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TheJournal of
The American Society of Hematology
BLOOD
SEPTEMBER 1,1995
VOL 86, NO 5
REVIEW ARTICLE
Chemoattractant Signaling and Leukocyte Activation
By Gary M. Bokoch
N
EUTROPHILS AND RELATED phagocytic leukocytes perform the important function of clearing the
body of hazardous invading organisms and inflammatory
debris. These same cells can also produce and/or exacerbate
inflammatory disease states, both acute and chronic. Both
activities can occur as a consequence of the potent systems
evolved in these cells for the purpose of microbial killing; in
the latter case, these activities may occur in an inappropriate
situation or to an excessive degree, resulting in tissue destruction and the inflammatory disease process.
Neutrophils are attracted to inflammatory sites and/or sites
of infection through the production at these sites of chemoattractant mediators. Many of these chemoattractants (ie, Nformylated peptides [ W ] , the fifth component of complement C5a, leukotriene BA.interleukin-8 [IL-81, Rantes, etc)
bind to heptahelical, G-protein-coupled cell surface receptors on the leukocyte. As a result of chemoattractant receptor
activation, neutrophils are stimulated to move, adhere and
de-adhere, rearrange their cytoskeleton, and, ultimately, to
phagocytize infectious microorganisms, secrete granule contents containing degradative enzymes and antimicrobial
agents, and activate the NADPH oxidase to generate toxic
metabolites of oxygen. The importance of these activities is
evident from the existence of disease states in which these
activities are deficient, resulting in recurrent severe infections of the afflicted individuals. Such disorders include various forms of chronic granulomatous disease (NADPH oxidase), Chediak-Higashi syndrome (secretion), neutrophil
adhesion deficiencies, and neutrophil motility disorders. The
signaling mechanisms and responses used by chemoattractant receptors are thus of paramount importance in determining how these cells will respond to any given biologic situation. In this review, I will describe how views of
chemoattractant signaling in neutrophils and other phagocytic leukocytes have changed dramatically from only a few
years ago. This includes a renewed appreciation for the complexities of chemoattractant signaling pathways, as well as
recognition of the roles of the guanosine triphosphate (GTP)binding proteins of the Ras superfamily in controlling many
of the basic phagocyte activities that allow them to perform
their roles as critical components of the immune response.
THECLASSICAL VIEW OF CHEMOAlTRACTANT
SIGNALING
The now"classical"view
of chemoattractant receptor
signaling (Fig 1) stemmed from the recognition of the imporBlood, Vol 86, No 5 (September l), 1995: pp 1649-1660
tance of protein kinase C activity in neutrophil activation,
as well as the finding that the ability of chemoattractants to
stimulate neutrophil function was blocked by pertussis toxin
and that chemoattractant receptors were coupled to heterotrimeric GTP-binding proteins (G proteins). The reader is
referred to several reviews of this early ~ 0 r k . l . ~
Avarietyof chemoattractant receptors have nowbeen
cloned, and these exhibit the seven transmembrane-spanning
structure typical of G-protein-coupled
The binding of a chemoattractant to its receptor results in the activation of the associated G protein. The majority of neutrophil
responses induced by chemoattractants can be inhibited by
pertussis toxin, and, consistent with this, Gi2 and Gig are the
primary transduction partners associated with these receptors. It has been observed that the C5a receptor can couple
to Gal6, a myeloid-specific and pertussis-toxin-insensitive G
protein, when both are cotransfected into Cos cells, although
the physiologic significance of this is questionable.' Upon
activation by the ligand-bound receptor, Gi dissociates into
the GTP-bound G a subunit and the
subunit complex."
Although the Gia subunit was originally thought to interact
with the effector enzyme phospholipase C, surprisingly, it
is actually the GP? complex that regulates phospholipase
CO isoforms in le~kocytes.""~Phospholipase CB activation
results in the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP,) to form inositol triphosphate (IP,) and diacylglycerol (DAG). Both have important roles as intracellular
second messengers, with I P 3 acting to mobilize Ca2+ from
intracellular stores and DAG acting in conjunction with Caz+
to activate various isoforms of protein kinase C (PKC). Activation of PKC, as well as various Ca2+-sensitive protein
kinases, catalyzes protein phosphorylation, and this was believed to account for activation of the various neutrophil
functions.'4"7 The ability of chemoattractants to stimulate
phospholipase A2 and phospholipase D was also known, but
whether these were downstream events resulting from PKC
Fromthe Departments of Immunology and Cell Biology, The
Scripps Research Institute, La Jolla, CA.
Submitted January 20, 1995; accepted May 12, 1995.
Supported by National Institutes of Health Grants No. GM39434,
GM44428, and HL48008.
Address reprint requests to Gary M. Bokoch, PhD, Departments
of Immunology and Cell Biology-IMMl4, The Scripps Research Institute, I0666 N Torrey Pines Rd, La Jolla, CA 92037.
0 1995 by The American Sociery of Hematology.
oooS-4971/95/8605-0145$3.00/0
1649
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GARY M. BOKOCH
1650
IP,+ DAG f- PA
Cell Activation
activation and Ca" mobilization or events regulated by distinct pathways was ~ncertain.~.""~
AN UPDATEDVIEW OF CHEMOATTRACTANT
SIGNALING
Although this classical view of neutrophil signaling provided substantial insight into neutrophil responses to chemoattractants. there were clear indications that this might not
explain all aspects of the activation process. A number of
studies provided evidence for PKC- and @'-independent
mechanisms of leukocyte
The existence of
additional signaling mechanisms began to be discerned, and
this has continued to expand into the recognition that chemoattractant signaling is more complex than originally envisioned, making use of alternate signaling pathways involving
kinases and phosphatases, adapter molecules. and small
GTP-binding proteins.
T\.rosine Phosphonlrtion
Many studies have now shown that neutrophil responses to
chemoattractants can be blocked with inhibitors of tyrosine
phosphorylation, indicating that tyrosine phosphorylation
plays an important role in chemoattractant signaling.'""
NFP and other chemotactic agents increase the tyrosine
phosphorylation of a number of proteins in human neutrophils. This appears to be caused by both the activation of
tyrosine kinases as well as by inhibition of tyrosine phosphatases.'5 The phosphatases and kinases involved have not been
well characterized. A number of nonreceptor tyrosine ki-
Fig 1. Classical chemoattractant receptor signaling. Schematic representation of effector pathways
involved in chemoattractant receptor signaling
based on earlier "classical" studies. The receptor is
indicated as the 7-transmembrane-spanning structure at the cell surface within the lipid bilayer. PLC,
phospholipase C; PLD, phospholipase D; PLA2, phospholipase A,; PKC, protein kinase C; PTX, pertussis
toxin; AA, arachidonic acid; DAG, diacylglycerol;IP3,
inositol trisphosphate; PA, phosphatidic acid.
nases of the Src family have recently been shown to participate in myeloid cell signaling responses to growth factors
and to IgG binding to Fc receptors. These include Lyn, Yes.
Hck, Fgr. c-Src.'"-3" as well as the non-Src kinase. Syk.3"''
The levels of many of these enzymes are regulated during
the course of myeloid cell differentiation."^'".'"'" In light of
the importance of these kinases in leukocyte activation by
growth factors and Fc receptors, and the demonstrated existence of multiple signaling pathways that involve such kinases, it is perhaps not surprising that chemoattractant receptors also appear to be able to use these mechanisms for
regulating cell function (Fig 2). The NFP receptor has recently been shown to activate the Lyn tyrosine kinase, stimulating autophosphorylation and activity toward exogenous
target^.'^.'^ One of these phosphorylation targets is the Shc
adapter protein. Shc is a member of a growing family of
adapter molecules made up largely of SH2 and SH3 proteinbinding motifs that serve to link together other proteins involved in cellular signaling.3"Associated with the Lyn-Shc
complex formed during chemoattractant stimulation is PI 3kinase, an important signaling enzyme (see below) whose
activity may be regulated by this interaction.3x.'"Shc is also
known to link receptors and Src-related kinases to a second
adapter molecule, Grb2. This is of interest because a fraction
of Grb2 in cells is normally constitutively bound to the mammalian Ras guanine nucleotide exchange regulator termed
mSOS (the mammalian homologue of the yeast Son of Sevenless
Thus, the interaction of chemoattractant
receptors with Shc can potentially lead to activation of the
small GTPase, Ras.
7Receptor
bE:ther
Fig 2. Activation of Src-related kinases by chemoattractant receptors. The interaction of chemoattractant receptors with various Src-related kinases,
as well as additional kinases and phosphatases, is
shown schematically. A defined linkage to the Shc
adapter protein and its possible downstream targets
is indicated, as described in the text.
