Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA
* Author for correspondence (e-mail: kdemali{at}med.unc.edu)
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Summary |
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Key words: Adhesion, Protrusion, Actin polymerization, Phagocytosis
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Introduction |
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Protrusion driven by actin polymerization
The driving force for the formation and extension of membrane protrusions
is the polymerization of actin. The leading edge of a lamellipodium is rich in
actin filaments arranged in a highly branched and dendritic network with the
barbed ends of the filaments concentrated close to the plasma membrane where
they are primed for the addition of actin monomers
(Svitkina and Borisy, 1999;
Svitkina et al., 1997
). A
dendritic model for lamellipodial extension has been proposed. In this model,
the entire actin filament network treadmills, growing at the filament barbed
ends and later shrinking at the lamellipodial base
(Svitkina and Borisy,
1999
).
How these actin filaments assemble in this highly branched dendritic
network in the seconds required for membrane protrusion to occur has been
extensively studied. The Arp2/3 complex has been identified as a potent
nucleator of actin polymerization, which initiates the formation of actin
filaments in lamellipodia of moving cells
(Machesky et al., 1994;
Mullins et al., 1998
;
Svitkina and Borisy, 1999
).
The Arp2/3 complex comprises seven subunits and is assembled
post-translationally (Welch and
Mitchison, 1998
). It is highly concentrated at the leading edge of
migrating cells (Bailly et al.,
2001
; Machesky et al.,
1997
; Mullins et al.,
1998
; Svitkina and Borisy,
1999
; Welch et al.,
1997
), where it undergoes a conformational change to provide a
template for the polymerization of actin filaments
(Robinson et al., 2001
;
Volkmann et al., 2001
). Actin
subunits then add to the new free barbed end, and the membrane is pushed
outward.
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Adhesion-stimulated protrusion |
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The Rho family of GTPases
Rac and Cdc42
How might adhesion stimulate protrusion? Adhesion to an extracellular
matrix, engagement of integrins and cell-cell adhesion all activate members of
the Rho family of GTPases (Clark et al.,
1998; Cox et al.,
2001
; Del Pozo et al.,
2000
; Kim et al.,
2000b
; Nakagawa et al.,
2001
; Noren et al.,
2001
; Price et al.,
1998
). Two of these family members, Cdc42 and Rac, stimulate the
formation of protrusions at the leading edge: Rac controls extension of
lamellipodia and Cdc42 controls extension of filopodia. Members of the WASP
family of proteins, including WASP and its ubiquitously expressed homolog
N-WASP bind to active Cdc42 through its GBD/CRIB (GTPase-binding domain/Cdc42
and Rac interactive binding) motif. Binding of WASP/N-WASP to Cdc42 relieves
an intramolecular interaction between the C-terminal VCA (verprolin and
cofilin acidic) domain and the GBD/CRIB motif
(Kim et al., 2000a
). This
unmasks the VCA domain, allowing it to bind and activate the Arp2/3 complex
(Fig. 1). In contrast to Cdc42,
Rac triggers activation of the Arp2/3 complex through a relative of WASP,
WAVE/Scar (Machesky and Insall,
1998
; Miki et al.,
1998
). WAVE does not have a GBD/CRIB motif, and direct binding of
WAVE to Rac has not been detected (Miki et
al., 1998
). Two alternative mechanisms for how Rac activates WAVE
have been described. Miki and colleagues provided evidence that IRSp53 acts as
an adaptor protein that links WAVE-2 to active Rac
(Miki et al., 2000
). However,
other work indicates that IRSp53 binds to Cdc42 rather than Rac
(Krugmann et al., 2001
).
WAVE-1 exists in a heterotetrameric complex that includes orthologues of human
p53-inducible messenger RNA (PIR121), a NCK-associated protein (Nap125) and
HSPC300 (Fig. 1). Active Rac
and NCK disrupt this WAVE complex, causing it to disassemble and release the
active WAVE protein in association with HSPC300
(Eden et al., 2002
). Active
WAVE binds to and activates the Arp2/3 complex, providing the driving force
for membrane protrusion at sites of integrin engagement.
|
Rho activity
In many cells, ß1 integrin engagement initially results in a decrease
in Rho activity (Arthur et al.,
2000; O'Connor et al.,
2000
; Ren et al.,
1999
). This decrease involves Src, FAK and the activation of
p190RhoGAP (Arthur et al.,
2000
; Ren et al.,
2000
). The transient dip in RhoA activity may contribute to
membrane extension, because, in many situations, high RhoA activity appears to
antagonize protrusion (Arthur and Burridge,
2001
; Cox et al.,
2001
; Kozma et al.,
1997
; Nobes and Hall,
1999
; Rottner et al.,
1999
). Inhibition of the Rho effector Rho kinase (ROCK) stimulates
cell migration (Nobes and Hall,
1999
) and promotes membrane protrusion
(Rottner et al., 1999
;
Tsuji et al., 2002
;
Worthylake and Burridge,
2003
), which lead to the idea that high Rho activity antagonizes
membrane extension through ROCK. Because ROCK activity stimulates myosin
contractility, there has been a presumption that the antagonism of membrane
protrusion by Rho is due to excessive contractility. However, other pathways
may also be important. In addition to stimulating myosin activity, ROCK acts
on and stimulates LIM kinase, which phosphorylates and inhibits cofilin
(Maekawa et al., 1999
). Active
cofilin severs and depolymerizes actin filaments (reviewed in
Bamburg, 1999
). Inhibiting
cofilin blocks growth-factor-induced actin polymerization and the extension of
lamellipodia (Chan et al.,
2000
; Zebda et al.,
2000
). These results have been interpreted to be caused by active
cofilin severing actin filaments, the resulting free barbed ends promoting
polymerization, and the newly polymerized filaments recruiting the Arp2/3
complex (Condeelis, 2001
). The
Arp2/3 complex bound to the sides of the newly polymerized filaments will
nucleate more filaments and give rise to the branched dendritic organization
observed at the leading edge of cells. Consequently, the inhibition of cofilin
by RhoA, via ROCK and LIM kinase, will tend to stabilize actin filaments and
inhibit cofilin's role in promoting nucleation of actin polymerization.
