resulting in dysfunction of
left-right coordination of spinal central pattern generator
neurons (378). The described alteration in motoric control
is a phenocopy of that observed earlier in EphA4- or ephrinB3-deficient animals (77, 99, 191, 192). Recrossing of
the midline was restricted to motoric axons, whereas proprioceptive sensory projections (which depend on plexinA1
guidance) have not been disturbed in ␣2-chimerin⫺/⫺ animals (31).
Neurons isolated from the motor cortex of wild-type animals show typical growth cone collapse upon treatment
with ephrinB3. Ephrin-induced growth cone collapse was
significantly impaired in cultured neurons of ␣2-chimerin⫺/⫺ animals (31, 378). This phenomenon could be reproduced in cultured neurons by silencing ␣2-chimerin expression or by expressing GAP-deficient ␣2-chimerin (327). The
need of local reduction in Rac activity for growth cone
collapse is consistent with the general observation that activated Rac induces axon outgrowth (see above).
The remarkable phenotype of ␣2-chimerin⫺/⫺ animals developed in spite of the undisturbed expression of ␣1-chimerin (31). Taken together, the presented data suggest that the
specific interaction between EphA4 receptor and the RacGAP ␣2-chimerin results in spatially restricted downregu-
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GNBPs suggested that axon growth, guidance, and branching are organized by different signaling pathways (255).
LIGETI ET AL.
lation of Rac that is inevitable for correct transmission of
repellent cues presented by ephrinB3 gradient in the midline
of the spinal cord.
The role of ␣2-chimerin in axon guidance has been supported by observations made in cases of a human disorder,
the Duane’s retraction syndrome (233). This congenital eye
movement disturbance is caused by aberrant innervation of
extraocular muscles by axons of the abducens and oculomotor nuclei resulting typically in restricted outward gaze
(FIGURE 10). In several patients, hypoplasia of the given
nerves could be demonstrated by magnetic resonance imaging. Genetic analysis of the affected families located the
mutation to CHN1, the gene of ␣-chimerins. Three of the
FIGURE 10. Typical disturbance of eye movements in Duane’s
retraction syndrome. [From Miyake et al. (233), with permission
from The American Association for the Advancement of Science.]
seven identified mutations localized to the segment that is
only present in ␣2-chimerin; thus the probable cause of the
disease is a misfunction of ␣2-chimerin. In vitro analysis of
the proteins with the identified mutations revealed that all
of them were gain-of-function mutations, mainly due to
increased binding to membrane phospholipids via the C1
DAG-binding domain of ␣2-chimerin. Overexpression of
␣2-chimerin in the oculomotor neurons of developing chick
embryos resulted in premature termination of the oculomotor axons. In which receptor pathway is ␣2-chimerin involved in cranial nerves has to be determined later. However, the observations are consistent with previous findings
on genetically modified mice: loss-of-function of ␣2-chimerin caused aberrant axon growth and loss of sensitivity to
repellant cues, whereas gain-of-function mutations of ␣2chimerin cause premature termination of axon growth.
Most recent data have revealed the role of the RapGAP
TSC2 (or tuberin) in downstream signaling of neuronal
EphA receptors (257). Stimulation of retinal ganglion cells
(RGCs) with ephrinA1 decreased the ERK1/2-dependent
phosphorylation of TSC2 with consequent increase of the
GAP activity and inactivation of the small GTPase Rheb.
Activity of Rheb is a crucial factor in regulation of the
mTOR pathway and protein synthesis. Increased mTOR
signaling was demonstrated in RGC upon expression of a
constitutively active mutant of Rheb (257). The role of local
protein synthesis in growth cone dynamics and axon guidance has been demonstrated earlier (50, 208). Growth cone
collapse induced by ephrinA1 was reduced in RGCs both by
expression of a constitutively active mutant of Rheb and by
silencing of TSC2. These data suggest that a decrease of
local protein synthesis as a consequence of decreased activity of the Rheb-mTOR pathway contributes to the growth
cone collapse induced by EphA receptor ligands. In line
with this hypothesis, Tsc-deficient mice (Tsc⫹/⫺) had in-
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FIGURE 9. Change of locomotion pattern in ␣2-chimerin-deficient mice. [From Beg et al. (31), with permission from Elsevier.]
GAPs IN SIGNAL TERMINATION
creased mTOR activity in the axons of the RGCs and
aberration of the axonal projection to the lateral geniculate nucleus has been detected (257). Mutations of TSC2
have been observed in patients suffering from tuberous
sclerosis. In addition to tumor growth (see sect. VII),
these patients also develop epilepsy, autism, or intellectual disabilities, which may be consequences of aberrant
axon guidance.
2. Semaphorin signaling
Semaphorin-induced responses were shown to depend on
the expression of p190A (GRLF1 in FIGURE 8), a GAP acting on Rac/Rho family small GTPases (23). By coimmunoprecipitation, a direct interaction between p190A and both
plexin A1 and B1 could be demonstrated. Furthermore,
stimulation of the cells by semaphorin ligand transiently
increased the association of p190A with the plexin receptor
consistent with a transient decrease in the level of active
RhoA (23). Depletion of cellular p190A levels impaired or
prevented several semaphorin-stimulated responses such as
the neurite outgrowth in PC12 neuroblasts, collapse and
integrin-mediated adhesion of fibroblasts, migration of
SKBR3 mammary carcinoma cells, or repulsion of primary
endothelial cells. Collapse and adhesion of fibroblasts could
be rescued by expression of wild-type but not GAP-deficient
p190A in p190A⫺/⫺ cells, indicating that the GAP activity
of p190A was required for the semaphorin-dependent response.
Impairment of neurite outgrowth in p190A-deficient cells is
consistent with findings in p190A knockout animals (52).
This mutation is perinatally lethal due to multiple and serious disturbance of neural development. Histological analysis of the embryos indicated inhibition of neurite outgrowth
and guidance at several locations of the brain such as subcortical and cortical axons or the anterior commissure
where projections do not reach and do not cross the midline
(51, 52).
3. Axon branching
While Rho-family members control the assembly of cytoskeletal structures during neural development, Ras-like
GNBPs seem to be responsible for the promotion of signal
transduction pathways involved in axonogenesis (136). The
consequences of the loss of RasGAPs in neuronal development have been investigated specifically with neurofibromin and SynGAP in mice. Loss of neurofibromin in neurons
leads to increased axon collateral branching after dorsal
252
B. Formation of Dendritic Spines
Mental retardation has been shown to be related to disturbance of the size and density of dendritic spines, the major
sites for excitatory synapses in the central nervous system
(280). The first gene associated with X-linked mental retardation was OPHN1, which codes the protein oligophrenin-1, a GAP for Rho family small GNBPs (34). All the
mutations identified in OPHN1 are loss-of-function alterations with decreased or absent expression of oligophrenin-1
(401).