\ Lvn
kinases, phosphatases
Shc
P13K
p145
Grb2
SOS
? Ras
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PHAGOCYTE SIGNALING MECHANISMS
Fig 3. Chemoattractant receptor stimulation of
Ras signalingpathways.The interaction of chemoattractant receptors with the Ras/Map kinase cascade
is indicated schematically. The mechanism of coupling to the Ras pathway is not yet well defined;
details are described in the text. GAP, GTF'ase activating protein for Ras; GEF, guanine nucleotide exchange factor;SOS,mammalian homolog of the Drosophih Son of Sevenless Ras exchange factor; Vav,
a proto-oncogone reported to have GEF activity for
Ras; Shcand Grb2,mammalian adapter proteins;143-3 refers to the 14-3-3 protein class.
The Ras/MAPK Cascade
Ras is indeed activated acutely in chemoattractant-stimulated human neutrophils, with the level of GTP-bound active
Ras rapidly increased in stimulated cells.43." Activation of
Ras is a component of normal cell growth in response to
growth factors, as well as of malignant growth induced by
oncogenes. Ras activation in these instances leads to stimulation of a cascade of protein kinases that ultimately phosphorylate and activate mitogen-activated protein kinase (MAPK;
also known as ERK, extracellular signal-regulated kinase).45
Proteins of -42 to 44 kD whose level of tyrosine phosphorylation increased in stimulated neutrophils were shown by
immunologic and biochemical methods to be the 42- and
44-kD forms of MAPK.43-47
Ras that has been activated either by tyrosine kinase receptors and/or G-protein-coupled receptors can initiate the
MAPK cascade by binding to the serine/threonine kinase
Raf. Recent work has determined that active Ras serves to
translocate Raf from the cytoplasm to the plasma membrane,48.49 whereit becomes activated in a Ras-independent
manner, possibly through interactions with the 14-3-3 family
of proteins.m*'' A second family of kinases, termed MEKKs
(MAPKERK kinase kinase), can also be regulated through
aprocessinvolvingRasandheterotrimeric
G
BothRafandMEKKphosphorylateandactivateMEK
(MAPKERK kinase), which is a dual-function kinase that in
turn catalyzes both the threonine and tyrosine phosphorylation
of MAPKERK itself, activating the enzyme and leading
to
the regulation of a variety of downstream targets. The basic
components of this kinasecascadehavebeenshownto
be
present and acutely activated during chemoattractant stimulation (Fig 3). Raf-l and B-Raf were both activated by the C5a
receptor, but with distinct time courses." Activation of both
enzymes led to phosphorylation
of MEK-1.Neutrophils express
both MEK-1 and MEK-2,but onlyMEK-l has been established
to undergo activation by ~hemoattractants."~~'~'~
Activation of
MEK-I was rapid and transient, occurring within
30 seconds
and peaking by 2 to 3 minutes. The time course of RafMEK
activation
paralleled
increases
phosphorylation
in
of
Activation of all of these responses is blocked in
neutrophils by pertussistoxin,indicatingthey
are mediated
through Gi. Both PKC-dependent and -independent activation
1651
l?/
Other
[Shc, GrbP]
3-
MAPK
' \hCytoskeleton
Transcription H
Factors Other
cPLA2
Sernhr Kinases
mechanisms appear to be operative,"3~"~'5although the exact
mechanism through which Ras becomes activated remains to
be elucidated. h4EKK hasbeenshownto
be stimulatedby
tumor necrosis factor a in mouse rnacrophage~?~
although its
activationinresponsetochemoattractantshasnotyetbeen
reported. The indication is that chemoattractant receptors can
acutely regulate activity of the Ras/MAPK pathway, and that
this regulation is likely to play an important role in the early
signaling events leading to cell activation.
Other Serineffhreonine Kinases
Neutrophil stimulation by N-formyl peptides induces the
rapid and transient activation of a group of ser/thr kinases
of approximate molecular masses 40, 49, 63, and 69 kD?'*O
These kinases exhibit the ability to be renatured after polyacylamide gel electrophoresis, and retain their activation state
under these circumstances. Activation is inhibited by pertussis toxin, but is not induced by phorbol myristate acetate
(PMA) or blocked by staurosporine. Interestingly, activation
of these kinases is also blocked
by
wortmannin and
Ly294002, inhibitors of PI 3-kinase, suggesting that the activities of the renaturable kinases may be dependent on the
lipid messengers generated by PI 3-kina~e.~'
The renaturable
kinases remain incompletely characterized, with their structure and regulatory properties still unknown. The identification of neutrophil p21-activated kinases (Paks) as members
of this group of renaturable kinases6Ia suggests thatlowmolecular-weight GTP-binding proteins (LMWG) are involved in the regulation of these signaling enzymes. The
close correlation between activation of the renaturable kinases and acute leukocyte stimulation by chemoattractants
makes it likely that they are participants in regulating early
events in pathways leading to activation of the respiratory
burst, cytoskeletal assembly and motility, and possibly vesicle secretion.
Phosphatidylinositol 3-Kinase (PI3K)
The enzyme PI3K catalyzes the addition of a phosphate
group to the D3-position of phosphatidylinositol lipids, ie,
phosphorylation of ph~sphatidylinositol~~'
bisphosphate
(PIP,) generates phosphatidylin~sitol~~~
trisphosphate (PIP3).
The lipid products of PI3K have been implicated in signaling
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1652
GARY M. BOKOCH
CtX
Receptor
Gia
Novel
Lyn/Shc
Ras
(p85/pl10)
PIP3
1
[? Rac Activation]
NADPH
Other
Cytoskeleton
Targets
Oxidase
Fig 4. Chemoattractant receptor activationof phosphatidyl- inositol 3-kinase(s). Pathways leading
to theformation of the 3-phosphorylated inositol lipids in human neutrophils,and their possible link to
Rac and NADPH oxidase activation (see text for details).
appears to result from these binding interactions. The mechanism by which P13K is controlled by G-protein-coupled
chemoattractant receptors does not appear to be a result of
direct recruitment of PI3K or tyrosine pho~phorylation.~~.~~
Regulation may involve the Src-related tyrosine kinase Lyn,
which is activated by the fMLP receptor and which binds
P13K.38 It has also been recently demonstrated that PI3K
activity can be regulated by Ras, with GTP-bound Ras binding directly to the 110-kD catalytic sub~nit.’~.’~
A role for
Ras in leukocyte PDK activation may account for the stimulation of Ras-GTP observed upon leukocyte activation (see
above). Interactions of PI3K with other small GTP-binding
proteins have also been d e ~ c r i b e d . ~Very
& ~ ~recently, myeloid cells and platelets have been shown to contain a novel
form ofP13K that does not have the classical p85/p110
s t r u c t ~ r eThe
. ~ ~activity
~ ~ ~ of this enzyme was stimulated by
the B y subunits of heterotrimeric G proteins, suggesting
that the G-protein-coupled chemoattractant receptors might
directly regulate the activity of this PI3K. The relative contributions of the multiple forms of P13K to leukocyte activation
remain to be defined.
TARGETS OF CHEMOATTRACTANTSIGNALING
PATHWAYS
It is clear from the preceding discussion that the signaling
responses generated on leukocyte activation by chemoattractants are complex (summarized in Fig 5). As a result of
these signaling events, the activation and regulation of those
leukocyte functions that are important for them to perform
their role in host defense against microbial invasion occurs.86
These activities include chemotaxis itself, whereby the leukocytes are directed to the sites of infection. Chemotaxis
requires highly developed motile responses involving actin
polymerizatioddepolymerization,adhesion events mediated
by integrins, and changes in cell shape. Once at the site
of infection, bacteria must be taken into the cell through
phagocytosis, a second process involving adhesion, cytoskeleta1 rearrangements, and membrane fusion events. Moreover,
phagocytosis must be tightly coordinated with bacterial killing events involving secretion of microbicidal granules and
generation of toxic oxidants by the NADPH oxidase. All
of these events, while particularly developed in phagocytic
leukocytes, involve many of the same basic cellular biologic
processes known to be important for regulating membrane
reshaping and fusion, cell motility, actin assembly, protein
trafficking and secretion, and protein-protein interactions in
all cells. Such processes are now known to be regulated by
the action of a variety of LMWG (low-molecular-weight
GTPases) of the Ras ~uperfamily.~~-~’
We would suggest
that, like in many other cells, signaling pathways used by
chemoattractant receptors have developed to regulate the
function of these LMWG, probably by controlling the activity of proteins that modulate whether these LMWG are in
active or inactive conformations.
pathways leading to cell growth and cytoskeletal assembly.62
In the human neutrophil (Fig 4), P13K activity is rapidly
stimulated by chem~attractants!~.~~
Formation of PIP3correlates with actin assembly in NW-stimulated neutrophils and
it has been suggested to be involved in this process.“ More
recently, the availability of inhibitors of P13K (wortmannin,
Ly294002) has enabled its role in neutrophil activation to
be more well defined. The major effect of PI3K inhibition
appears to be blockade of chemoattractant stimulation of
the NADPH
Overall actin polymerization is not
blocked, but wortmannin can induce oscillations in F-actin
content.70In many other cells, PI3K appears to be involved
with the cytoskeletal changes accompanying membrane ruffling, a process involving the localized polymerization of
actin filaments to produce short, highly cross-linked membrane-associated fib er^.""^ Wortmannin blocked NFF”stimulated granule secretion in neutrophils” and PI3K also appears to be necessary for receptor-mediated secretion in rat
basophilic leukemia cells.75
PI3K is a heterodimeric protein made up of an 85-kD
regulatory subunit and a 110-kDcatalytic s ~ b u n i t . The
~~.~~
REGULATION OF LMWG ACTIVITY
mechanisms of regulation are not well defined. PI3K is reRas is a widely expressed 189-amino acid GTP-binding
cruited to phosphotyrosine residues on activated tyrosine
protein that is a key regulator of eukaryotic cell growth and
kinase receptors and other tyrosine-phosphorylated proteins
development. More than 50 mammalian Ras-related GTPvia the SH2 motifs present on the p85
Activation
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1653
PHAGOCYTE SIGNALINGMECHANISMS
signaling:
PLC
P&
Fig 5. An updated view of chemoattractant receptor
Multiple pathways.
diaSchematic
gram summarizing the known signaling pathways
and effectors used by leukocyte chemoattractant recaptors, as
the
described in
text.