Expression of constitutively active cofilin results in inappropriate
lamellipodial extensions (Worthylake and
Burridge, 2003
).
The conclusion that Rho universally antagonizes membrane extension, while
appealing, is too simplistic. There are multiple examples, particularly in the
case of epithelial cells, in which the converse has been observed. For
example, Rho activity was shown to be critical in membrane ruffling induced by
PMA (Nishiyama et al., 1994).
Subsequent work confirmed the role of Rho and ROCK in PMA-induced membrane
ruffling and identified adducin as a relevant target in the cell cortex
(Fukata et al., 1999
). Working
with clone A colon carcinoma cells, O'Connor and colleagues provided evidence
that RhoA promotes lamellipodial extension and noted that dominant-negative
Rac constructs do not affect membrane extension in this system
(O'Connor et al., 2000
). A
different type of apical membrane protrusion is induced by RhoA activation in
NIH 3T3 fibroblasts (Shaw et al.,
1998
). This extension correlated with phosphorylation of ERM
proteins that were recruited to these projections
(Shaw et al., 1998
). That Rho
has been associated both with the generation of protrusive structures and with
their inhibition indicates a complexity that is not yet understood. Some of
this complexity may derive from different signaling pathways operating in
different cell types, but other factors are also likely to be involved and
merit further investigation.
Adhesion-mediated signaling
Numerous signaling pathways are initiated downstream of cell-matrix and
cell-cell adhesion (reviewed in Juliano,
2002) (Yap and Kovacs,
2003
; Zamir and Geiger,
2001a
; Zamir and Geiger,
2001b
). Some of these pathways affect the activity of Rho family
proteins and cell migration. One of the best characterized is activation of
focal adhesion kinase (FAK) (reviewed in
Schaller, 2001
). FAK
activation initiates multiple other signaling cascades. At least two of the
tyrosine phosphorylated proteins associated with FAK, p130cas (Cas) and
paxillin, have been linked to activation of Rac and therefore to lamellipodial
extension. Tyrosine phosphorylated Cas assembles a complex with the adaptor
protein Crk (Cary et al.,
1998
; Klemke et al.,
1998
; Vuori et al.,
1996
), which associates with DOCK180
(Hasegawa et al., 1996
;
Matsuda et al., 1996
). DOCK180
acts as a guanine nucleotide exchange factor (GEF) for Rac, even though it
lacks the DH/PH domain signature motif of most Rho GEFs
(Brugnera et al., 2002
;
Cote and Vuori, 2002
).
Paxillin also forms a multi-component complex with the proteins PKL and PIX,
the latter being another Rac GEF (Bagrodia
et al., 1998
; Manser et al.,
1998
; Turner et al.,
1999
). Assembly and activation of these two signaling complexes
depends on the tyrosine phosphorylation of FAK, Cas and paxillin. Not
surprisingly, therefore, protein tyrosine phosphatases have key regulatory
roles. Deletion of the phosphatase PTP-PEST results in increased spreading and
membrane protrusion (Angers-Loustau et al.,
1999
), and both Cas and paxillin are substrates for PTP-PEST
(Garton et al., 1996
;
Shen et al., 1998
). Moreover,
PTP-PEST regulates the level of active Rac
(Sastry et al., 2002
).
Deletion of the phosphatase PTP
produces a decrease in cell migration
and membrane protrusion (Zeng et al.,
2003
). This tyrosine phosphatase appears to be acting further
upstream in the pathway, such that cells lacking PTP
have decreased Src
family kinase activity and diminished FAK tyrosine phosphorylation and
activation (Zeng et al.,
2003
). Decreasing the activation of FAK would be predicted to
suppress the downstream activation of both Cas and paxillin and result in
depressed Rac activity. Cell adhesion thus triggers numerous signaling
pathways that may stimulate membrane protrusion.
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Physical coupling between cell adhesion molecules and the protrusive machinery |
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|
In addition to vinculin, two proteins have been implicated in linking the
actin polymerization machinery to integrins: N-WASP and cortactin. The
ß1-integrin subunit can be co-immunoprecipitated with N-WASP
(Sturge et al., 2002). It will
be interesting to learn whether this association stimulates the nucleation
activity of the Arp2/3 complex and the role this has in integrin-mediated
events. Cortactin, a c-Src substrate that binds to the Arp2/3 complex and
stimulates its ability to nucleate actin polymerization, represents another
potential link between integrins and the Arp2/3 complex
(Uruno et al., 2001
;
Weed et al., 2000
). Cortactin
becomes tyrosine phosphorylated in response to integrin-mediated adhesion,
which suggests a close association with integrins, but it is not seen in focal
adhesions and its mode of linkage to integrins remains to be determined
(Vuori and Ruoslahti,
1995
).
An intriguing link between adhesion and protrusion was suggested by recent
work on myosin X. This unconventional myosin is found at the tips of filopodia
and in phagocytic cups (Berg and Cheney,
2002; Cox et al.,
2002
). Overexpression of this myosin promotes formation of
filopodia, whereas expression of truncated forms inhibits phagocytosis. Both
results suggest a role for myosin X in generating membrane protrusions.
Interestingly, this myosin possesses three PH domains, one of which binds to
phosphatidylinositol 3,4,5-trisphosphate [Ptd(3,4,5)P3],
as well as a FERM domain. The latter have been implicated in binding to the
cytoplasmic domains of membrane proteins. Little is known about the components
of filopodial tips, but ß1 integrins and Mena have been identified at
this site (Grabham et al.,
2000
; Lanier et al.,
1999
), raising the possibility that myosin X may function together
with these proteins, thereby coupling integrin-mediated adhesion to extension
of the filopodial membrane.