Oligophrenin-1 shows a widespread distribution in the nervous system, present both in dendritic shafts and spines and
axons and axon terminals (127). Downregulation of oligophrenin-1 in CA1 pyramidal neurons in hippocampal slices
resulted in shortening of dendritic spines that was detectable already 48 h following the treatment. After 8 days also
the spine density was significantly decreased (245). Both the
morphological and the behavioral and memory changes
could be reproduced in oligophrenin-1 knockout mice
(176).
Oligophrenin-1 is able to enhance both in vitro and in vivo
the GTPase activity of all three major members of the Rho/
Rac family, i.e., RhoA, Rac1, and Cdc42 (127). However,
the fact that the observed morphological changes of dendritic spines could be mimicked with constitutively activated Rho and prevented by inhibitors of Rho kinase, suggests that oligophrenin-1 affects spine morphology by reduction of local Rho activity.
In a recent study, oligophrenin-1 was shown to be enriched
in dendritic spines upon activation of NMDA receptors and
to be coimmunoprecipitated with the AMPA receptor subunits GluR1 and GluR2 but not with the NMDA receptor
subunit NR1. Overexpression of oligophrenin-1 enhanced
whereas its downregulation decreased synaptic transmission via AMPA but not NMDA receptors. At the same time,
an increase or a decrease of spine density could also be
observed. All these effects depended on the intact GAP activity of oligophrenin-1. Both expression of oligophrenin-1
and inhibition of Rho kinase decrease the NMDA-induced
internalization of the AMPA receptor subunit GluR2. Thus
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Another family of axon guidance molecules is constituted
by semaphorins that interact with plexin receptors. The
biological function of semaphorins is not restricted to the
nervous tissue, as they also play a role in shape changes and
migration of epithelial and endothelial cells.
root injury, correlating with elevated activation of the
MAPK pathway (294). Neurofibromin is also required for
barrel formation in the mouse somatosensory cortex (210),
and its ablation in neurons induces abnormal development
of the cerebral cortex and reactive gliosis in the brain (408).
While in those studies the importance of the RasGAP was
primarily derived from upregulation of the MAPK pathway, a study in PC12 cells demonstrated the role of the GAP
domain of neurofibromin for neurite outgrowth by overexpressing an inactive mutant, which resulted in impaired
neurite outgrowth (400).
LIGETI ET AL.
the surface of the cerebellar cortex below the Purkinje cell
layer. This migration is dependent on processes of the Bergmann glial cells.
In addition to its effects on the postsynaptic side, important
function has been ascribed to oligophrenin-1 also in presynaptic axon terminals (247). It has been demonstrated that
reduction or lack of oligophrenin-1 impairs recycling of
synaptic vesicles by delaying endocytosis. At high-frequency stimulation, an impairment of synaptic transmission could be revealed when oligophrenin-1 was downregulated in the presynaptic neurons.
At birth, the cellular structure of the cerebellar cortex did
not differ in wild-type and Bcr plus Abr double null mutant
animals. However, serious differences occurred within the
first 2 weeks: in double null mutant animals, granule cells
were found in an aberrant location on the cerebellar surface
(167). Bcr and Abr are normally expressed both in Purkinje
and granule cells as well as in astrocytes. Detailed morphological investigation could not reveal any alteration in Purkinje cells or granule cells of double null animals, whereas
serious defects were observed in organization of cerebellar
glial cells. These cells showed also an increased level of
activated, GTP-bound Rac. Consistent with Rac hyperactivation, double null astrocytes showed increased spreading,
had elevated basal phosphorylation of p38 MAP kinase,
and reacted more vigourously to activation by lipopolysaccharide (LPS) or EGF. How far these morphological and
biochemical alterations affect electrophysiological activity
and coordination of cerebellar functions has not yet been
determined.
Taken together, presently available information indicates
that downregulation of Rho activity by oligophrenin-1 allows replenishing of synaptic vesicles on the presynaptic
side and stabilizes AMPA receptors on the postsynaptic
side. Both effects are critical for efficient synaptic transmission. Reduced expression of oligophrenin-1 in case of mutation of the OPHN1 gene impairs transmission and synaptogenesis in developing brain, explaining (at least partially) the appearance of mental retardation.
Morphology of dendritic spines is also affected by ␣1-chimerin (60). This shorter splice variant appears during embryogenesis at a later timepoint than its longer homolog,
␣2-chimerin, but its expression is continued in adulthood.
Expression of ␣1-chimerin is strongly dependent on neuronal activity as pharmacological blockade can decrease its
expression by 60% within 48 h (60). Activation of mGluRs
or mAChRs induced detectable translocation of ␣1-chimerin to the plasma membrane. This effect depended on phospholipase C (PLC)-␤ activity and the ability of ␣1-chimerin
to bind to DAG. Overexpression of ␣1-chimerin in Purkinje
cells resulted in ⬃50% reduction in dendritic length and
branching points (60), and this pruning effect depended on
both the GAP activity and ability of DAG binding. In contrast, downregulation of ␣1-chimerin increased the length
of dendritic protrusions. These findings are consistent with
the general observation that activated Rac increases spine
generation (see above). Although the functional consequences of alteration of dendritic morphology are not
known, ␣1-chimerin seems to be involved in regulation of
the ratio of active Rac and Rho at defined locations in the
nervous system.
C. Development of the Vestibular System
Mice lacking both Bcr and Abr, two similar RacGAPs (see
details in sect. VB1), exhibit a distinct phenotype characterized by clumsy movements, hyperactivity, and persistent
circling (167, 168). Disturbances have been detected both in
cerebellar and in vestibular development.
In mice, cerebellar development occurs within the first 3
postnatal weeks when granule cells typically migrate from
In addition to cerebellar disturbances, also a dysgenesis of
the vestibular system has been observed in Abr plus Bcr
double null mutant animals (168). The neuroepithel was
found to be detached from the underlying connective tissue
in the utriculus and sacculus, and otoconia were absent in
the double null animals. No similar alteration was observed
either in the semicircular canals or in the cochlea, and the
animals were not deaf.
Further research is needed to clarify whether there is any
common cellular mechanism underlying the two detected
aberrations affecting movement coordination in Bcr⫺/⫺ ⫻
Abr⫺/⫺ animals. However, it is striking that in spite of
general expression of both RacGAPs in various parts of the
nervous system, specifically two locations related to motor
coordination are affected by defective expression of both
Bcr and Abr.
D. Cognitive Functions
Since the MAPK pathway has been demonstrated to be
involved in the regulation of memory formation and synaptic plasticity (220, 377), it is probably not surprising that
deficiency of the RasGAP neurofibromin (encoded by the
tumor suppressor gene NF1) is associated with impairment
of cognitive functions such as learning and memory (3, 283,
298). Mice in which one neurofibromin copy has been genetically inactivated show clear deficits in spatial learning in
a water maze (332). Furthermore, mice in which the alternatively spliced exon 23a has been deleted show learning
deficits but normal development and tumor predisposition
(79). Exon 23a encodes a 23-residue insertion within the
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oligophrenin-1, by local downregulation of Rho activity,
seems to stabilize AMPA receptors and thereby it promotes
synaptic transmission and spine formation (245).