R=
PLD
1
1
/
PKC
binding proteins of MW -18 to 26 kDwhich regulate a
variety of fundamental cellular processes have been described over the past several
Based on sequence
and structural similaritie~,~"~".~~
they can be grouped into
five subfamilies: Ras, Rho, Rab, Arf, and Ran. The LMWG
function as molecular switches that are regulated by their
association with guanine nucleotides (Fig 6). When bound
to GTP, these switches are active, whereas conversion of
GTP to guanosine diphosphate (GDP) by hydrolysis results
in inactivation. The activity of each LMWG is determined
by exogenous proteins that modulate this GTPIGDP cycle."
Guanine nucleotide exchange factors (GEFs; also termed
GDP dissociation stimulators, GDSs) stimulate the exchange
of GTP for GDP on LMWGs and are necessary for activation
in the presence of cytosolic Mgz+concentrations that inhibit
nucleotide release. GTPase activating proteins (GAPs) stimulate the low intrinsic rate of GTP hydrolysis, converting
the active GTP-bound LMWG to the inactive GDP-bound
form. In addition, certain families of LMWG bind to cytosolic regulatory proteins termed GDP dissociation inhibitors
(GDIs). GDIs can be important determinants of the overall
/"
GAPs
l
LMWG
.
1
1
Phosphatase8
Phosphata8ea
PIP3
AA
MAPKDAG IPS,
Inacfive
\
GbP
GTP~GEFS
GTP
LMWG
GDls
Fig 6. Regulatory cycle for the LMW GTP-binding proteins. The
activity of LMWG iscontrolled by regulation of its GTP- versus GDPbound state by associated regulatory proteins. GAPs, GTPaseactivating proteins; GEFs, guanine nucleotide exchange factors; GDls, GDP
dissociation inhibitors.
PMK
1
Tyr
Kinand
Ser/rhr
KTnaww
and
\
Cax
function of an LMWG because they not only inhibit GDP
dissociation, but can also prevent GTP hydrolysis and maintain LMWG in soluble (cytosolic) forms.
There exists an increasingly wide variety of such regulatory proteins, which may be specific for a single LMWG or
which can act on multiple substrates. Additionally, many of
these regulatory molecules have multiple functional domains
that allow them to interact with other signaling molecules.8'
Hormone and autocoid receptors generally control the activity of LMWG by targeting these regulatory proteins; the
chemoattractant receptors are likely to be no exception to
this paradigm.
REGULATION OF CELL FUNCTION BY LMWG
The Rab Subfamily: Vesicular Secretion and Phagocytosis
Biochemical and genetic studies (in yeast) of the Rab
GTP-binding proteins have demonstrated their role in regulating the vesicular trafficking of proteins from the endoplasmic reticulum through the Golgi apparatus and to/from the
cell surface in mammalian cells. This includes the processes
of constitutive and regulated secretion and endocytic events.
Such a function for Rabs is supported by their localization
to distinct subcompartments of the endocytic and exocytic
pathways. The interested reader is referred to several excellent reviews on the field.92-95
Leukocytes undergo both phagocytic processes and regulated granule secretion. These events are regulated by GTPbinding proteins, based on their sensitivity to GTP and its
Rab proteins have been identified in human
phagocytes."' Levels of Rab lAp, 2p, 4p, and 6p were all
shown to increase when precursor cell lines were differentiated into macrophage- or neutrophil-like cells. Interestingly,
none was shown to be present in specific or azurophilic
granules purified from human neutrophils. Other st~dies"'~~~''~
have determined that these granules do contain distinct subsets of LMWG, although none have yet been identified as
Rabs. A candidate as a regulator of phagosome-lysosome
fusion is RabS, and depletion of RabS using antisense oligonucleotides inhibited the phagocytosis of antibody-coated
particles in U937 cells (Alverez C, Stahl PD: personal communication, January 1995).
Rab3A and/or 3B have been shown to localize to secretory
vesicles in adrenal chromaffin,'" anterior pit~itary,"~and
neuronal synapses,'06 and Rab3A and/or3Bcan modulate
vesicle secretion in these systems. A role for Rab3 in leukocyte secretion is suggested by the observation that an Rab3A
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1654
effector domain peptide could specifically stimulate degranulation from mast cells.'" Thus, although the role of Rab
proteins in leukocyte vesicular trafficking has not yet been
clearly established, the importance of protein trafficking,
granule secretion, and phagocytic processes to leukocyte
function suggests that further investigation into the action
of Rab proteins in these events is warranted.
The ARF Subfamily: Vesicular Trajjicking and
Phospholipase D
The Arf proteins (at least six distinct forms) have also
been implicated in the regulation of intracellular vesicle
t r a n ~ p o r t . 9 Arf
~ ~ ~was
~ ~ 'originally
~~
identified as a protein
cofactor necessary for efficient cholera toxin-catalyzed adenosine diphosphate (ADP)-ribosylation of the (Y subunit of
Gs, the stimulatory component of adenylyl cyclase (hence
the name ADP-ribosylationfictor).'w,''nArf is located in the
Golgi apparatus in mammalian cells and is a major component of non-clathrin-coated vesicles, where it is thought to
be involved in vesicle formation, coat assembly, and intraGolgi transport."'-''3 A role for Arfin vesicle transport
from the endoplasmic reticulum to the Golgi has also been
demonstrated,'14 and Arf can regulate endosome-endosome
fusion in vitro."'
Although the existence of Arf in human neutrophils has
been demonstrated immunologically: its role in vesicular
trafficking regulated by chemoattractants in these cells remains speculative. However, Arf was recently identified as
a GTP-dependent cytosolic regulator of phospholipase D
activity in the HL60 myelocytic cell line."6,"7 Arf appears
to directly regulate phospholipase D (PLD) activity, and
there is considerable speculation that this effect might contribute to the well-known ability of Arf to regulate membrane
traffic.I18 Activated PLD cleaves phosphatidylcholine (PC)
to produce phosphatidic acid (PA) and choline. Ethanol and
similar alcohols can compete with water in the cleavage
reaction to inhibit PA formation, and a role for PA in NWor C5a-stimulated secretion was inferred from the ability of
ethanol to partially inhibit secretion from neutrophils and
HL60 cells.'19,12n
Evidence for PA as a mediator of chemoattractant-stimulated NADPH oxidase activation has also been
~ b t a i n e d . ' ~ 'It" ~is~of interest that PA may also indirectly
regulate the activity of other LMWG involved in regulating
leukocyte function.Iz4Details of PLD regulation as it relates
to chemoattractant-modulatedArf function in leukocytes remain to be elucidated; a relationship to Rho function appears
to be emerging as well (see the following section).
The Rho Subfamily: Cytoskeleton, PLD, Oxidant
Production
The Rho proteins. The three closely related forms of
Rho, termed A, B, C, serve as efficient substrates for ADPribosylation by the C3 ADPribosyltransferase of Clostridium
botulinum. Early studies had shown that treatment of cells
with C3 exoenzyme caused marked changes in cell morphology correlated with disassembly of actin
microfilaments.'25.'26Microinjection of mutationally activated Rho
protein (Val14 Rho; equivalent to the position 12 activating
mutation of Ras) induced dramatic stimulation of actin stress
GARY M. BOKOCH
fibers and focal adhesions, and this effect was inhibited by
prior ADPribosylation of
These elegant experiments established that serum growth factor(s) can regulate
the assembly of focal adhesions andactin stress fibers in
fibroblasts through activation of Rho. The interested reader
is referred to the excellent reviews on this
The mechanisms by which Rho modulates the polymerization of actin (ie, regulation of nucleation sites, regulation of
monomer sequestration, etc) remain to be determined. A
number of proteins that can control filamentous actin levels
in cells (ie, profilin, gelsolin) are regulated by the binding
of the lipid PIP2.13n,131
It was recently shown that Rho can
regulate the activity of a kinase that phosphorylates phosphatidylinositol4-phosphate (PIP) on the 5 position to generate
Potentially, modulation of this PI 5-kinase by Rho
provides a mechanism to explain the regulation of stress
fiber and focal adhesion-associated F-actin by Rho. A chemoattractant receptor-, GTP-regulated PI 5-kinase activity
has been described in human neutrophils."'