Direct linkages between the actin polymerization machinery and adhesion
molecules are also emerging in the context of cell-cell adhesion. E-cadherin
colocalizes and co-immunoprecipitates with the Arp2/3 complex
(Kovacs et al., 2002). This
interaction localizes actin polymerization to sites of cadherin engagement.
Although more work is needed to determine the effect of perturbing this
interaction, these findings represent another example of a physical link
between membrane protrusion and cell adhesion. Interestingly, vinculin, which
acts as a link between integrins and the Arp2/3 complex, is also present at
sites of cadherin-mediated adhesion and may represent another mechanism by
which the Arp2/3 complex is localized to sites of cadherin engagement.
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Lessons from phagocytosis |
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Phagocytosis provides a means by which cells clear particulate material and
pathogens from their immediate vicinity. However, it is also a route of entry
into cells for many intracellular bacterial pathogens. The entry of these
bacteria into host cells typically exploits cell adhesion molecules on the
surface of the cells and is accompanied by phagocytosis either by the trigger
or zippering mechanisms. Listeria monocytogenes adheres to host cells
via the binding of a bacterial surface protein internalin (InlA) to E-cadherin
(Fig. 3) (reviewed in
Braun and Cossart, 2000)
(Dramsi and Cossart, 1998
).
This induces phagocytosis via the zippering mechanism. Uptake involves actin
polymerization as demonstrated by its inhibition with cytochalasin D
(Gaillard et al., 1987
). It
also requires tyrosine phosphorylation and activation of PI3 kinase
(Ireton et al., 1996
).
Yersinia pseudotuberculosis exploits a different adhesive interaction
to enter cells. This bacterium expresses a protein, invasin, on its surface
that binds with high affinity to ß1 integrins
(Cornelis, 2002
;
Isberg et al., 2000
).
Yersinia uptake requires Rac1 and the Arp2/3 complex, but some
disagreement exists as to the role of N-WASP in this process
(Alrutz et al., 2001
;
McGee et al., 2001
;
Wiedemann et al., 2001
).
|
Association of enteropathogenic Escherichia coli (EPEC) with cells
is a special example because it is not internalized but bound to a host cell
protrusion known as a pedestal. Attachment to the pedestal is mediated via the
translocated intimin receptor (TIR), a receptor secreted by EPEC, and inserted
into the host cell plasma membrane, where it acts as a receptor for intimin
expressed on the bacterial surface (Fig.
3) (Kenny et al.,
1997). TIR shares a number of similarities with integrins in that
it clusters, stimulates tyrosine phosphorylation of effector proteins and
recruits several cytoskeletal proteins, including
-actinin, talin,
cortactin, ezrin, VASP, villin and fimbrin to the site of EPEC attachment
(Freeman et al., 2000
;
Huang et al., 2002
) (reviewed
in Goosney et al., 2001
).
Adhesion via TIR stimulates actin reorganization to form the pedestal
structure that can extend up to 10 µm beneath the pathogen
(Rosenshine et al., 1996
).
WASP and the Arp2/3 complex are recruited to the actin pedestal beneath the
bacterium (Kalman et al.,
1999
; Lommel et al.,
2001
). Interference of WASP with dominant-negative constructs or
cells lacking N-WASP prevents pedestal formation
(Kalman et al., 1999
;
Lommel et al., 2001
).
Interestingly, cortactin, which binds and activates the Arp2/3 complex, is
directly recruited to TIR, and dominant-negative mutants of cortactin block
F-actin accumulation beneath the attached bacteria
(Cantarelli et al., 2000
;
Cantarelli et al., 2002
).
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Adhesion and the inhibition of protrusion |
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In mature tissues, there is abundant adhesion either to other cells or to
the matrix, and yet membrane protrusive activity is usually suppressed. The
stimulation of membrane extension by adhesion appears to be largely the result
of new adhesions occurring. The time course of activation of Rac and Cdc42 in
response to adhesion to the ECM (Cox et
al., 2001; Del Pozo et al.,
2000
; Price et al.,
1998
) is consistent with this interpretation. An initial
stimulation that occurs during the first few minutes declines to a baseline
level over a period of hours. Similarly, the recruitment of the Arp2/3 complex
to vinculin is a transient phenomenon. In this case, it lasts only for a few
minutes following matrix adhesion and is then suppressed in mature adhesive
structures such as focal adhesions (DeMali
et al., 2002
). Hence, the timing of interactions as well as the
extracellular environment may dictate whether adhesion stimulates or inhibits
protrusion.
Some of the best-characterized examples of adhesion molecules either
inhibiting or stimulating membrane protrusive activity and cell migration
occur in the nervous system. Several families of adhesion molecules have been
identified that affect neurite outgrowth and growth cone guidance either in a
positive or in a negative way (Dickson,
2002). Space limitations prevent discussion of this topic, but it
is relevant that many of these receptors in nerve growth cones initiate
signals that regulate the activities of Rho family proteins (reviewed in
Giniger, 2002
).
Just as lessons can be learned from bacteria that trigger phagocytosis
through their adhesion to host cells, so too information can be acquired from
bacteria that inhibit phagocytosis to promote their survival. Intriguing
examples involve the enteropathic bacteria Yersinia
pseudotuberculosis and Y. enterocolitica. Within the host
intestine, these bacteria initially promote their own phagocytosis by M cells
in the intestinal epithelium. Entry into these cells provides their passage
out of the intestinal lumen and into the body. These bacteria then adhere to
immune cells within Peyer's patches but inhibit phagocytosis that otherwise
would lead to their destruction in phagolysosomes. As mentioned above, the
Yersinia bacteria use adhesins (as well as other bacterial surface
proteins) to adhere very strongly to ß1 integrins on host cells (reviewed
in Isberg and Barnes, 2001).