GAPs IN SIGNAL TERMINATION
IV. ROLE OF GAPs IN THE ACTION OF
INSULIN ON GLUCOSE UPTAKE
One of the most characteristic effects of insulin is the stimulation of glucose uptake into fat and muscle cells. This
action is achieved by enrichment of a specific isoform of
glucose transporters, the GLUT4 in the plasma membrane
(FIGURE 11). Prevalence of GLUT4 on the cell surface depends on the dynamic equilibrium of exocytosis and endocytosis of specialized membrane vesicles. Insulin accelerates
the exocytosis and impairs the endocytosis of GLUT4-containing vesicles. In skeletal muscle cells also physical exercise is able to mobilize GLUT4 transporters to the cell surface.
Although the above facts were known for decades, the signaling pathway from insulin receptors to mobility of
GLUT4-containing vesicles remained an enigma until the
21st century. Several elements such as insulin receptor substrate (IRS), PI3K, and Akt, a PI3K-dependent serine/threonine kinase, have been identified in the signaling pathway
from insulin receptor to GLUT4 translocation. The breakthrough came in 2002–2005 with the discovery of AS160,
an Akt substrate of 160 kDa present in adipocytes and
muscle cells (171). AS160 was shown to become phosphorylated by Akt on several amino acids, and this phosphorylation (especially that of T642) was essential for insulindependent translocation of GLUT4 (306, 402). The most
intriguing discovery was that AS160 proved to be a GAP for
several Rab proteins, and the GAP activity was required for
insulin-dependent regulation of glucose transport (103,
306). Knocking down of AS160 (or TBC1D4 in the RabGAP nomenclature; referred to in this review as AS160/
254
TBC1D4) resulted in an increase of GLUT4 on the surface
of resting cells and a decrease in insulin-stimulated exocytosis of GLUT4 and glucose uptake (103). This phenotype
could be reversed by expression of wild-type AS160/
TBC1D4 but not by a protein where the critical arginine of
the GAP domain has been mutated (103). Expression of a
protein where the Akt phosphorylation sites were mutated
to alanine prevented the insulin action, but this could be
compensated by a GAP-domain mutant (306).
AS160/TBC1D4 was shown to be an efficient GAP for Rabs
2A, 8A, 10, and 14, and all these Rabs have been detected in
GLUT4-containing vesicles (198, 225). In adipocytes,
Rab10 seems to be the major target of AS160/TBC1D4 on
GLUT4 vesicles as overexpression of hydrolysis-deficient
Rab10 increased, whereas knocking down endogenous
Rab10 decreased the insulin-stimulated translocation of
GLUT4 (305, 307). Interestingly, in skeletal muscle cells,
Rab8A seems to be the substrate of AS160/TBC1D4 (156,
157).
With the effect of insulin and other agonists of GLUT4
translocation (such as adiponectin, ALCAR [5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside], berberine or
physical exercise) taken together, the picture arises (FIGURE
11) that constant RabGAP activity of AS160/TBC1D4 is
required for retaining GLUT4 vesicles in the cell. Insulin
stimulation results in phosphorylation of AS160/TBC1D4
on the critical T642 and several other amino acids by Akt.
Apparently, phosphorylation by itself is sufficient for a decrease of the RabGAP activity of AS160/TBC1D4 (341).
Promoting the GTP-bound, active form of critical Rab proteins (Rab10 in adipocytes, Rab8A in muscle cells) allows
the correct docking and promotes fusion of GLUT4-containing vesicles with the plasma membrane.
Two recent clinical observations support the above
mechanism. On one hand, it is well known that glucocorticoids induce insulin resistance and impair glucose uptake. In human and murine adipocytes, it has been found
that dexamethasone treatment reduced the insulin-stimulated phosphorylation of AS160/TBC1D4 at T642 and
brought about a proportional impairment of GLUT4
translocation and glucose uptake. No change could be
detected in insulin-induced phosphorylation or activity
of Akt (256).
Finally, in a genetic screen of severely insulin-resistant patients, a truncation mutation of AS160/TBC1D4 has been
found in a young patient characterized by acanthosis nigricans and extreme postprandial hyperinsulinemia (88). The
RabGAP domain of AS160/TBC1D4 has been deleted due
to the truncation. Expressing this mutant protein in adipocytes resulted in increased basal and reduced insulinstimulated GLUT4 levels on the cell surface. The clinical
findings support the physiological importance of the Rab-
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GAP domain (258) that has been reported to affect the GAP
activity in vitro (14). These results suggested that modulation of GAP activity by the presence of exon 23a encoded
sequence is responsible for the observed learning deficits
(79). Decreasing Ras activity by genetic or pharmacological
manipulation in vivo rescues the learning deficits caused by
the deletion of exon 23a, supporting the idea that indeed
hyperactive Ras is responsible for these defects (78). Lack of
neurofibromin in Drosophila is also associated with a learning phenotype (131). The importance of the GAP-related
domain and its activity for long-term memory in flies has
been demonstrated by testing known GAP-impairing NF1
mutations in long-term memory tests (151). Specifically,
learning deficits in mice are due to increases in ERK activation leading to increased levels of synapsin phosphorylation, enhanced GABA release, and as a consequence inhibition of LTP and learning deficits (81). Apart from neurofibromin, the brain specific SynGAP has also been
demonstrated to regulate ERK/MAPK signaling, synaptic
plasticity, and learning (182), consistent with both neurofibromin and SynGAP being components of the NMDAreceptor proteome (155).
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FIGURE 11. Involvement of RabGAP in insulin-stimulated glucose uptake. Glucose uptake is stimulated by
insulin, which activates a signaling cascade starting at the insulin receptor and results in the phosphorylation
of Akt. Activated Akt triggers several cellular responses which include the activation of Rab at GLUT4containing vesicles via the inhibition of the RabGAP AS160, leading to exocytosis of GLUT4 glucose transporters (88, 115, 154). AKT (PKB), murine thymoma viral oncogene; aPKC, atypical protein kinase C; AS160, AKT
substrate of 160 kDa; GLUT4, glucose transporter type 4; Ins, insulin; IR, insulin receptor; IRS1, insulin
receptor substrate 1; mTORC1, mammalian target of rapamycin complex 1; mTORC2, mammalian target of
rapamycin complex 2; PDK, phosphoinositide-dependent kinase; PI3K, phosphatidylinositol-3-kinase; PI345P3,
phosphatidylinositol-3,4,5-trisphosphate; PI45P2, phosphatidylinositol-4,5-bisphosphate; PTEN, phosphatase
and tensin homolog; Rab, ras-related in brain.
GAP activity of AS160/TBC1D4 in regulation of GLUT4
trafficking and cellular glucose uptake.
been genetically associated with obesity (342) indicates its
physiological relevance.