Thus, the evidence is quite strong that Rho can regulate
actin microfilament organizatiodassembly, although this has
notbeen established in chemoattractant-stimulated leukocytes. Polymerization of neutrophil actin can be induced by
guanine nucleotides in permeabilized cells,134and both Rho
and Rac have been shown to regulate the state of the actin
cytoskeleton in mast cells.'35The latter have also been implicated in regulation of the secretory responses of mast cells,
although it is unclear whether this activity is distinct from
the actin regulatory
The existence of Rho as a C3
substrate in leukocytes has been s h o ~ n ~ ~ * .and
" ~ .the
" ~ ability of C3 ADPribosyltransferase to inhibit neutrophil chemotaxis, but not superoxide production or degranulation, has
been r e ~ 0 r t e d . IRho
~ ~ is apparently essential for motility of
fibroblastsImand sperm.I4' Rho also exerts regulatory effects
on leukocyte adhesion, where it may play some role in controlling integrin affinity. Inhibition of Rho function by C3
transferase blocked phorbol ester-induced, CD1 IdCD 18-dependent B-lymphocyte aggregation,I4* and a similar effect
on platelet integrin GP IIb-IIIa-mediated platelet aggregation was 0 b ~ e r v e d . l Because
~~
such activities are critical
determinants of leukocyte margination and chemotaxis to
inflammatory sites, Rho may play an important role in regulating leukocyte responsiveness.
Very recently, a role for Rho as a regulator of PLD activity
has been described. A GTP-sensitive PLDactivityinrat
liver plasma membranes waspartially inhibited (-50%)
when Rho was extracted from the membranes with RhoGDI,
and activity could be restored by the addition of recombinant
Rho.14;' This result would be consistent withthe previous
observation that PLD activity in differentiated HL60 cells
was inhibited by R ~ O G D I .Rho
' ~ ~appears to act directly on
PLD and is synergistic with Arfin activating the partially
purified enzyme.'46However, it is of interest that PIP2 has
been shown to be a required cofactor for PLD activity in
vitro."' The ability of Rho to regulate cellular PIP2formation
may have regulatory implications for PLD activity as well.
The RUCproteins. Regulatory activity for theRacproteins in at least two biologic systems has been established:
actin filament formation associated with membrane ruffling/
larnellip~dia,'~~'~~~'~~
and activation of the phagocyte NADPH
oxidase.I4'
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PHAGOCWE SIGNALING
The microinjection of an activated mutant form of Racl
(Gly12 Val) into fibroblasts induced the accumulation of
macropinocytotic vesicles and membrane ruffling, accompaniedby formation of polymerized actin in rufflesat the
plasma mernb~ane.~'
Membrane ruffling induced by growth
factors and activated Ras were both blocked by microinjection of a dominant negative mutant form of Racl. Therefore,
Ras maybe an upstream regulator of RacRho. Raclwas also
shown to induce stress fiber formation in an Rho-dependent
manner, indicating communication between the Rho and Rac
signaling pathways. As with Rho-induced actin assembly in
stress fibers, little is known about the mechanisms by which
Rac regulates actin assembly associated with rufflinghell
motility, and there is no evidence yet that Rac has similar
effects in chemoattractant-stimulated leukocytes. However,
the changes in F-actin distribution accompanying phagocytosis are reminiscent of the cytoskeletal responses to Rac, and
there is an evident coordination between the phagocytic process and oxidant production by the Rac-regulated NADPH
oxidase (see below). It is of interest that periodic oscillations
in actin assembly/disassembly induced in neutrophils by the
PI 3-kinase inhibitor wortmannin are exactly paralleled by
changes in NADPH oxidase activity7'-is this due to a coordinate regulation of both events by Rac?
The second system regulated byRac protein@) is the
NADPH oxidase found in phagocytic leukocytes (neutrophils, macrophages, eosinophils). The NADPH oxidase consists of at least one membrane component, cytochrome bSB8,
and the cytosolic proteins p47phox and p67phox, which interact at the level of the plasma membrane upon phagocyte
activation. The Rac proteins form a third required cytosolic
component of the system, with Racl identified as the active
component in guinea pig macrophage^'^^ and Rac2 in human
ne~trophi1s.l~~
Cell-free studies in which cytosol could be
replaced with recombinant p47phox,p67phox. and Rac2
proved that Rac was required for human neutrophil superoxide p r o d ~ c t i o n . ' ~Cells
~ " ~ ~in which rac gene activity was
blocked by antisense oligonucleotides were unable to produce superoxide, demonstrating the requirement for Rac in
intact cell oxidase function.ls3 Rac only stimulates oxidant
production when bound with GTP and the formation of superoxide by leukocytes can be regulated by controlling Rac
activity through the various regulatory GEFs and GAPS inv01ved.I~~
The observation that the Rac GAP Bcr functions
as a regulator of the oxidative burst in vivo as a result of its
ability to modulate Rac
suggests that inflammatory diseases such as bacterial septicemia, adult respiratory
distress syndrome, and rheumatoid arthritis, among others,
may involve defects in Rac regulation.
Rac dissociates from a cytoplasmic complex with GDI
upon activation of neutrophils by chemoattractant and translocates from the cytosol to associate with the plasma membrane and/or the submembranous cytoskeleton.'s5"57Translocation appears to
be
determined by the stimulated
exchange of GTP for GDP on Rac,Is8probably as a result of
chemoattractant receptor-initiated signals. Chemoattractantstimulated Rac translocation canbe enhanced by protein
phosphatase inhibitors and blocked by tyrosine kinase inhibitors, suggesting that tyrosine phosphorylation may control
proteins able to modulate the GTP/GDP state of Rac.Is9The
1655
connections with upstream signaling mechanisms still remain to be worked out, but Rac activation may require the
activity of the Src-related and/or other tyrosine kinases
whose activity is triggered by chemoattractants.
The Ras Subfamily: NADPH Oxidase, P U 2 , PI3K
The Rasproteins. The activation of Ras as an early event
in leukocyte Stimulation by chemoattractants has been discussed in a previous section. Despite this knowledge, the
role of Ras in the acute leukocyte responses during chemotaxis are unclear, since Ras controls growth and development
inmost cells. Although such regulation occurs mainly at
level of transcription through the phosphorylation of transcription factors by MAPK and associated kinases, the likelihood of other phosphorylation targets more relevant to
short-term cellular responses is high. One such target whose
activity can be regulated via phosphorylation is phospholipase A2(PLA2).ImPhosphorylation-dependent PLA2 activation has been observed in macrophages16' andHL60 granuloc y t e ~ . ' ~ It
~ . is
' ~ possible
~
that a primary role of Ras in
chemoattractant-stimulatedleukocytes is to regulate the formation of arachidonic acid and the subsequent generation of
eicosanoids and leukotrienes. An additional possibility is
regulation of PI3K by active Ras, as discussed previously.
Clearly a tremendous amount of work needs to be done to
define the role(s) of Ras in leukocyte function.
The Rap proteins.
Closely related to Ras structurally
(-50% homology overall) but distinct in their function are
the Rap proteins (RaplA, RaplB,RapZA, Rap2B). Rapl has
been shown to antagonize Ras action in vitro by competing
for effector targets via an effector domain (amino acids 30
through 40) that is identical to that of Ras.'" RaplA is a
very abundant protein in human neutrophil^'^' and has been
localized to the plasma membrane and granule membranes,
where itappears to be closely associated with the cytochrome
bssBsubunit of the NADPH ~ x i d a s e . ' ~Indeed,
~ . ' ~ ~Rapl has
been shown to copurify with and bind to cytochrome
bs58r'67,168
suggesting it might be involved with the process
of oxidant production. Immunodepletion of Rapl led to a
loss of superoxide formation in a cell-free systemi69 and
expression of Rapl dominant negative and dominant positive
mutants in Epstein-Barr virus-transformed B lymphocytes
inhibited oxidant formation.I7'
Although a functional and physical interaction of RaplA
with the NADPH oxidase has been established, it is not yet
clear what its role in the system might be. RaplA is clearly
not necessary for basic superoxide formation by the NADPH
oxidase in a cell-free system and its function may be modulatory rather than required. RaplA is phosphorylated by cyclic
adenosine monophosphate (CAMP)-dependentprotein kinase in neutrophils and could play some role in the inhibitory
effects of CAMP on oxidant prod~ction.'~'
CONCLUSIONAND
PERSPECTIVE
Our current view of chemoattractant signaling indicates a
rich diversity in the structural elements and pathways involved. The regulation of these signaling pathways and their
relationships to regulation of the LMWG and other proteins
important for controlling the ultimate functional responses
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1656
GARY M. BOKOCH
of leukocytes still largely remain to be elucidated. In view
of the critical importance of leukocytes (neutrophils, eosinophils, macrophages and monocytes, mast cells, and lymphocytes) in host defense and in a variety of disease states, the
potential reward for understanding the signaling paradigms
involved certainly justifies the intense interest in this topic.