The tightly adhering Yersinia use a protein delivery system to inject
into the host cell a series of proteins that function to inhibit both
phagocytosis and the development of the host's immune response. Three of the
proteins (Yops) delivered by Yersinia affect, directly or indirectly,
the Rho family GTPases. YopE is a RhoGAP that acts on RhoA, Rac and Cdc42 to
promote their hydrolysis of GTP and consequent inactivation
(Black and Bliska, 2000
;
Von Pawel-Rammingen et al.,
2000
). YopT is a protease that cleaves RhoA, Rac and Cdc42 close
to their C-terminal prenylation site (Shao
et al., 2003
). Their prenyl groups link these small G-proteins to
cell membranes and so this cleavage will release them from their sites of
action. YopH is a potent tyrosine phosphatase that inhibits phagocytosis
(Rosqvist et al., 1988
;
Zhang et al., 1992
).
Introduction of this phosphatase into the cells disrupts the organization of
the actin cytoskeleton (Schneider et al.,
1998
), and this phosphatase targets many of the tyrosine
phosphorylated proteins in focal adhesions such as FAK and p130cas
(Black and Bliska, 1997
;
Persson et al., 1997
). Given
that FAK and p130cas signal downstream to Rac activation, this suggests at
least one way by which this tyrosine phosphatase will depress Rac activity. An
additional Yop protein, YopO (also known as YpkA, for Yersinia
protein kinase A) is a serine-threonine protein kinase that binds RhoA, Rac
and actin (Dukuzumuremyi et al.,
2000
; Galyov et al.,
1993
; Juris et al.,
2000
). This probably also contributes to the inhibition of
phagocytosis, but the mechanism is not fully understood. The work on how
Yersinia inhibits phagocytosis emphasizes once again the critical
role Rho GTPases play in regulating membrane protrusion in response to
adhesion.
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Conclusions |
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Acknowledgments |
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References |
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---|
Abercrombie, M. (1967). Contact inhibition: the phenomenon and its biological implications. Natl. Cancer Inst. Monogr. 26,249 -277.[Medline]
Aderem, A. and Underhill, D. M. (1999). Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17,593 -623.[CrossRef][Medline]
Alrutz, M. A., Srivastava, A., Wong, K. W., D'Souza-Schorey, C., Tang, M., Ch'Ng, L. E., Snapper, S. B. and Isberg, R. R. (2001). Efficient uptake of Yersinia pseudotuberculosis via integrin receptors involves a Rac1-Arp 2/3 pathway that bypasses N-WASP function. Mol. Microbiol. 42,689 -703.[CrossRef][Medline]
Angers-Loustau, A., Cote, J. F., Charest, A., Dowbenko, D.,
Spencer, S., Lasky, L. A. and Tremblay, M. L. (1999). Protein
tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and
cytokinesis in fibroblasts. J. Cell Biol.
144,1019
-1031.
Arthur, W. T. and Burridge, K. (2001). RhoA
inactivation by p190RhoGAP regulates cell spreading and migration by promoting
membrane protrusion and polarity. Mol. Biol. Cell
12,2711
-2720.
Arthur, W. T., Petch, L. A. and Burridge, K. (2000). Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism. Curr. Biol. 10,719 -722.[CrossRef][Medline]
Aznavoorian, S., Stracke, M. L., Parsons, J., McClanahan, J. and
Liotta, L. A. (1996). Integrin alphavbeta3 mediates
chemotactic and haptotactic motility in human melanoma cells through different
signaling pathways. J. Biol. Chem.
271,3247
-3254.
Bagrodia, S., Taylor, S. J., Jordon, K. A., van Aelst, L. and
Cerione, R. A. (1998). A novel regulator of p21-activated
kinases. J. Biol. Chem.
273,23633
-23636.
Bailly, M., Ichetovkin, I., Grant, W., Zebda, N., Machesky, L. M., Segall, J. E. and Condeelis, J. (2001). The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension. Curr. Biol. 11,620 -625.[CrossRef][Medline]
Bamburg, J. R. (1999). Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu. Rev. Cell Dev. Biol. 15,185 -230.[CrossRef][Medline]
Barry, O. P., Pratico, D., Savani, R. C. and FitzGerald, G.
A. (1998). Modulation of monocyte-endothelial cell
interactions by platelet microparticles. J. Clin.
Invest. 102,136
-144.
Berg, J. S. and Cheney, R. E. (2002). Myosin-X is an unconventional myosin that undergoes intrafilopodial motility. Nat. Cell Biol. 4,246 -250.[CrossRef][Medline]
Black, D. S. and Bliska, J. B. (1997).
Identification of p130Cas as a substrate of Yersinia YopH (Yop51), a bacterial
protein tyrosine phosphatase that translocates into mammalian cells and
targets focal adhesions. EMBO J.
16,2730
-2744.
Black, D. S. and Bliska, J. B. (2000). The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence. Mol. Microbiol. 37,515 -527.[CrossRef][Medline]
Braun, L. and Cossart, P. (2000). Interactions between Listeria monocytogenes and host mammalian cells. Microbes Infect. 2,803 -811.[CrossRef][Medline]
Bray, D. (1992). Migration of cells over surfaces. In Cell Movements. pp.17 -29. New York: Garland Publishing Co.
Brugnera, E., Haney, L., Grimsley, C., Lu, M., Walk, S. F., Tosello-Trampont, A. C., Macara, I. G., Madhani, H., Fink, G. R. and Ravichandran, K. S. (2002). Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex. Nat. Cell Biol. 4,574 -582.[Medline]
Cantarelli, V. V., Takahashi, A., Akeda, Y., Nagayama, K. and
Honda, T. (2000). Interaction of enteropathogenic or
enterohemorrhagic Escherichia coli with HeLa cells results in
translocation of cortactin to the bacterial adherence site. Infect.