Following the discovery of TBC1D4, a homolog has also
been revealed. The new protein, TBC1D1, is ⬃50% identical to AS160/TBC1D4 and has the same specificity for Rabs
(291); however, it is preferentially expressed in skeletal
muscle (63). Phosphorylation by Akt or by AMP-dependent
protein kinase (AMPK) reduces the RabGAP activity of
TBC1D1 and increases GLUT4 translocation to the plasma
membrane (274, 291). The importance of TBC1D1 seems
to be minor in adipocytes, but it may have a more significant
role in skeletal muscles in transmitting signals on physical
activity (63). The fact that R125W variant of TBC1D1 has
Thus both the results of physiological experiments and the
available clinical observations support the critical role of
RabGAPs in controling glucose uptake into adipocytes and
muscle cells even if the two major metabolic compartments
carry out their basically similar job with partially different
molecular sets.
V. ROLE OF GAPs IN BLOOD CELLS
Formation and physiological function of blood cells require
correct adhesion and migration, processes where the in-
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GAPs IN SIGNAL TERMINATION
volvement of small GTPases of the Rho/Rac family has been
amply documented (160, 241, 359) (and sect. IIB2). In human blood peripheral granulocytes, three different GAPs acting on Rho family GTPases (p50GAP, Bcr, and p190GAP) have
been identified at the protein level (122). In addition, the
presence of ␤2-chimerin has been documented in T lymphocytes (61, 330, 331). Information on the potential function
of these proteins derives from experiments on genetically
modified animals or cell lines.
A. Hematopoiesis
Genetic deletion of p50GAP resulted in mice of remarkably
small body and organ size at birth and high (⬃90%) perinatal lethality, although the ratio of homo- and heterozygous animals followed the Mendelian distribution (373). In
the investigated tissues, the active form of Cdc42 was elevated, whereas the level of active Rac or Rho has not been
altered, suggesting that the major substrate of p50GAP was
Cdc42 (346, 373, 374).
An analysis of the hematopoietic system of the p50GAPdeficient animals revealed serious disturbance of hematopoiesis with remarkable decrease in total bone marrow cell
number as well as in the number of stem and progenitor
cells. Erythropoietic precursors were more seriously affected than granulocytic precursors, although in peripheral
blood, both red cells, neutrophils, and platelets were decreased (374).
Investigating the potential reasons of disturbed hematopoiesis, it was revealed that the cell cycle progression of wildtype and p50GAP-deficient cells did not differ, but the deficient cells showed significantly enhanced apoptosis. Increased apoptosis depended on the elevated activity of JNK
in p50GAP-deficient cells and could be prevented by downregulation of JNK.
A mixture of hematopoietic stem and progenitor cells isolated from p50GAP⫺/⫺ animals showed impairment in adhesion to fibronectin through integrin ␣4␤1 and in migration directed by chemoattractive gradient. In line with defective adhesion and migration, hematopoietic stem and
progenitor cells of p50GAP-deficient animals showed significantly reduced engraftment when transplanted into irradiated wild-type animals (374).
Peripheric granulocytes deficient in p50GAP also showed
decreased chemotactic migration, whereas their random
256
B. Phagocytic Functions
1. Bcr and Abr
These two GAPs have significant similarity: both possess a
COOH-terminal RhoGAP domain and a tandem DH-PH
domain characteristic for Rac/Rho family GEFs (FIGURE 8).
In fact, the isolated DH domain of both Bcr and Abr was
shown to have moderate GEF activity toward Cdc42, Rac,
and Rho (69). The gene of Bcr is subject to crossing over
with the gene of Abl tyrosine kinase forming the Philadelphia chromosome, and the fusion protein is involved in
development of leukemia (91, 144, 370). However, the
GAP activity of the proteins is not related to the development of leukemia.
Bcr was the first Rac/Rho family GAP that has been studied
in a genetically deficient animal model (371). Under neutral
conditions, the mice had no significant phenotype. However, they reacted vigorously upon exposition to LPS: they
developed serious intestinal necrosis, lost weight, and died
within a few days. An increase of reactive oxygen species
(ROS) production and tissue damage due to agressive
phagocytes underlie the clinical symptoms. In contrast to
Bcr, Abr-deficient mice had no overt phenotype (68).
Phagocytes represent a double-edged sword: their activity is
inevitable for elimination of bacteria, but in the case of
pathological activation, they are able to cause serious damage to the host. Hyperactivity of phagocytic cells underlies
the development of sepsis. The remarkable phenotype of
Bcr⫺/⫺ animals initiated the detailed investigation of the
role of the two related GAPs in phagocytic cells. In phagocytes, both migration, engulfment of particles, and granule
exocytosis depend on extensive and precisely organized
changes of the actin cytoskeleton mediated by Rac/Rho
family small GTPases. Furthermore, production of superoxide, the precursor to all other reactive oxygen species, is
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The protein of 50 kDa molecular mass named alternatively
as Cdc42GAP or p50RhoGAP (or RhoGAP1 in FIGURE 8)
was the first Rho/Rac family GAP that had been cloned (24,
196). When tested with prenylated small GTPases, it reacts
under in vitro conditions equally well with Rac, Rho, or
Cdc42 (235). It will be referred to in this review as p50GAP.
motility has been increased (346). In a gradient of chemotactic agent, wild-type cells formed membrane protrusions
at the leading edge and moved towards the source of the
stimulant, whereas p50GAP-deficient granulocytes formed
multiple extensions in various directions and moved in random directions, often away from the chemoattractant
source (346). These findings are consistent with previous
observations on the key role for Cdc42 in orientation of the
cells. In the absence of p50GAP, spatiotemporal control of
Cdc42 activation is apparently disturbed, resulting in randomized orientation attempts of the cell. In contrast to disturbance of orientation, p50GAP-deficient PMN showed
no alteration in adhesion to fibrinogen, and their directed
migration on fibrinogen surface or across the epithelial
layer has even been increased (347). These findings indicate
that organization of cell orientation and motility depends
on different signaling processes.
LIGETI ET AL.
carried out by the NADPH oxidase, and one of the essential
subunits of this enzyme is Rac (depending on the cell type
and species Rac1 or Rac2) (368).
Under in vitro conditions, the isolated GAP domain of Bcr
and Abr reacted with Rac and Cdc42 (69, 145) and in case
of prenylated GTPases, also with Rho (207). However,
when overexpressed in cells, both Bcr and Abr reduced the
amount of active, GTP-bound Rac but did not affect either
RhoGTP or Cdc42GTP (68) indicating that in phagocytes,
both GAPs recognize mainly Rac as a substrate.
These alterations of phagocytic functions contribute to the
development of the serious clinical condition that was observed in these animals in two different models of sepsis.
Administration of Escherichia coli LPS induced acute inflammation of the lungs, characterized by cellular infiltration and fluid leakage. The same symptoms occurred in
both single-knockout animals, but they were more serious
in double knockouts. Whereas in wild-type and singleknockout animals a tendency of recovery was evident after
48 h, the double knockouts showed no sign of recovery and
died within 2 days. Similar enhancement of intestinal damage has been observed after cecum ligation and puncture of
(Bcr ⫻ Abr)⫺/⫺ animals (83).