ACKNOWLEDGMENT
I thank Drs Eric Prossnitz and Mark T. Quinn for comments on
the manuscript and Drs Quinn and Gregory Downey for providing
prepublication copies of reviews they hadprepared. I am grateful for
the assistance of Dr Richard D. Ye in preparing Figure I . Antonette
Lestelle provided excellent editorial assistance. Finally, I would note
that no attempt has been made to make the already long reference
list exhaustive; I apologize to those authors whose relevant reports
were omitted.
REFERENCES
1. Snyderman R, Pike MC: Transductional mechanisms of chemoattractant receptors on leukocytes. Contemp Top Immunobiol 14:I ,
1984
2. Omann GM, Allen RA, Bokoch GM, Painter RG, Traynor
AE, Sklar LA: Signal transduction and cytoskeletal activation in the
neutrophil. Physiol Rev 67:285, 1987
3. Sha'afi RI, Molski T F P Activation of the neutrophil. Prog
Allergy 42:1, 1988
4. Bokoch GM: Signal transduction by GTP-binding proteins during leukocyte activation: Phagocytic cells, in Grinstein S, Rotstein
OD (eds): Current Topics in Membranes and Transport, 1991, p 65
S . Cockroft S: G protein regulated phospholipases C,D, and Azmediated signalling in neutrophils. Biochim Biophys Acta 1113:135,
1992
6. Murphy PM: The molecular biology of leukocyte chemoattractant receptors. Annu Rev Immunol 12:593, 1994
7. Thelen M, Dewald B, Baggiolini M: Neutrophil signal transduction and activation of the respiratory burst. Physiol Rev 73:797,
1993
8. Gerard C, Gerard NP: The pro-inflammatory seven-transmembrane segment receptors of the leukocyte. Curr Opin Immunol6: 140,
1994
9. Amatruda TT, Gerard NP, Gerard C, Simon MI: Specific interactions of chemoattractant receptors with G-proteins. J Biol Chem
268:10139, 1993
10. Gilman AG: G proteins: Transducers of receptor-generated
signals. Ann Rev Biochem 56:615, 1987
11. Camps M, Hou C, Sidiropoulos D, Stock JB, Jakobs KH,
Gierschik P: Stimulation of phospholipase C by guanine-nucleotidebinding protein py subunits. Eur J Biochem 206:821, 1992
12. Blank JL, Brattain K A , Exton JH: Activation of cytosolic
phosphoinositide phospholipase C by G-protein py subunits. J Biol
Chem 267:23069, 1992
13. Katz A, Wu D, Simon HI: Subunits p b of heterotrimeric G
protein activate p2 isoform of phospholipase C. Nature 360:686,
1992
14. Rossi F: The 0,"forming NADPH oxidase of the phagocytes:
Nature, mechanisms of activation and function. Biochim Biophys
Acta 853:65, 1986
15. Lew PD: Receptors and intracellular signaling in human neutrophils. Am Rev Respir Dis 141:5127, 1990
16. Billah MM: Phospholipase D and cell signaling. Curr Opin
Immunol 5:114, 1993
17. Thelen M, Wirthmueller U: Phospholipases and protein kinases during phagocyte activation. Curr Opin Immunol 6:106, 1994
18. McPhail LC, Clayton CC, Snyderman R: The NADPH oxi-
dase of human polymorphonuclear leukocytes: Evidence for regulation by multiple signals. J Biol Chem 2595768, 1984
19. Watson F, Robinson J, Edwards SW: Protein kinase C-dependent and -independent activation of the NADPH oxidase of human
neutrophils. J Biol Chem 266:7432, 1991
20. Della Bianca V, Grzeskowiak M, Dusi S, Rossi F: Fluoride
can activate the respiratory burst independently of Ca2+ stimulation
of phosphoinositide turnover and protein kinase C translocation in
primed human neutrophils. Biochem Biophys Res Commun 150:955.
1988
21. Dewald B, Thelen M, Baggiolini M: Two transduction sequences are necessary for neutrophil activation by receptor agonists.
J Biol Chem 263:16179, 1988
22. Gomez-Camhronero J, Huang C-H, Bonak VA, Wang E, Casnellie J-E, Shiraishi T, Sha'afi RI: Tyrosine phosphorylation in human neutrophils. Biochem Biophys Res Commun 162:1478, 1989
23. Naccahe PH, Gilbert C, Caon AC, Gaudry M, Huang C-K,
Bonak VA, Umezawa K, McColl SR: Selective inhibition of human
neutrophil functional responsiveness by erbstatin, an inhibitor of
tyrosine protein kinase. Blood 76:2098, 1990
24. Kusunoki T, Higashi H, Hosoi S, Hata D, Sugie K, Mayumi
M, Mikawa H: Tyrosine phosphorylation and its possible role in
superoxide production by human neutrophils stimulated with FMLP
and IgG. Biochem Biophys Res Commun 183:789, 1992
25. Berkow RL, Dodson RW: Alterations in tyrosine protein kinase activities upon activation of human neutrophils. J Leukoc Biol
49599, 199I
26. Corey S, Equinoa A, Puyana-Theall K,Bolen JB, Cantley
L, Mollinedo F, Jackson TR, Hawkins PT, Stephens LR: GMCSF
stimulates both association and activation of phosphoinositide 30Hkinase and src-related tyrosine kinase(s) in human myeloid derived
cells. EMBO J 12:2681, 1993
27. Willman CL, Stewart CC, Longacre TL, Head DK,Habbersett
R, Ziegler SF, Perlmutter RM: Expression of the c-fgr andhck
protein tyrosine kinases in acute myeloid leukemia blasts is associated with early commitment and differentiation events in the monocytic and granulocytic lineages. Blood 77:726, 1991
28. Zhou M, Lubin DM, Link DC, Brown EJ: Distinct tyrosine
kinases associate with FcyRII and FcyRIIIB in human polymorphonuclear leukocytes: Implications for immune complex activation of
the respiratory burst. J Biol Chem 270:13553, 1995
29. Berton G, Fumagalli L, Laudanne C, Sori0 C: p2 Integrindependent protein tyrosine phosphorylation and activation ofthe
FGRprotein tyrosine kinase inhuman neutrophils. J Cell Biol
126:l I 1 I , 1994
30. Gee CE, Griffin J, Sastre L, Miller LJ, Springer TA, PiwnicaWorms H, Roberts TM: Differentiation of myeloid cells is accompanied by increased levels of pp60,.sx protein and kinase activity. Proc
Natl Acad Sci USA 83:5131, 1986
31. Aganval A, Salem P, Robbins KC: Involvement of
a protein tyrosine kinase, in Fc receptor signalling. J BiolChem
268: 15900, 1993
32. Hamada F, Aoki M, Ahyama T, Toyoshima K: Association
of immunoglobin G Fc receptor I1 with Src-like protein tyrosine
kinase Fgr in neutrophils. Proc Natl Acad Sci USA 90:6305, 1993
33. Durden DL, Liu YB: Protein-tyrosine kinase ~72"' in FcyRl
receptor signaling. Blood 84:2102, 1994
34. Ravetch JV: Fc receptors: Rubor redux. Cell 78553, 1994
35. Notario V, Gutkind JS, Imaizumi M, Katamine S, Robbins
KC: Expression of the f g r protooncogene product as a function of
myelomonocytic cell maturation. J Cell Biol 109:3129, 1989
36. Meier RW, Bielke W, Chen T, Niklaus G, Triis RR, Tobler
A: Lyn, a src-like tyrosine-specific protein kinase, is expressed in
HL60 cells induced to monocyte-like or granulocyte-like cells. Biochem Biophys Res Commun 185:91, 1992
37. Stephens L. Equinoa A,Corey S, Jackson T. Hawkins T:
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PHAGOCYTESIGNALING MECHANISMS
Receptor-stimulated accumulation of phosphatidylinositol (3,4,5)triphosphate by G protein mediated pathways in human myeloid
derived cells. EMBO J 12:2265, 1993
38. Ptasznik A, Traynor-Kaplan A, Bokoch GM: G-protein-coupled chemoattractant receptors regulate Lyn tyrosine kinase-Shc
adaptor protein signaling complexes. J Biol Chem 1995 (in press)
39. Pawson T, Schlessinger J: SH2 and SH3 domains. Curr Biol
3:434, 1993
40. Pleiman CM, Hertz W M , Cambier JC: Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85
subunit. Science 263:1609, 1994
41. Rozakis-Adcock M, McGlade J, Mbamaln G, Pelicci G , Daly
R, Li W, Batzer A, Thomas S, Brugge J, Pelicci PG, Schlessinger
J, Pawson T: Association of the Shc and GrbUSem5 SH2-containing
proteins is implicated in activation of the Ras pathway by tyrosine
kinases. Nature 360:689, 1992
42. Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland
AM, Weinberg RA: Association of SOS Ras exchange protein with
Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature 363:45, 1993
43. Worthen CS, Avdi N, Buhl AM, Suzuki N, Johnson GL:
FMLP activates Rac and Raf in human neutrophils. J Clin Invest
94:815, 1994
44. Buhl AM, Audio N, Worthen GS, Johnson GL: Mapping of
the C5a receptor signal transduction network in human neutrophils.