Immun. 68,382
-386.
Cantarelli, V. V., Takahashi, A., Yanagihara, I., Akeda, Y.,
Imura, K., Kodama, T., Kono, G., Sato, Y., Iida, T. and Honda, T.
(2002). Cortactin is necessary for F-actin accumulation in
pedestal structures induced by enteropathogenic Escherichia coli
infection. Infect. Immun.
70,2206
-2209.
Caron, E. and Hall, A. (1998). Identification
of two distinct mechanisms of phagocytosis controlled by different Rho
GTPases. Science 282,1717
-1721.
Cary, L. A., Han, D. C., Polte, T. R., Hanks, S. K. and Guan, J.
L. (1998). Identification of p130Cas as a mediator of focal
adhesion kinase-promoted cell migration. J. Cell Biol.
140,211
-221.
Chan, A. Y., Bailly, M., Zebda, N., Segall, J. E. and Condeelis,
J. S. (2000). Role of cofilin in epidermal growth
factor-stimulated actin polymerization and lamellipod protrusion.
J. Cell Biol. 148,531
-542.
Clark, E. A., King, W. G., Brugge, J. S., Symons, M. and Hynes,
R. O. (1998). Integrin-mediated signals regulated by members
of the rho family of GTPases. J. Cell Biol.
142,573
-586.
Condeelis, J. (1998). The Biochemistry of Animal Cell Crawling. In Motion Analysis of Living Cells, pp. 85-100. New York: Wiley-Liss.
Condeelis, J. (2001). How is actin polymerization nucleated in vivo? Trends Cell Biol. 11,288 -293.[CrossRef][Medline]
Cornelis, G. R. (2002). Yersinia type III
secretion: send in the effectors. J. Cell Biol.
158,401
-408.
Cote, J. F. and Vuori, K. (2002). Identification of an evolutionarily conserved superfamily of DOCK180-related proteins with guanine nucleotide exchange activity. J. Cell Sci. 115,4901 -4913.[CrossRef][Medline]
Cox, D., Berg, J. S., Cammer, M., Chinegwundoh, J. O., Dale, B. M., Cheney, R. E. and Greenberg, S. (2002). Myosin X is a downstream effector of PI(3)K during phagocytosis. Nat. Cell Biol. 4,469 -477.[Medline]
Cox, E. A., Sastry, S. K. and Huttenlocher, A.
(2001). Integrin-mediated adhesion regulates cell polarity and
membrane protrusion through the Rho family of GTPases. Mol. Biol.
Cell 12,265
-277.
Del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D. and
Schwartz, M. A. (2000). Adhesion to the extracellular matrix
regulates the coupling of the small GTPase Rac to its effector PAK.
EMBO J. 19,2008
-2014.
DeMali, K. A., Barlow, C. A. and Burridge, K.
(2002). Recruitment of the Arp2/3 complex to vinculin: coupling
membrane protrusion to matrix adhesion. J. Cell Biol.
159,881
-891.
Dickson, B. J. (2002). Molecular mechanisms of
axon guidance. Science
298,1959
-1964.
Dramsi, S. and Cossart, P. (1998). Intracellular pathogens and the actin cytoskeleton. Annu. Rev. Cell Dev. Biol. 14,137 -166.[CrossRef][Medline]
Dukuzumuremyi, J. M., Rosqvist, R., Hallberg, B., Akerstrom, B.,
Wolf-Watz, H. and Schesser, K. (2000). The Yersinia protein
kinase A is a host factor inducible RhoA/Rac-binding virulence factor.
J. Biol. Chem. 275,35281
-35290.
Eden, S., Rohatgi, R., Podtelejnikov, A. V., Mann, M. and Kirschner, M. W. (2002). Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418,790 -793.[CrossRef][Medline]
Freeman, N. L., Zurawski, D. V., Chowrashi, P., Ayoob, J. C., Huang, L., Mittal, B., Sanger, J. M. and Sanger, J. W. (2000). Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins. Cell Motil. Cytoskeleton 47,307 -318.[CrossRef][Medline]
Fukata, Y., Oshiro, N., Kinoshita, N., Kawano, Y., Matsuoka, Y.,
Bennett, V., Matsuura, Y. and Kaibuchi, K. (1999).
Phosphorylation of adducin by Rho-kinase plays a crucial role in cell
motility. J. Cell Biol.
145,347
-361.
Gaillard, J. L., Berche, P., Mounier, J., Richard, S. and Sansonetti, P. (1987). In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect. Immun. 55,2822 -2829.[Medline]
Galyov, E. E., Hakansson, S., Forsberg, A. and Wolf-Watz, H. (1993). A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature 361,730 -732.[CrossRef][Medline]
Garton, A. J., Flint, A. J. and Tonks, N. K. (1996). Identification of p130(cas) as a substrate for the cytosolic protein tyrosine phosphatase PTP-PEST. Mol. Cell Biol. 16,6408 -6418.[Abstract]
Giniger, E. (2002). How do Rho family GTPases direct axon growth and guidance? A proposal relating signaling pathways to growth cone mechanics. Differentiation 70,385 -396.[CrossRef][Medline]
Goosney, D. L., DeVinney, R. and Finlay, B. B.
(2001). Recruitment of cytoskeletal and signaling proteins to
enteropathogenic and enterohemorrhagic Escherichia coli pedestals.
Infect. Immun. 69,3315
-3322.
Grabham, P. W., Foley, M., Umeojiako, A. and Goldberg, D. J.
(2000). Nerve growth factor stimulates coupling of beta1 integrin
to distinct transport mechanisms in the filopodia of growth cones.
J. Cell Sci. 113,3003
-3012.