The detailed investigation of phagocytic functions in singleknockout animals indicated that Bcr and Abr participate in
similar reactions; however, slight differences have been observed both in superoxide production and in enzyme release. Thus the function of the two similar GAPs seems to be
only partially overlapping, and both molecules are required
to keep the reactivity of phagocytic cells under control and
prevent extensive tissue damage in case of bacterial infection.
2. p190A
p190A [also known as glucocorticoid receptor DNA binding factor 1 (Grlf1, FIGURE 8) or p190RhoGAP] is one of
two (p190A and p190B) homologous GAPs acting on Rac/
Rho family GTPases (318), yet regulating different functions.
Expression of p190A has been shown in neutrophilic granulocytes both at the mRNA and at the protein level (122,
251). Translocation to the plasma membrane has been detected in stimulated neutrophils (101), and superoxide production was decreased by p190 under in vitro conditions
(149). In human neutrophils, Src-dependent translocation
and activation of p190A was observed upon ␤2-integrin
stimulation (95). However, in contrast to these findings, a
careful investigation of the phenotype of mice transplanted
with p190A⫺/⫺ bone marrow has not revealed any significant alteration in the in vitro or in vivo properties or activity
of neutrophils or in the development of autoimmune arthritis (251). These data suggest a cell-type or species specific
role of p190A in integrin signaling.
C. T-Cell Activation
Expression and RacGAP activity of ␤2-chimerin has been
shown in Jurkat cells, a T lymphocyte cell line. Stimulation
of the cells with high concentration of PMA induced a redistribution of ␤2-chimerin from cytosolic localization to
the cell membrane, and this redistribution depended on
binding of the chimerin C1 domain to membrane DAG
(330). A detailed investigation revealed an important role of
␤2-chimerin in signal transduction following stimulation of
the T-cell receptor (TCR) (61, 331). Overexpression of ␤2chimerin inhibited both adhesion and interfered with activation of the key regulator NF-AT upon stimulation of
TCR. These effects depended on the ability of ␤2-chimerin
to downregulate RacGTP (61, 330). Upon activation of the
TCR, ␤2-chimerin was shown to be tyrosine-phosphorylated by Lck, a receptor-dependent Src family tyrosine kinase. This phosphorylation interferes with the binding of
the C1 domain of ␤2-chimerin to the membrane phospholipid DAG and reduces the RacGAP activity of ␤2-chimerin.
Lack of phosphorylation of ␤2-chimerin results in enhanced
RacGAP activity with consequent decrease of the level of
active RacGTP and functional alterations such as reduced
activation of NF-AT and decreased IL-2 production upon
TCR stimulation (331). Hyperactivated forms of ␤2-chimerin (either due to blocked tyrosine phosphorylation or release of autoinhibition) interfere with formation of the immunologic synapse between T-cell and antigen-presenting
cells contributing to impaired TCR stimulation.
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Macrophages isolated from double knockout (Abr ⫻
Bcr)⫺/⫺ animals showed a remarkable, elongated morphology and significant increase in directed motility both in vitro
and in vivo. In addition, their phagocytic capacity has been
increased, and prolongation of the prevalence of RacGTP
was observed (68). Also, PMA-stimulated superoxide production and LPS-induced release of the proteolytic enzyme
matrix myeloperoxidase 9 has been elevated (83). Neutrophilic granulocytes of double knockout animals showed
enhanced release of primary granules, but no change was
observable in release of secondary granules or secretory
vesicles (83).
P190A is phosphorylated by Src-family tyrosine kinases
upon stimulation by growth factor receptors or adhesion
proteins (52, 104, 246), and this phosphorylation promotes
the association of p190A with p120RasGAP directing its
relocalization from the cytosol to the cell periphery (47,
153, 237). P190A is an ubiquitous protein (319), and it was
shown to be involved in decreasing Rho activity upon integrin signaling in fibroblasts (17, 27, 147, 246). It also
plays a central role in directing axon outgrowth of neurons
(51, 52), and its association with semaphorins has been
detailed above (see sect. IIIA2).
GAPs IN SIGNAL TERMINATION
Interestingly, TCR stimulation, via Lck, also contributes to
activation of Vav1, a GEF for Rac (6, 137, 391). Thus
phosphorylation via Lck influences the level of active
RacGTP on two parallel pathways: both by activation of
the RacGEF Vav1 and by inhibition of the RacGAP ␤2chimerin. The rapid elevation of local [RacGTP] is apparently essential for full activation of T lymphocytes, and
interference with one of the two parallel Rac-activating
pathways results in detectable deficit of the biological function (331).
VI. ROLE OF GAPs IN HOST-PATHOGEN
INTERACTIONS
A. Pathogenic Yersinia Species
Three species of the Yersinia genus are pathogenic for humans and animals: Y. enterocolitica and Y. pseudotuberculosis cause enterocolitis and mesenteric lymphadenitis,
whereas Y. pestis is the pathogenic agent of plague. All three
pathogenic Yersinia species contain a virulence plasmid
(pYv) that encodes the constituents of a type III secretion
system (TTSS). TTSS are characterized by a needlelike projection of the bacteria that contacts the host cell. The proteins secreted via the TTSS are elements of a translocation
channel or effector proteins injected into the cytoplasm of
the host cell. Pathogenic Yersinia species secrete six different effector proteins, named Yersinia outer protein (Yop) E,
T, O, H, J, and M. All of the Yops have remarkable functions within the host cell, affecting mostly the cytoskeleton.
In addition to YopE, several other Yersinia toxins target the
actin cytoskeleton via small GTPases. YopT is a cysteine
protease that is able to remove the prenyl tail mainly from
RhoA and to a lesser degree also from Rac and Cdc42
(322). As a result, YopT induces the release of RhoA from
the plasma membrane and disruption of stress fibers in infected cells (5, 409). YopO was shown to have serine/threonine kinase activity, and on this basis, it has been named
also Yersinia protein kinase A (YpkA). Recently, the crystal
structure of the COOH-terminal part of YopO revealed a
domain homologous to RhoGDIs, and this region of YopO
was shown to bind to and inhibit exchange on Rac1 and
RhoA (278). Thus the concerted action of YopE, YopT, and
YopO is able to inactivate the Rho-family GTPases, remove
them from the plasma membrane, and sequester them in a
protein complex in the cytosol.