Proc Natl Acad Sci USA 91:9190, 1994
45. Davis RI: The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553, 1993
46. Torres M, Hall FL, O'Neill K: Stimulation of human neutrophils with fMLP induces tyrosine phosphorylation and activation of
two distinct mitogen-activated protein kinases. J Immunol 150:1563,
1993
47. Thompson L, Shiroo M, Saklatuala J: The chemotactic factor
f Metleuphe activates microtubule-associated protein 2 (MAP) kinase and a Map kinase kinase in polymorphonuclear leucocytes.
Biochem J 290483, 1993
48. Stokoe D, MacDonald SG, Cadwallader K, Symons M, Hancock J P Activation of Raf as a result of recruitment to the plasma
membrane. Science 264:1463, 1994
49. Leevers SJ, Patterson HR, Marshall CJ: Requirement for Ras
in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369:411, 1994
50. Freed E, Symons M, MacDonald SG, McCormick F, Ruggieri
R Binding of 14-3-3 proteins to the protein kinase Raf and effects
on its activation. Science 265:1713, 1994
5 I . hie K, KotohY, Yashar BM, Errede B, Nishida E, Matsumoto
K: Stimulatory effects of yeast and mammalian 14-3-3 proteins on
the raf protein kinase. Science 265:1716, 1994
52. Winitz S, Russell M, Qian N-X, Gardner A, Dwyer L, Johnson GL: Involvement of Ras and Raf in the G,-coupled acetycholine
muscarinic M2 receptor activation of mitogen-activated protein
(MAP) kinase kinase and MAP kinase. J Biol Chem 269:19196,
1993
53. Johnson GL, Gardner AM, Lange-Carter C, Qian NX, Russell
M, Winitz S: How does the G protein, Gi.2rtransduce mitoogenic
signals? J Cell Biochem 54:415, 1994
54. Howe LR, Marshall CJ: Lysophosphatidic acid stimulates mitogen-activated protein kinase activation via a G-protein-coupled
pathway requiring p21" and ~74""'. J Biol Chem 268:20717, 1993
55. Grinstein S, Butler JR, Furaya W, L'Allemain G , Downey
GP: Chemotactic peptides induce phosphorylation and activation of
MEK-I in human neutrophils. J Biol Chem 269: 19313, 1994
56. Fialkow L, Chen C-K, Rosa D, Grinstein S, Downey GP:
Activation of the mitogen-activated protein kinase in neutrophils:
Role of oxidants. J Biol Chem 1995 (in press)
57. Winston BW, Lange-Carter CA, Gardner AM, Johnson GL,
1657
Riches DWH: TNFa rapidly activates the MEWMAP kinase cascade
in an MEKK-dependent, C-raf-l-independent fashion in mouse macrophages. Proc Natl Acad Sci USA 92:1614, 1995
58. Grinstein S , Furuya W, Butler JR, Tseng J: Receptor-mediated
activation of multiple serineheronine kinases in human leukocytes.
J Biol Chem 268:20223, 1993
59. Ding J, Badwey JA: Neutrophils stimulated with a chemotactic peptide or a phorbol ester exhibit different alterations in the
activities of a battery of protein kinases. J Biol Chem 268:5234,
1993
60. Ding J, Badwey JA: Stimulation of neutrophils with a chemoattractant activates several novel protein kinases that can catalyse
the phosphorylation of peptides derived from the 47-KDa protein
component of the phagocyte oxidase and myristoylated alanine-rich
C kinase substrate. J Biol Chem 268:17326, 1993
61. Ding J, Badwey JA: Wortmannin and l-butanol block activation of a novel family of protein kinases in neutrophils. FEBS Lett
348: 149, 1994
61a. Knaus UG, Moms S, Dong H-J, Chemoff J, Bokoch GM:
Regulation ofhuman leukocyte p21-activated kinases through G
protein-coupled receptors. Science 1995 (in press)
62. Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani
A, Kapeller R, Soltoff S: Oncogenes and signal transduction. Cell
64:281, 1991
63. Traynor-Kaplan AE, Hams AL, Thompson BL, Taylor P,
Sklar LA: An inositoltetrakisphosphate-containingphospholipid in
activated neutrophils. Nature 334:353, 1988
64. Traynor-Kaplan AE, Thompson BL, Hams AL, Taylor P,
Omann GM, Sklar LA: Transient increase in phosphatidylinositol
3.4-bisphosphate and phosphatidylinostol trisphosphate during activation of human neutrophils. J Biol Chem 264:15668, 1989
65. Stephens LR, Hughes KT, Irvine R F Pathway of phosphatidylinositol (3,4,5) trisphosphate synthesis in activated neutrophils.
Nature 351:33, 1991
66. Eberle M, Traynor-Kaplan AE, Sklar LA, Norgauer J: Is there
a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils? J Biol Chem 265:16725,
1990
67. Arcaro A, Wymann MP: Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: The role of phosphatidylinositol 3,4,5trisphosphate in neutrophil responses. Biochem J 296:297, 1993
68. Okada T, Sakuma L, Fukui Y, Hazeki 0, Ui M: Blockage of
chemotactic peptide-induced stimulation of neutrophils by Wortmannin asa result of selective inhibition of phosphatidylinositol 3-kinase.
J Biol Chem 269:3563, 1994
69. Vlahos CJ, Matter WF, Brown W, Traynor-Kaplan AE,
Heyworth PG, Prossnitz ER, Ye RD, Marder P, Schelm JA, Rothfuss
KJ, Serlin BS, Simpson PJ: Investigation of neutrophil signal transduction using a specific inhibitor of phosphatidylinositol 3-kinase.
J Immunol 1995 (in press)
70. Wymann MP, Kermen P, Deranleau DA, Baggiolini M: Respiratory burst oscillations in human neutrophils and their correlation
with fluctuations in apparent cell shape. J BiolChem 264:15829,
1989
71. Ridley A J , Paterson HF, Johnston CL, Diekmann D, Hall A:
The small GTP-binding protein rac regulates growth factor-induced
membrane ruffling. Cell 70:401, 1992
72. Kotani K, Yonezawa K, Hara K, Ueda H, Kitamura Y, Adno
A, Chavanieu A, Calas B, Grigorescu F, Nishiyama M, Waterfield
MD, Kasuga M: Involvement of phosphoinositide 3-kinase in insulin- or IGF-l-induced membrane ruffling. EMBO J 13:22313, 1994
73. Wennstrom S, Hawkins P, Crooke F, Hara K, Yonezawa K,
Kasuga M, Jackson T, Claesson-Welsh L, Stephens L: Activation
of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Cum Biol 4:385, 1994
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1658
74. Bretscher A: Microfilament structure and function in the cortical cytoskeleton. Annu Rev Cell Biol 7:337, 1991
75. Yano H, Nakanishi S, Kimura K, Hanai N, Saitoh Y, Fukui
Y, Nonomura Y, Matsuda Y: Inhibition of histamine secretion by
Wortmannin through the blockade of phosphatidylinositol 3-kinase
in RBL-2H3 cells. J Biol Chem 268:25846, 1993
76. Koch CA, Anderson D, Moran MF, Ellis C, Pawson T: SH2
and SH3 Domains: Elements that control interactions of cytoplasmic
signalling proteins. Science 252:668, 1991
77. Vlahos CJ, Matter WF: Signal transduction in neutrophil activation: Phosphatidylinositol 3-kinase is stimulated without tyrosine
phosphorylation. FEBS Lett 309:242, 1992
78. Rodriquez-Viciana P, Warne PhD, Dhand R, Vanhaesebroeck
B, Gout I, Fry MJ, Waterfield MD, Downward J: Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370:527, 1994
79. Kodaki T, Woscholski R, Hallberg B, Rodriquez-Viciana P,
Downward J, Parker PJ: The activation of PI3-kinase by Ras. Curr
Biol 4:798, 1994
80. Zheng Y, Bagrodia S, Cerione RA: Activation of phosphoinositide 3-kinase activity by CDC42Hs binding to p85. J Biol Chem
269: 18727, 1994
81. Zhang J, King WC, Dillon S, Hall A, Feig L, Rittenhouse
SE: Activationof platelet phosphatidylinositide 3-kinase requires
the small GTP-binding protein Rho. J Biol Chem 268:22251, 1993
82. Kumagai N, Moriii N, Fujisawa K, Nemoto Y, Narumiya S:
ADPribosylation of rho p21 inhibits lysophosphatidic acid-induced
protein tyrosine phosphorylation and phosphatidylinositol 3-kinase
activation in cultured Swiss 3T3 cells. J Biol Chem 268:24535, 1993
83. Bokoch GM, Vlahos CJ, Knaus UG: (submitted for publication)
84. Stephens L, Smrcka A, Cooke I T , Jackson TR, Sternweis PC,
Hawkins PT: A novel phosphoinositide 3 kinase activity in myeloidderived cells is activated by G protein By subunits. Cell 77:83, 1994