Hasegawa, H., Kiyokawa, E., Tanaka, S., Nagashima, K., Gotoh, N., Shibuya, M., Kurata, T. and Matsuda, M. (1996). DOCK180, a major CRK-binding protein, alters cell morphology upon translocation to the cell membrane. Mol. Cell. Biol. 16,1770 -1776.[Abstract]
Huang, L., Mittal, B., Sanger, J. W. and Sanger, J. M. (2002). Host focal adhesion protein domains that bind to the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). Cell Motil. Cytoskeleton 52,255 -265.[CrossRef][Medline]
Ireton, K., Payrastre, B., Chap, H., Ogawa, W., Sakaue, H.,
Kasuga, M. and Cossart, P. (1996). A role for
phosphoinositide 3-kinase in bacterial invasion.
Science 274,780
-782.
Isberg, R. R. and Barnes, P. (2001). Subversion
of integrins by enteropathogenic Yersinia. J. Cell
Sci. 114,21
-28.
Isberg, R. R., Hamburger, Z. and Dersch, P. (2000). Signaling and invasin-promoted uptake via integrin receptors. Microbes Infect. 2, 793-801.[CrossRef][Medline]
Juliano, R. L. (2002). Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annu. Rev. Pharmacol. Toxicol. 42,283 -323.[CrossRef][Medline]
Juris, S. J., Rudolph, A. E., Huddler, D., Orth, K. and Dixon,
J. E. (2000). A distinctive role for the Yersinia protein
kinase: actin binding, kinase activation, and cytoskeleton disruption.
Proc. Natl. Acad. Sci. USA
97,9431
-9436.
Kalman, D., Weiner, O. D., Goosney, D. L., Sedat, J. W., Finlay, B. B., Abo, A. and Bishop, J. M. (1999). Enteropathogenic E. coli acts through WASP and Arp2/3 complex to form actin pedestals. Nat. Cell Biol. 1,389 -391.[CrossRef][Medline]
Kenny, B., DeVinney, R., Stein, M., Reinscheid, D. J., Frey, E. A. and Finlay, B. B. (1997). Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell 91,511 -520.[Medline]
Kim, A. S., Kakalis, L. T., Abdul-Manan, N., Liu, G. A. and Rosen, M. K. (2000a). Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. Nature 404,151 -158.[CrossRef][Medline]
Kim, S. H., Li, Z. and Sacks, D. B. (2000b).
E-cadherin-mediated cell-cell attachment activates Cdc42. J. Biol.
Chem. 275,36999
-37005.
Klemke, R. L., Leng, J., Molander, R., Brooks, P. C., Vuori, K.
and Cheresh, D. A. (1998). CAS/Crk coupling serves as a
"molecular switch" for induction of cell migration. J.
Cell Biol. 140,961
-972.
Kovacs, E. M., Goodwin, M., Ali, R. G., Paterson, A. D. and Yap, A. S. (2002). Cadherin-directed actin assembly. E-cadherin physically associates with the arp2/3 complex to direct actin assembly in nascent adhesive contacts. Curr. Biol. 12,379 -382.[CrossRef][Medline]
Kozma, R., Sarner, S., Ahmed, S. and Lim, L. (1997). Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol. Cell. Biol. 17,1201 -1211.[Abstract]
Krugmann, S., Jordens, I., Gevaert, K., Driessens, M., Vandekerckhove, J. and Hall, A. (2001). Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex. Curr. Biol. 11,1645 -1655.[CrossRef][Medline]
Krukonis, E. S., Dersch, P., Eble, J. A. and Isberg, R. R.
(1998). Differential effects of integrin alpha chain mutations on
invasin and natural ligand interaction. J. Biol. Chem.
273,31837
-31843.
LaFlamme, S. E., Thomas, L. A., Yamada, S. S. and Yamada, K. M. (1994). Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly. J. Cell Biol. 126,1287 -1298.[Abstract]
Lanier, L. M., Gates, M. A., Witke, W., Menzies, A. S., Wehman, A. M., Macklis, J. D., Kwiatkowski, D., Soriano, P. and Gertler, F. B. (1999). Mena is required for neurulation and commissure formation. Neuron 22,313 -325.[Medline]
Leong, J. M., Fournier, R. S. and Isberg, R. R. (1990). Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein. EMBO J. 9,1979 -1989.[Abstract]
Lommel, S., Benesch, S., Rottner, K., Franz, T., Wehland, J. and
Kuhn, R. (2001). Actin pedestal formation by enteropathogenic
Escherichia coli and intracellular motility of Shigella flexneri are
abolished in N-WASP-defective cells. EMBO Rep.
2, 850-857.
Machesky, L. M. and Insall, R. H. (1998). Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Curr. Biol. 8,1347 -1356.[Medline]
Machesky, L. M., Atkinson, S. J., Ampe, C., Vandekerckhove, J. and Pollard, T. D. (1994). Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose. J. Cell Biol. 127,107 -115.[Abstract]
Machesky, L. M., Reeves, E., Wientjes, F., Mattheyse, F. J., Grogan, A., Totty, N. F., Burlingame, A. L., Hsuan, J. J. and Segal, A. W. (1997). Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins. Biochem. J. 105,105 -112.
Maekawa, M., Ishizaki, T., Boku, S., Watanabe, N., Fujita, A.,
Iwamatsu, A., Obinata, T., Ohashi, K., Mizuno, K. and Narumiya, S.
(1999). Signaling from Rho to the actin cytoskeleton through
protein kinases ROCK and LIM-kinase. Science
285,895
-898.
Manser, E., Loo, T. H., Koh, C. G., Zhao, Z. S., Chen, X. Q., Tan, L., Tan, I., Leung, T. and Lim, L. (1998). PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell 1,183 -192.[Medline]
Matsuda, M., Ota, S., Tanimura, R., Nakamura, H., Matuoka, K.,
Takenawa, T., Nagashima, K. and Kurata, T. (1996).
Interaction between the amino-terminal SH3 domain of CRK and its natural
target proteins. J. Biol. Chem.