Concerted action of the Yops results in inhibition of the
respiratory burst; impairment of phagocytosis; killing of
pathogenic Yersinia species by macrophages and neutrophils (140, 302); inhibition of secretion of pro-inflammatory mediators such as IL-8, tumor necrosis factor (TNF)-␣,
or interferon (IFN)-␥ (53, 367); and induction of apoptosis
of macrophages and dendritic cells (301, 405). As a result,
enteropathogenic Yersinia species colonize typically the
Peyer’s patches and mesenteric lymph nodes where they
replicate in an extracellular form. Highly virulent strains
are also able to disseminate systemically and reach the
spleen or liver. Isolated deficiency in YopE decreased virulence and systemic spreading of the bacteria more vigorously than deficiency in YopT or YopO (366). Thus the
GAP activity of YopE towards Rac/Rho family GTPases
significantly contributes to the overall pathogenicity of the
Yersinia species.
B. Pseudomonas aeruginosa
YopE has in vitro GAP activity toward RhoA, Rac1, and
Cdc42 but not other small GTPases (35, 369). In vivo GAP
activity towards RhoA, Rac1, Cdc42, and RhoG has been
confirmed in HeLa and human umbilical vein endothelial
cells (HUVEC) (9, 295). GAP activity of YopE depends on
the critical arginine in position 144 (35, 369).
Pseudomonas aeruginosa is an opportunistic pathogen bacterium that causes severe infections such as pneumonia typically in patients with cystic fibrosis, burn wounds, or immunodeficiency (37). When investigated in cell culture, P.
aeruginosa was shown to exert a cytotoxic effect characterized by rounding and detachment of epithelial cells, disruption of the actin cytoskeleton, decrease of viability, and
impairment of phagocytosis (114).
Injection of YopE into monolayers of cultured HeLa or
HUVEC results in disruption of the actin filaments, round-
Toxicity of P. aeruginosa depends on four effector proteins
(ExoU, ExoS, ExoT, and ExoY) secreted via a TTSS similar
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The infectious capacity of a potentially pathogenic microorganism depends on its ability to enter and invade epithelial layers and to succumb or evade various protective mechanisms of the innate and adaptive immune system. Small
GTPases of the Rho/Rac family play key roles in maintaining the integrity of the epithelial layer, in phagocytosis, and
in production of ROS or cytokines, all being functions that
determine the fate of an invading microorganism. It is thus
no surprise that microorganisms have evolved a broad variety of effective methods that target small GTPases, and
several of these molecules have GEF or GAP activity for
members of the Rho/Rac family. Remarkably, the GAP or
GEF modules encoded by these proteins employ similar
regulatory mechanisms like their eukaryotic counterparts
yet implemented in structural entities unrelated to those of
the eukaryotic Rho family regulators (116, 238, 338, 393).
ing up and eventually detachment of the cells (15, 35, 369).
At the same time, uptake of the bacteria is seriously impaired (35). All these effects were shown to depend on the
GAP activity of YopE as they are absent if the catalytically
inactive R144 mutant toxin is injected and could be prevented by expression of constitutively activated Rac1 and
RhoA (35, 369).
LIGETI ET AL.
to that of Yersinia species. The biochemical activity of all
four exotoxins has been determined: ExoU has phospholipase A2 activity, ExoY functions as an adenylate cyclase,
whereas ExoS and ExoT are bifunctional toxins. Both ExoS
and ExoT have ADP-ribose transferase (ADPRT) activity
localized to the COOH-terminal part of the molecules,
whereas the NH2-terminal part of both ExoS and ExoT was
shown to have in vitro GAP activity for Rho, Rac, and
Cdc42 GTPases (124, 185). In vivo GAP activity of ExoS
has been confirmed both in epithelial type cells (186, 404)
and macrophages (292) and for ExoT in HeLa cells (174).
Plasma membrane localization is essential for the in vivo
GAP activity of ExoS (404), and cooperation was shown
between ExoS GAP activity and intracellular RhoGDI in
effects upon the actin cytoskeleton (345).
C. Salmonella Species
The effect of Salmonella species on intestinal epithelial cells
provides an interesting example of sequential activation
and deactivation of Rho-family GTPases.
Infection with different Salmonella species occurs by oral
ingestion and depends on uptake of the microorganisms
into epithelial cells of the small intestine. Entry of Salmonella into nonphagocytic cells requires profound rearrangement of the cytoskeleton. Membrane ruffles and lamellipodia are formed at the site of pathogen-host interaction, and
macropinocytosis is increased leading to gradual engulfment of the microorganism and sequestration into membrane-bound vesicles [Salmonella containing vesicles (SCV)]
(272). Parallelly, MAP kinases (p38 and JNK) are activated,
and local inflammation is initiated resulting in increased
secretion and diarrhea. Internalization of Salmonella into
epithelial cells absolutely requires injection of specific effector proteins by the TTSS encoded by the Salmonella pathogenicity island 1 (SPI-1). Three of these bacterially synthetized proteins turned out to affect small GTPases in the
epithelial cells.
In epithelial cells in the first phase following infection, activation of the small GTPases Rac and Cdc42 occurs, initi-
However, these cytoskeletal changes are reversible, and
they are typically reversed in 2–3 h (130, 272). The reversal
phase depends on another bacterial protein injected via the
TTSS into the host cell. The critical protein SptP has a
double function: the NH2-terminal part is an active GAP for
Rac and Cdc42, whereas the COOH-terminal part has tyrosine phosphatase activity (116, 117). The GAP activity of
SptP allows reversibility of the cytoskeletal rearrangement
of the host cell, as recovery of the cellular morphology was
equally low when cells were infected with Salmonella
strains lacking SptP or expressing a GAP-deficient mutant
SptP (116). In addition to the cytoskeletal changes, SptP
also reversed the activation of JNK. In this effect, both the
GAP and the tyrosine kinase activity of the protein has a
role (116, 242).
SptP possesses a critical arginine and accelerates the GTP
hydrolysis on the target small GTPases by a mechanism
similar to mammalian GAPs (116). Despite the lack of significant sequence or structural homology between SopE and
eukaryotic RhoGEFs, SopE appears to act on Cdc42 also in
a similar way to its eukaryotic counterparts (55).
Although SopE and SptP, the GEF and GAP for Rac and
Cdc42, respectively, are delivered at the same time in equivalent amount to the host cell, there is a time shift in the
activation and inactivation of the small GTPases. The puzzle has been solved by demonstrating a significantly different half-life for the two regulatory proteins in the host cell
(189). Both SopE and SptP are degraded by the proteasomal
pathway, but with largely different speed: SopE cannot be
detected 30 min after injection, whereas SptP remained detectable for more than 3 h (189). Intracellular stability of
the proteins is controlled by the NH2-terminal secretion
domain and the half-life of chimeric proteins (NH2-terminal SopE ⫹ COOH-terminal SptP or NH2-terminal SptP ⫹
COOH-terminal SopE) correlated with the identity of the
NH2-terminal part. Inhibition of proteasomal degradation
of SopE prevented cellular recovery after Salmonella infection (189).