85. Thomason PA, James SR, Casey PI, Downes CP: A G-protein
By-subunit-responsive phosphoinositide 3-kinase activity in human
platelet cytosol. J Biol Chem 269:16525, 1994
86. Baggiolini M, Boulay F, Badwey JA, Curnutte JT: Activation
of neutrophil leukocytes: Chemoattractant receptors and respiratory
burst. FASEB J 7:1OW, 1993
87. Bourne HR, Sanders DA, McCormick F: The GTPase superfamily: A conserved switch for diverse cell functions. Nature
348: 125, 1991
88. Bokoch GM, Der CJ: Emerging concepts in the ras superfamily of GTP-binding proteins. FASEB J 7:750, 1993
89. Hall A: The cellular functions of small GTP-binding proteins.
Science 249:635, 1990
90. Kahn RA, Der CJ, Bokoch GM: The ras superfamily of GTPbinding proteins: Guidelines on nomenclature. FASEB J 6:2512,
I992
91. Valencia A, Chardin P, Wittinghofer A, Sander C: The ras
protein family: Evolutionary tree and role of conserved amino acids.
Biochemistry 30:4637, 1991
92. Nuoffer C, BalchWE: GTPases: Multifunctional molecular
switches regulating vesicular traffic. AnnuRevBiochem
63:949,
1994
93. Ferro-Novick S, Novick P: The role of GTP-binding proteins
in transport along the exocytic pathway. Annu Rev Cell Biol 9575,
1993
94. Pfeffer S: Rab GTPases: Master regulators of membrane trafficking. Curr Opin Cell Biol 6:522, 1994
95. Donaldson JG, Klausner RD: Arf: A key regulatory switch in
membrane traffic andorganelle structure. Curr Opin Cell Biol6:527,
I994
96. Brown EJ, Newell AM, Gresham HD: Molecular regulation
of phagocyte function: Evidence for the involvement of a guanosine
GARY M. BOKOCH
triphosphate-binding protein in opsonin-mediated phagocytosis by
monocytes. J lmmunol 139:3777, 1987
97. Gomperts BD: Involvement of guanine nucleotide-binding
protein in the gating of Ca2-t by receptors. Nature 30654, 1983
98. Barrowman MM, Cockroft S, Gomperts BD: Two roles for
guanine nucleotides in the stimulus-secretion sequence of neutrophils. Nature 319504, 1986
99. Smolen JE, Stoehr SJ: Guanine nucleotides reduce the free
calcium requirement for secretion of granule constitutents from permeabilized human neutrophils. Biochim Biophys Acta 889: I7 I , 1986
100. Femandez JM, Lindau M, Eckstein F: Intracellular stimulation of mast cells with guanine nucleotides mimic antigenic stimulation. FEBS Lett 216:89, 1987
101. Maridonneau-Parini I, Yang CZ, Bomens M, Goud B: Increase in the expression of a family of small GTP-binding proteins,
kab proteins, during induced phagocyte differentiation. J Clin Invest
87:901, 1991
102. Philips MR, Abramson SB, Kolasinski SL, HainesKA,
Weissman G, Rosenfeld MC: Low molecular weight GTP-binding
proteins in human neutrophil granule membranes. J BiolChem
266: 1289, 1991
103. Dexter D, Rubins JB, Manning EC, Khachatrian L, Dickey
BF: Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules. J Immunol 145:1845,
I990
104. HolzRW,Brondyk
WH, Senter RA, Kuizon L, Macara
IC: Evidence for the involvement of Rab3Ain Cat'-dependent
exocytosis from adrenal chromaftin cells. J Biol Chem 269:10229,
1994
105. Liedo P-M, Vernier P, Vincent J-D, Mason WT, Zorec R:
Inhibition ofRab3B expression attenuates &"-dependent
exocytosis in rat anterior pituitary cells. Nature 364540, 1993
106. Matteuli M, Takei K, Cameron R, Hurlbut P, Johnston PA,
Sudhof TC, Jahn R, de Camilli P: Association of Rab3A with synapticvesiclesatlate
stages ofthe secretory pathway. .ICellBiol
I15:625, 1991
107. Oberhauser AF, Monck JR. Balch WE, Fernandez JM: Exocytotic fusion is activated by Rab3a peptides. Nature 360:270, 1992
108. Boman A, Kahn A: Arf proteins: Beyond regulators of membrane traffic. Trends Biol Sci 1995 (in press)
109.KahnRA, Gilman AG: Purification of a protein cofactor
required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxins. J Biol Chem 259:6228,
1984
I IO. Kahn RA, Gilman AG: The protein cofactor necessary for
ADP ribosylation of Gs by cholera toxin is itself a GTP binding
protein. J Biol Chem 261:7906, 1986
1 1 I , Steams T, Willingham MC, Botstein D, KahnRA: Arf is
functionally and physically associated with the Golgi complex. Proc
Natl Acad Sci USA 87:1238, 1990
1 12. Seratini T, Orci L, Amerdt M, Brunner M, Kahn RA, Rothman JE: Arf is a subunit of the coat of Golgi-derived COP-coated
vesicles: A novel role for a GTP-binding protein. Cell 67:239, 1991
I 13. Taylor TC, Kahn RA, Melancon P: Two distinct members
of the Arf family of GTP-binding proteins regulate cell-free intraGolgi transport. Cell 70:69, 1992
114. Balch WE, Kahn RA, Schwaninger R:Arf isrequired for
vesicular trafficking between the endoplasmic reticulum and the cisGolgi compartment. J Biol Chem 267: 13053, 1992
1 15.Lenhard JM, Kahn RA, Stahl PD: Evidence for Arf as a
regulator of in vitro endosome-endosome fusion. J BiolChem
267: 13047, I992
CR.
Slaughter C,
116.
Brown
HA,
Gutowski S, Moomau
Sternweis PC: ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell 75:1137,
1993
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PHAGOCYTE SIGNALINGMECHANISMS
117. Cockroft S, Thomas GMH, Fensome A, Geny B, Cunningham E, Gout I, Hiles I, Totty NF, Truong 0,Hsuan JJ: Phospholipase
D: A downstream effector of ARF in granulocytes. Science 263523,
1994
118. Kahn RA, Yucel JK, Malhotra V: ARF Signaling: A potential
role for phospholipase D in membrane traffic. Cell 75:1045, 1993
119. Kanaho Y, Kanoh H, Saitch K, Nozawa Y: Phospholipase
D activation by PAF, LTB,, and FMLP in rabbit neutrophils. J
Immunol 146:3536, 1991
120. Stutchfield J, Cockroft S: Correlation between secretion and
phospholipase D activation in differentiated HL60 Cells. Biochem
J 293:1, 1993
121. Pai J-K, Siege1 MI, Egan RW, Billah MM: Phosphilipase D
catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes. J Biol Chem 263:12472, 1988
122. Rossi F, Grzeskowiak M, Della-Bianca V, Calzetti F, Gandini
G: Phosphatidic acid and not diacylglycerol generated by phospholipase D is functionally linked to the activation of theNADPH oxidase
by FMLP in human neutrophils. Biochem Biophys Res Commun
168:320, 1990
123. Agwu DE, McPhail LC, Sozzani S, Bass DA, McCall CE:
Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. J Clin Invest 88531, 1991
124. Chuang T-H, Bohl BP, Bokoch GM: Biologically active
lipids are regulators of Rac:GDI complexation. J Biol Chem
268:36206, 1993
125. Chardin P, Boquot P, Madaule P, Popoff MR. Rubin EJ, Gill
DM: The mammalian G protein rho C is ADP-ribosylated by C.
botulinum exoenzyme C, and affects actin microfilaments in Vero
cells. EMBO J 8:1087, 1989
126. Paterson HF, Self A J , Garret MD, Just I, Aktories K, Hall
A: Microinjection of recombinant Rho induces rapid changes in cell
morphology. J Cell Biol 11 1:1001, 1990
127. Ridley AJ, Hall A: The small GTP-binding protein rho regulates the assembly of focal adhesion and actin stress fibers in response to growth factors. Cell 70:389, 1992
128. Hall A: Ras-related GTPases and the cytoskeleton. Mol Biol
Cell 3:475, 1992
129. Hall A: Small GTP-binding proteins and the regulation of
the actin cytoskeleton. Annu Rev Cell Biol 10:31, 1994
130. Stossel TP: From signal to psendopid: How cells control
cytoplasma actin assembly. J Biol Chem 264:18261, 1989
131. Theriot JA, Mitchison TJ: The three faces of profilin. Cell
75:835, 1993
132. Chong L, Traynor-Kaplan A, Bokoch GM, Schwartz MA:
The small GTP-binding protein Rho regulates a phosphatidylinositol
4-phosphate 5-kinase in mammalian cells. Cell 79:507, 1994
133. Stephens L, Jackson T, Hawkins PT: Activation of PI (43) P2
supply by agonists and non-hydrolyzable GTP analogues. Biochem J
295:48 1, 1993
134. Themen S, Naccache PH: Guanine nucleotide-induced polymerization of actin in electropermeabilized human neutrophils. J Cell
Biol 109:1125, 1989
135. Norman JC, Price LS, Ridley AJ, Hall A, Koffer A: Actin
filamant organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins. J Cell Biol 126:1005, 1994
136. Price L, Norman J, Ridley A, Koffer A: The small GTPases,
Racand Rho, as regulators of secretion in mast cells. Curr Biol
5:68, 1995
137. Bokoch GM, Parkos CA, Mumby SM: Purification and characterization of the 22,000 Da. GTP binding substrate for ADP-ribosylation by botulinum toxin, G22K. J Biol Chem 263:16744, 1988
138. Quilliam LA, Lacal J-C, Bokoch GM: Identification of rho
as a substrate for botulinum toxin C3-catalyzed ADP-ribosylation.