271,14468
-14472.
May, R. C., Caron, E., Hall, A. and Machesky, L. M. (2000). Involvement of the Arp2/3 complex in phagocytosis mediated by FcgammaR or CR3. Nat. Cell Biol. 2, 246-248.[CrossRef][Medline]
McGee, K., Zettl, M., Way, M. and Fallman, M. (2001). A role for N-WASP in invasin-promoted internalisation. FEBS Lett. 509,59 -65.[CrossRef][Medline]
Mengaud, J., Ohayon, H., Gounon, P., Mege, R. M. and Cossart, P. (1996). E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell 84,923 -932.[Medline]
Miki, H., Suetsugu, S. and Takenawa, T. (1998).
WAVE, a novel WASP-family protein involved in actin reorganization induced by
Rac. EMBO J. 17,6932
-6941.
Miki, H., Yamaguchi, H., Suetsugu, S. and Takenawa, T. (2000). IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature 408,732 -735.[CrossRef][Medline]
Mullins, R. D., Heuser, J. A. and Pollard, T. D.
(1998). The interaction of Arp2/3 complex with actin: nucleation,
high affinity pointed end capping, and formation of branching networks of
filaments. Proc. Natl. Acad. Sci. USA
95,6181
-6186.
Nakagawa, M., Fukata, M., Yamaga, M., Itoh, N. and Kaibuchi,
K. (2001). Recruitment and activation of Rac1 by the
formation of E-cadherin-mediated cell-cell adhesion sites. J. Cell
Sci. 114,1829
-1838.
Nishiyama, T., Sasaki, T., Takaishi, K., Kato, M., Yaku, H., Araki, K., Matsuura, Y. and Takai, Y. (1994). rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells. Mol. Cell. Biol. 14,2447 -2456.[Abstract]
Nobes, C. D. and Hall, A. (1999). Rho GTPases
control polarity, protrusion, and adhesion during cell movement. J.
Cell Biol. 144,1235
-1244.
Noren, N. K., Niessen, C. M., Gumbiner, B. M. and Burridge,
K. (2001). Cadherin engagement regulates Rho family GTPases.
J. Biol. Chem. 276,33305
-33308.
O'Connor, K. L., Nguyen, B. K. and Mercurio, A. M.
(2000). RhoA function in lamellae formation and migration is
regulated by the alpha6beta4 integrin and cAMP metabolism. J. Cell
Biol. 148,253
-258.
Persson, C., Carballeira, N., Wolf-Watz, H. and Fallman, M.
(1997). The PTPase YopH inhibits uptake of Yersinia, tyrosine
phosphorylation of p130Cas and FAK, and the associated accumulation of these
proteins in peripheral focal adhesions. EMBO J.
16,2307
-2318.
Price, L. S., Leng, J., Schwartz, M. A. and Bokoch, G. M.
(1998). Activation of Rac and Cdc42 by integrins mediates cell
spreading. Mol. Biol. Cell
9,1863
-1871.
Ren, X. D., Kiosses, W. B. and Schwartz, M. A.
(1999). Regulation of the small GTP-binding protein Rho by cell
adhesion and the cytoskeleton. EMBO J.
18,578
-585.
Ren, X. D., Kiosses, W. B., Sieg, D. J., Otey, C. A., Schlaepfer, D. D. and Schwartz, M. A. (2000). Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover. J. Cell Sci. 113,1679 -1684.
Robinson, R. C., Turbedsky, K., Kaiser, D. A., Marchand, J. B.,
Higgs, H. N., Choe, S. and Pollard, T. D. (2001). Crystal
structure of Arp2/3 complex. Science
294,1679
-1684.
Rosenshine, I., Ruschkowski, S., Stein, M., Reinscheid, D. J., Mills, S. D. and Finlay, B. B. (1996). A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation. EMBO J. 15,2613 -2624.[Abstract]
Rosqvist, R., Bolin, I. and Wolf-Watz, H. (1988). Inhibition of phagocytosis in Yersinia pseudotuberculosis: a virulence plasmid-encoded ability involving the Yop2b protein. Infect. Immun. 56,2139 -2143.[Medline]
Rottner, K., Hall, A. and Small, J. V. (1999). Interplay between Rac and Rho in the control of substrate contact dynamics. Curr. Biol. 9,640 -648.[CrossRef][Medline]
Sastry, S. K., Lyons, P. D., Schaller, M. D. and Burridge,
K. (2002). PTP-PEST controls motility through regulation of
Rac1. J. Cell Sci. 115,4305
-4316.
Schaller, M. D. (2001). Biochemical signals and biological responses elicited by the focal adhesion kinase. Biochim. Biophys. Acta 1540, 1-21.[Medline]
Schneider, G. B., Gilmore, A. P., Lohse, D. L., Romer, L. H. and Burridge, K. (1998). Microinjection of protein tyrosine phosphatases into fibroblasts disrupts focal adhesions and stress fibers. Cell Adhes. Commun. 5,207 -219.[Medline]
Shao, F., Vacratsis, P. O., Bao, Z., Bowers, K. E., Fierke, C.
A. and Dixon, J. E. (2003). Biochemical characterization of
the Yersinia YopT protease: Cleavage site and recognition elements in Rho
GTPases. Proc. Natl. Acad. Sci. USA
100,904
-909.
Shaw, R. J., Henry, M., Solomon, F. and Jacks, T.
(1998). RhoA-dependent phosphorylation and relocalization of ERM
proteins into apical membrane/actin protrusions in fibroblasts.
Mol. Biol. Cell 9,403
-419.
Shen, Y., Schneider, G., Cloutier, J. F., Veillette, A. and
Schaller, M. D. (1998). Direct association of
protein-tyrosine phosphatase PTP-PEST with paxillin. J. Biol.
Chem. 273,6474
-6481.