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Both enzyme activities contribute to the toxic effects of
ExoS and ExoT of P. aeruginosa. ADP-ribosylation of small
GTPases and other proteins playing a role in organization
of the actin cytoskeleton seems to be responsible for impairment of the barrier function of epithelial surfaces. Antiphagocytic effects allowing the bacteria to avoid intracellular killing and degradation seem to be more attributable
to the GAP activity of the exotoxins on Rho family GTPases. In an animal model of acute pneumonia, the persistence of P. aeruginosa in the lungs was impaired both by
deficiency of the GAP and ADPRT activity of ExoS (323),
supporting the bifunctional biological effect of the exotoxin.
ating actin polymerization and branching via the Arp2/3
complex, leading to formation of membrane ruffles and
lamellipodia and activation of JNK. These cytoskeletal
changes are dependent on two proteins secreted via the
TTSS of the bacteria: SopE and SopB. SopE turned out to be
a GEF for Rac1, Rac2, Cdc42, and a few other members of
the Rho subfamily, but not for Ras or Ran proteins (139). A
homolog of SopE (SopE2) has also been identified, and it
was shown that one of the two SopE proteins was expressed
by all pathogenic Salmonella strains (339). SopB is an inositol phosphate polyphosphatase that contributes to activation of Cdc42 (272). Mutant strains lacking both SopE
proteins and SopB are completely unable to initiate cytoskeletal rearrangement and to invade the host cell (119).
GAPs IN SIGNAL TERMINATION
Transient activation of the host cell cytoskeleton is required
for the entry of Salmonella bacteria in the epithelial cells.
The following rapid recovery of the cell morphology ensures survival of the infected cell and, in this way, long-term
intracellular residence for the pathogen. The sequential activation and deactivation of the small GTPases responsible
for the cytoskeletal changes are achieved in case of Salmonella strains by simultaneous injection of two proteins with
opposing effects but largely different half-life.
VII. ROLE OF GAPs IN TUMORIGENESIS
Hyperactivation of small GNBPs can occur by mutation of
amino acids essential for GTP hydrolysis, rendering the protein constitutively active. As one of the most important
oncogenes, RAS genes are found mutated in a variety of
malignancies (214), with the highest incidence in pancreatic
(12), lung (293), and colorectal (43) tumors. Oncogenic
activation in most cases results in impaired and GAP-insensitive GTPase activity (4, 22, 357). Interestingly, somatic
gain-of-function mutations were only very rarely identified
in members of the Rac/Rho family (364). Alternatively,
overexpression of GEFs can result in increased activity of
small GTPases. Typically overexpression of different GEFs
for the Rac/Rho family GTPases has been associated with
development of cancer (54, 90, 105, 110, 227, 282, 406).
In the following we describe a few examples where lacking
GAP activity has been revealed as the cause of tumorigenesis.
A. Neurofibromatosis Type 1
(Neurofibromin)
Deregulation of cellular (normal) Ras by the inactivation of
relevant RasGAP proteins may lead to phenotypes characterized by upregulation of Ras, i.e., elevated levels of GTPbound Ras in cells with a number of implications. The most
prominent and probably best explored example is reflected
in a long known disorder, termed neurofibromatosis type 1
(NF1), also called von Recklinghausen neurofibromatosis.
Patients have an increased risk to develop typical neurofibromas (283), essentially benign tumors of the peripheral
nerve sheath that, however, can turn into malignancy in a
significant number of cases (18). In addition, the disease is
associated with numerous symptoms including pigment
260
NF1 encodes the cytoplasmic RasGAP neurofibromin (320
kDa) (92, 133, 134). Its RasGAP activity is located in a
central portion reported to comprise between 300 and 400
residues (21, 217, 394) that has been narrowed down to
230 residues sufficient for its enzymatic function (8). It is
followed by a bipartite phospholipid binding module composed of a glycerophospholipid binding Sec14-homology
and a pleckstrin homology (PH)-like domain (16, 84, 382)
(FIGURE 8), the funcion of which is yet unclear. Alternative
splice variants have been reported, with one of them leading
to the so-called type II transcript expressed in Schwann cells
(258) carrying a 21-residue insertion within the GAP domain biochemically associated with reduced GAP activity
(14). Of the reported NF1 associated alterations, the majority results in premature stop codons most likely leading
to truncated transcripts removed by the surveillance machinery. Ten percent of the alterations are missense mutations, single/double residue deletions, or peptide insertions
(324, 361). Provided that these mutations do not affect
protein stability or folding, they have a high potential to act
as functional missense mutants in the cell and are thus excellent tools to probe the molecular functions at high sequence resolution.
The role of neurofibromin’s RasGAP activity for tumorigenesis has been impressively demonstrated by a number of
studies that report increased levels of activated (i.e., GTP
bound) Ras and consequently a hyperactivated MAPK
pathway (FIGURE 3) in NF1-deficient tumor tissues derived
from Schwannomas and malignant peripheral nerve sheath
tumors (29, 40, 87, 93, 109, 129, 178, 199, 325, 358). In
addition, loss of NF1 has been demonstrated to result in
abnormal growth of hematopoietic cells along with activation of the Ras signaling pathway (40), as has been found in
types of myeloid leukemia (321). It is believed that controlling Ras activity defines neurofibromin’s primary role as a
tumor suppressor, although loss of heterozygosity has not
been established in all examples. Indeed, the RasGAP domain harbors a cluster of missense mutations that have been
studied in biochemical detail (7, 181). Several of these affect
the catalytic machinery and thus GAP activity (132, 181,
203, 317, 360). Most impressively, the mutation of the
catalytic arginine to proline, as found in an NF1-patient
family and has been associated with heavy tumor formation, virtually abolishes GAP activity (181). The hyperproliferative phenotype of NF1-depleted/deficient Schwann
cells can be rescued by the retroviral expression of the GAP
domain of neurofibromin in a RasGAP-dependent mechanism but not of that of the similar one of p120GAP (150,
354), suggesting that at least in those cells the catalytic
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As detailed in section II, small GNBPs regulate a diverse
array of cellular functions such as proliferation, survival or
apoptosis (Ras), adherence, migration, gene expression
(Rac/Rho), or vesicular traffic that determine the exposure
or internalization of plasma membrane constituents (Rab,
Arf). Evidently, hyperactivation of various small GTPases
may increase proliferation and metastatic development of
different cells.
anomalies of the skin (cafe au lait spots) or iris (Lish nodules), bone deformations, vascular and cardiac abnormalities, and learning disabilities (18, 283). Genetic alterations
in the tumor suppressor gene NF1 are responsible for the
pathogenesis of the disease (70).
LIGETI ET AL.
domain is equipped with the requirements to effectively
interact with activated Ras.
The earlier notion that the NF1 gene carries sporadic mutations in several types of tumors (315) has been reinforced
by recent cancer genome studies indicating that NF1 is mutated in a significant number of rather aggressive malignancies including glioblastoma (252, 270), lung adenocarcinoma (97, 379), ovarian cancers (304), and soft tissue sarcoma (26), suggesting that NF1 may act as a deregulated
tumor suppressor gene in these types of cancers.