FEBS Lett 247:221, 1989
139. Stasia M-J. Jovan A, Bourmeyster N, Boquet P, Vignais P:
1659
ADP-ribosylation of a small size GTP-binding protein in bovine
neutrophils by the C3 exoenzyme of Clostridium botulinum and
effect on the cell motility. Biochem Biophys Res Commun 180:615,
1991
140. Takaishi K, Kikuchi A, Kuroda S, Kotani K, Sasaki T, Takai
Y: Involvement of rho p21 and its inhibitory GDPIGTP exchange
protein (rho GDI) in cell motility. Mol Cell Biol 13:72, 1993
141. Hinsch K-D, Habermann B, Just I, Hinsch E, Pfisterer S,
Schill W-B, Aktories K: ADP-ribosylation of Rho proteins inhibits
sperm motility. FEBS Lett 334:32, 1993
142. Tominaga T, Sugie K, Morii N, Fukata J, Uchida A, Imura
H, Narumiya S: Inhibition of PMA-induced, LFA-l-dependent lymphocyte aggregation by ADP ribosylation ofthe small molecular
weight GTP binding protein, rho. J Cell Biol 120:1529, 1993
143. Morii N, Teru-uchi T, Tominaga T, Kumagai N, Kozak S,
Ushikubi F, Narumiya S: A rho Gene product in human blood platelets. J Biol Chem 267:20921, 1992
144, Malcolm KC, Ross AH, Qiu RG, Symons M,ExtonJH:
Activation of rat liver phospholipase D by the small GTP-binding
protein RhoA. J Biol Chem 269:25951, 1994
145. BowmanEP, Uhlinger DJ, Lambeth JD: Neutrophil phospholipase D is activated by a membrane-associated Rho family small
molecular weight GTP-binding protein. J Biol Chem 268:21509,
1993
146. Singer WD, Brown HA, Bokoch GM, Sternweis PC:Resolved phospholipase D activity is modulated by cytosolic factors
other than Arf. J Biol Chem 270:14944, 1995
147. Bokoch GM: Regulation of the human neutrophil NADPH
oxidase by the Rac GTP-binding proteins. Curr Opin Cell Biol6:212,
1994
148. Abo A, Pick E, Hall A, Totty N, Teahan CG, Segal AW:
Activation of the NADPH oxidase involves the small GTP-binding
protein ~21'""'.Nature 353:668, 1991
149. Knaus UG, Heyworth PG, Evans T, Curnutte JT, Bokoch
GM: Regulation of phagocyte oxygen radical production by the
GTP-binding protein Rac2. Science 254: 1512, 1991
150. Abo A, Boyhon A, West I,Thrasher AJ, Segal AW: Reconstitution of neutrophil NADPH oxidase activity in the cell-free system
by four components: p67-phox, 47-phox, p21 racl and cytochrome
b.245.J Biol Chem 267:16767, 1992
151. Heyworth PG, Knaus UG, Xu X, Uhlinger DJ, Conroy L,
Bokoch GM, Curnutte J T Requirement for post-translational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase. Mol Biol Cell 4:261, 1993
152. Mizuno T, Kaibuchi K, Ando S, Musha T, Hiraoka K, Takaishi K, Asada M, Nunoi H, Matsuda 1, Takai Y: Regulation of
the superoxide-generating NADPH oxidase by a small GTP-binding
protein and its stimulating and inhibitory GDP/GTP exchange proteins. J Biol Chem 267:10215, 1992
153. Dorseuil 0, Vazquez A, Lang P, Bertoglio J, Gacon G , Leca
G: Inhibition of superoxide production in B lymphocytes by rac
antisense oligonucleotides. J Biol Chem 267:20540, 1992
154. Voncken JW, van Schaick H, Kaartinen V, Deemer K, Coates
T, Landing B, Pattengale P, Dorseuil 0 , Bokoch GM, Groffen J,
Heisterkamp N: Increased neutrophil respiratory burst in BCR null
mutants. Cell 80:719, 1995
155. Quinn MT, Evans T, Loetterle LR, Jesaitis AJ, Bokoch GM:
Translocation ofRac correlates withNADPH oxidase activation:
Evidence for equimolar translocation of oxidase components. J Biol
Chem 268:20983, 1993
156. Abo A, Webb MR, Grogan A, Segal AW: Activation of
NADPH oxidase involves the dissociation of p21"' from its inhibitory GDPIGTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane. Biochem J 298:585, 1994
157. El Benna J, Ruedi JM, Babior BM: Cytosolic guanine nucleo-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1660
tide-binding protein Rac2 operates in vivo as a component of the
neutrophil respiratory burst oxidase. J Biol Chem 269:6729, 1994
158. Bokoch GM, Bohl BP, Chuang TH: Guanine nucleotide
exchange regulates membrane translocation of Racmho GTP-binding proteins. J Biol Chem 269:31674, 1994
159. Dorseuil 0, Quinn MT. Bokoch GM: Tyrosine kinase inhibitors dissociate membrane translocation of Rac from the cytosolic
oxidase components, p47phox and p67phox. J Leuk Biol 1995 (in
press)
160. Nemeroff RA, Winitz S, Qian N-X, Van Putten V, Johnson
GL, Heasley LE: Phosphorylation and activation of a high molecular
weight form of phospholipase A2 by p42 microtubule-associated
protein 2 kinase and protein kinase C. J Biol Chem 268:1960, 1993
161. Qui Z-H, de Carvalho MS, Leslie CC: Regulation of phospholipase A2activation by phosphorylation in mouse peritoneal macrophages. J Biol Chem 268:24506, 1993
162. Xing M, Mattera R Phosphorylation-dependent regulation
of phospholipage A2 by G-proteins and CaZi in HL60 granulocytes.
J Biol Chem 267:25966, 1992
163. Xing M, Wilkins PL, McConnell BK, Mattera R: Regulation
of phospholipase A2 activity in undifferentiated and neutrophil-like
HL60 Cells. J Biol Chem 269:3117, 1994
164. Bokoch GM: Biology of the Rap proteins, members of the
m s superfamily of GTP-binding proteins. Biochem J 289:17, 1993
GARY M. BOKOCH
165. Quinn MT, Mullen ML, Jesaitis AJ, Linner JG: Subcellular
distribution of the RaplA protein in human neutrophils. Colocalization and cotranslocation with cytochrome b(558). Blood 79:1563,
1992
166. Maridonneau-Panni I, de Gunzburg J: Association of rap1
and rap2 proteins with the specific granules of human neutrophils.
J Biol Chem 267:6396, 1992
167. Quinn MT, Parkos CA, Walker L, Orkin SH, Dinauer MC,
Jesaitis AJ: Association of Ras-related protein with cytochrome b
of human neutrophils. Nature 342:198, 1989
168. Bokoch GM, Quilliam LA, Bohl BP, Jesaitis AJ, Quinn MT:
Inhibition of binding of RaplA to cytochrome b558 of the NADPH
oxidase system by phosphorylation. Science 254:1794, 1991
169. Eklund EA, Marshall M, Gibbs JB, Crean CD, Gabig TG:
Resolution of a low molecular weight G protein in neutrophil cytosol
required for NADPH oxidase activation and reconstitution by recombinant Krev-l protein. J Biol Chem 266:13964, 1991
170. Maly FE, Quilliam LA, Dorseuil 0, Der CJ, Bokoch GM:
Activated or dominant inhibitory mutants of RaplA decrease the
oxidative burst of Epstein-Barr virus-transformed human B lymphocytes. J Biol Chem 269:18743, 1994
171. Quilliam LA, Mueller H, Bohl BP, Prossnitz V, Sklar LA,
Der CJ, Bokoch GM: RaplA is a substrate for CAMP-dependent
protein kinase in human neutrophils. J Immunol 147:1628, 1991
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1995 86: 1649-1660
Chemoattractant signaling and leukocyte activation
GM Bokoch
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