Sturge, J., Hamelin, J. and Jones, G. E.
(2002). N-WASP activation by a beta1-integrin-dependent mechanism
supports PI3K-independent chemotaxis stimulated by urokinase-type plasminogen
activator. J. Cell Sci.
115,699
-711.
Svitkina, T. M. and Borisy, G. G. (1999).
Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic
organization and treadmilling of actin filament array in lamellipodia.
J. Cell Biol. 145,1009
-1026.
Svitkina, T. M., Verkhovsky, A. B., McQuade, K. M. and Borisy,
G. G. (1997). Analysis of the actin-myosin II system in fish
epidermal keratocytes: mechanism of cell body translocation. J.
Cell Biol. 139,397
-415.
Tsuji, T., Ishizaki, T., Okamoto, M., Higashida, C., Kimura, K.,
Furuyashiki, T., Arakawa, Y., Birge, R. B., Nakamoto, T., Hirai, H. et
al., (2002). ROCK and mDia1 antagonize in Rho-dependent Rac
activation in Swiss 3T3 fibroblasts. J. Cell Biol.
157,819
-830.
Turner, C. E., Brown, M. C., Perrotta, J. A., Riedy, M. C.,
Nikolopoulos, S. N., McDonald, A. R., Bagrodia, S., Thomas, S. and Leventhal,
P. S. (1999). Paxillin LD4 motif binds PAK and PIX through a
novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal
remodeling. J. Cell Biol.
145,851
-863.
Uruno, T., Liu, J., Zhang, P., Fan, Y., Egile, C., Li, R., Mueller, S. C. and Zhan, X. (2001). Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nat. Cell Biol. 3,259 -266.[CrossRef][Medline]
Volkmann, N., Amann, K. J., Stoilova-McPhie, S., Egile, C.,
Winter, D. C., Hazelwood, L., Heuser, J. E., Li, R., Pollard, T. D. and
Hanein, D. (2001). Structure of Arp2/3 complex in its
activated state and in actin filament branch junctions.
Science 293,2456
-2459.
Von Pawel-Rammingen, U., Telepnev, M. V., Schmidt, G., Aktories, K., Wolf-Watz, H. and Rosqvist, R. (2000). GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: a mechanism for disruption of actin microfilament structure. Mol. Microbiol. 36,737 -748.[CrossRef][Medline]
Vuori, K. and Ruoslahti, E. (1995). Tyrosine
phosphorylation of p130Cas and cortactin accompanies integrin-mediated cell
adhesion to extracellular matrix. J. Biol. Chem.
270,22259
-22262.
Vuori, K., Hirai, H., Aizawa, S. and Ruoslahti, E. (1996). Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases. Mol. Cell Biol. 16,2606 -2613.[Abstract]
Weed, S. A., Karginov, A. V., Schafer, D. A., Weaver, A. M.,
Kinley, A. W., Cooper, J. A. and Parsons, J. T. (2000).
Cortactin localization to sites of actin assembly in lamellipodia requires
interactions with F-actin and the Arp2/3 complex. J. Cell
Biol. 151,29
-40.
Welch, M. D. and Mitchison, T. J. (1998). Purification and assay of the platelet Arp2/3 complex. Methods Enzymol. 298,52 -61.[Medline]
Welch, M. D., DePace, A. H., Verma, S., Iwamatsu, A. and
Mitchison, T. J. (1997). The human Arp2/3 complex is composed
of evolutionarily conserved subunits and is localized to cellular regions of
dynamic actin filament assembly. J. Cell Biol.
138,375
-384.
Wiedemann, A., Linder, S., Grassl, G., Albert, M., Autenrieth, I. and Aepfelbacher, M. (2001). Yersinia enterocolitica invasin triggers phagocytosis via beta1 integrins, CDC42Hs and WASp in macrophages. Cell Microbiol. 3, 693-702.[CrossRef][Medline]
Worthylake, R. A. and Burridge, K. (2003). RhoA
and ROCK promote migration by limiting membrane protrusions. J.
Biol. Chem. 278,13578
-13584.
Yap, A. S. and Kovacs, E. M. (2003). Direct
cadherin-activated cell signaling: a view from the plasma membrane.
J. Cell Biol 160,11
-16.
Ylanne, J., Huuskonen, J., O'Toole, T. E., Ginsberg, M. H.,
Virtanen, I. and Gahmberg, C. G. (1995). Mutation of the
cytoplasmic domain of the integrin beta 3 subunit. Differential effects on
cell spreading, recruitment to adhesion plaques, endocytosis, and
phagocytosis. J. Biol. Chem.
270,9550
-9557.
Zamir, E. and Geiger, B. (2001a). Components of cell-matrix adhesions. J. Cell Sci. 114,3577 -3579.[Medline]
Zamir, E. and Geiger, B. (2001b). Molecular complexity and dynamics of cell-matrix adhesions. J. Cell Sci. 114,3583 -3590.[Medline]
Zebda, N., Bernard, O., Bailly, M., Welti, S., Lawrence, D. S.
and Condeelis, J. S. (2000). Phosphorylation of ADF/cofilin
abolishes EGF-induced actin nucleation at the leading edge and subsequent
lamellipod extension. J. Cell Biol.
151,1119
-1128.
Zeng, L., Si, X., Yu, W. P., Le, H. T., Ng, K. P., Teng, R. M., Ryan, K., Wang, D. Z., Ponniah, S. and Pallen, C. J. (2003). PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration. J. Cell Biol. 160,137 -146.[CrossRef][Medline]
Zhang, Z. Y., Clemens, J. C., Schubert, H. L., Stuckey, J. A.,
Fischer, M. W., Hume, D. M., Saper, M. A. and Dixon, J. E.
(1992). Expression, purification, and physicochemical
characterization of a recombinant Yersinia protein tyrosine phosphatase.
J. Biol. Chem. 267,23759
-23766.