B. Colorectal Cancer (RASAL)
D. Liver Cancer (DLC1)
“Deleted in liver cancer” (DLC1/2; FIGURE 8) (also known
as ARHGAP7 or STARD12) has been identified as a
RhoGAP (67, 398) that inhibits the proliferation in hepatocellular carcinoma cells in a cellular context that requires
RhoGAP activity (389) but employs also RhoGAP-independent mechanisms (142). In the case of DLC1, tumor suppression did not occur if a catalytically inactive point mutation was introduced (389). An epigenetic mechanism that
involves silencing by hypermethylation of CpG islands has
been proposed contributing to gastric cancerogenesis (179,
388). Cellular studies in cell lines derived from a variety of
tumors suggest that DLC1/2 may play a tumor-suppresive
role beyond liver cancer (205).
E. Breast Cancer (␤2-Chimerin)
C. Tuberous Sclerosis (Tuberin)
Stimulation of epidermal growth factor receptors (EGFRs)
results in activation of both Ras and Rac (195), the latter
being mediated by the exchange factor Vav (236, 349)
(Figs. 3 and 4). Parallelly, PLC-␥ is recruited and activated
by the phosphorylated intracellular tail of EGFR (259,
299), resulting in production of cytosolic IP3 and membrane-attached DAG. It has been shown recently that EGF
induces the redistribution of ␤2-chimerin, a RacGAP with a
DAG-binding C1 domain (FIGURE 8), from the cytosol to
the plasma membrane where it interacts with activated Rac
(372). Downregulation of ␤2-chimerin resulted in significant prolongation of the prevalence of RacGTP, indicating
an essential role of ␤2-chimerin in termination of the EGFRinduced Rac signal (372).
Tuberous sclerosis is an autosomal dominant tumor syndrome characterized by deregulation of cell proliferation
that results in the formation of hamartoma-like neoplasias/
tumors in many organs (125). TSC2 is one of the two genetic determinants of the disease (277), encoding the Rhebspecific RapGAP tuberin (204, 215). Tuberin is a 200-kDa
protein with a COOH-terminal 160-residue domain homologous to RapGAP1 (FIGURE 8). Several missense mutations have been identified in the RapGAP domain of tuberin
that appears to be a target for such mutations in tuberous
sclerosis (213, 219). Mapping those mutations onto the
structure of the canonical RapGAP reveals the highly conserved catalytic asparagine (Asn290, Asn1643) (89) mu-
The biological significance of the downregulation of Rac
signaling is enlighted by the finding that the mRNA level of
␤2-chimerin is significantly decreased in several breast cancer cell lines as well as in 70% of the investigated breast
cancer tissues (396). Hyperactivation of Rac resulting in
enhanced proliferation has been reported earlier in human
breast cancer models (229). Expression of the full-length
protein in ␤2-chimerin-deficient MCF-7 breast cancer cells
resulted in decrease of proliferation and cell cycle arrest at
the G1/S phase. Parallel dose-dependent decrease was observed in the level of RacGTP and cyclin D1. All these
effects depended on the GAP activity of ␤2-chimerin (396).
In addition to breast cancer cells, downregulation of ␤2-
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261
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Apart from gain-of-function mutation in Ras or loss-offunction mutations in neurofibromin, epigenetic mechanisms have been demonstrated to be involved in cancer
development of certain tissues. Colorectal cancer (CRC)
progression is frequently associated with aberrant Ras activation, although not all CRC cells carry activating Ras
mutations. Based on these observations, researchers investigated CRC cells for expression of 12 RasGAPs and found
that the Ca2⫹-regulated RasGAP like protein (RASAL) that
decodes the frequency of Ca2⫹ oscillations is silenced in
CRC cells from multiple tumors, most likely by CpG methylation (164, 262). Similarly, another RasGAP, the human
DOC-2/DAB2 interactive protein (hDAB2IP), has been
demonstrated to be a tumor suppressor gene inactivated
also by aberrant methylation of CpG islands in a number of
malignancies including prostate (65, 226) and gastrointestinal (98) cancers. The respective phenotype can be rescued
by ectopic expression of RASAL in these cells. In RASALdepleted cells, Ras was indeed upregulated with respect to
normal cells correlating RASAL expression to the regulation of the MAPK pathway. The results suggest that RASAL
and DAB2IP act as tumor suppressor-like proteins in the
respective tissue cells and that epigenetic silencing of a GAP
represents a previously unknown mechanism of Ras activation in certain types of cancers.
tated to lysine (213) or isoleucine (19). The corresponding
RapGAP mutant was entirely inactive (or insoluble as in the
case of Asn1643Ile), implying that loss of RapGap activity
may be constitutional for TSC pathogenesis, similarly like
mutation of the catalytic arginine in neurofibromin has
been found mutated in an NF1-family that was affected
with heavy tumor development, resulting in an 8,000-fold
reduced GAP activity (181).
GAPs IN SIGNAL TERMINATION
chimerin has also been observed in high-grade gliomas
(399), and a progressive loss of ␤2-chimerin expression was
detected in microarrays of benign duodenal adenomas and
adenocarcinomas (396).
Taken together, ␤2-chimerin seems to play an essential and
specific role in termination of EGFR-induced Rac signaling,
and partial loss of this GAP is sufficient to enhance cell
proliferation.
ACKNOWLEDGMENTS
E. Ligeti is indebted to Professors András Spät, Anna
Faragó, and Péter Enyedi for stimulating discussions and
critical reading of the manuscript and to Roland CsépányiKömi for devoted editorial help.
Address for reprint requests and other correspondence:
E. Ligeti, Dept. of Physiology, Semmelweis University,
Tűzoltó u. 37– 47, H-1094 Budapest, Hungary (e-mail:
[email protected]).
VIII. CONCLUDING REMARKS
GRANTS
The large number of GAPs with identical or similar substrate preference has raised the question about specific or
overlapping functions. Although it is too early to give a final
answer, two lines of observations allow some speculations.
On one hand, the diverse multidomain structure of GAPs
suggests that these proteins participate in well-defined molecular complexes confined to specific sites within the cell.
Replacement of one multidomain protein by another complex protein with a similar GAP domain can probably be
regarded rather as an exception than the general pattern.
On the other hand, the very specific symptoms and unique
phenotype alterations arising in case of deficient GAP activity of one particular protein in spite of undisturbed expression of several other GAPs of the same or similar substrate
specificity, also suggest a nonredundant role for these proteins.
The examples of alteration of physiological functions detailed in this review support the view that GAPs represent
key elements in the precise spatiotemporal regulation of a
wide variety of cellular functions.
262
This work was supported by grants from the Hungarian
National Research Fund (OTKA K81277 and K75084) and
TÁMOP (grants 4. 2. 1/B-09/1/KMR-2010-0001 and 4. 2.
2/B10/1-2010-0013). S. Welti has been supported by the
Peter and Traudl Engelhorn Stiftung (Germany) and by
grants (to K. Scheffzek) from the Baden-Württemberg Stiftung and the Federal Ministry of Education and Research
(Germany).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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