From the Department of Cell Biology and Anatomy,
School of Medicine, the § Department of Prosthodontics,
School of Dentistry, and
Lineberger Comprehensive Cancer Center,
University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599 and ¶ McGill Cancer Centre, McGill
University, Montreal, Quebec H3G 1Y6, Canada
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ABSTRACT |
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Tyrosine phosphorylation of focal adhesion-associated proteins may be involved in the regulation of the cytoskeleton and in the control of signals for growth and survival. The focal adhesion kinase (FAK) functions in regulating tyrosine phosphorylation of several of these proteins, including paxillin, tensin, and p130cas. Protein- tyrosine phosphatases, the counterparts of protein-tyrosine kinases, also presumably regulate phosphorylation of these proteins. We have tested the hypothesis that FAK intimately associates with a protein-tyrosine phosphatase. Protein-tyrosine phosphatase activity associated with the recombinant C-terminal domain of FAK in vitro and could be coimmunoprecipitated with both FAK and paxillin from lysates of chicken embryo cells. However, the interaction with FAK appeared to be indirect and mediated via paxillin. The protein-tyrosine phosphatase was subsequently identified as protein-tyrosine phosphatase-PEST, a nonreceptor protein-tyrosine phosphatase. The C-terminal noncatalytic domain of protein-tyrosine phosphatase-PEST directly bound to paxillin in vitro. The association of both a protein-tyrosine kinase and a protein-tyrosine phosphatase with paxillin suggests that paxillin may play a critical role in the regulation of the phosphotyrosine content of proteins in focal adhesions.
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INTRODUCTION |
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The integrins were originally identified as cell surface receptors for extracellular matrix proteins. They participate in a variety of cellular events, including adhesion, migration, invasion, cytoskeleton organization, and the generation of cytoplasmic signals (1-3). Engagement of the integrins with the extracellular matrix results in the formation of protein complexes termed focal adhesions. In cultured fibroblasts, focal adhesions are specialized structures that link the actin cytoskeleton to extracellular matrix (4, 5). As a communication point between extracellular matrix and the inside of cells, focal adhesions perform both structural and signaling functions. One of the most important events after the engagement of integrins with their ligands is the rapid tyrosine phosphorylation of focal adhesion-associated proteins (2, 3, 6).
Tyrosine phosphorylation of focal adhesion-associated proteins may be controlled by the focal adhesion kinase (FAK),1 a nonreceptor tyrosine kinase that localizes principally in focal adhesions (2, 3, 6, 7). FAK is a major substrate for tyrosine phosphorylation following integrin-dependent cell adhesion and simultaneously becomes enzymatically activated (8-11). Autophosphorylation of FAK creates a binding site for Src family PTKs, and the assembly of this complex appears to be important for tyrosine phosphorylation of focal adhesion-associated proteins (12-14). p130cas and paxillin are, potentially, downstream components of FAK signaling. They are colocalized with FAK in focal adhesions, associate with FAK, and become tyrosine-phosphorylated in coordination with FAK (9, 15-20). Tyrosine phosphorylation of p130cas and paxillin by FAK and/or Src-like PTKs likely regulates the assembly of signaling complexes in focal adhesions (13, 21-23).
The results of a number of pharmacological experiments suggest that tyrosine phosphorylation may be involved in the regulation of focal adhesion and stress fiber formation (9, 24-27). Other evidence suggests that tyrosine phosphorylation may not contribute to the assembly of focal adhesions. Microinjection of a dominant-negative FAK protein ablated tyrosine phosphorylation in focal adhesions, yet the integrity of the focal adhesions was unaffected, and new focal adhesions could still form (28). Experiments using various strategies to perturb normal FAK signaling suggest that FAK controls cell spreading, the rate of focal adhesion formation, and cell migration (7, 28, 29). Recent evidence also suggests that FAK may function in the transmission of a cell adhesion-dependent signal that is required for cell survival (30, 31).
The phosphorylation of proteins on tyrosine can also be controlled by PTPases (32). The role of PTPases in the regulation of cytoskeletal structure and signaling via focal adhesions is only beginning to be addressed. Several candidate PTPases that may be involved in regulating tyrosine phosphorylation in focal adhesions have been identified; they include LAR, PTP1B, and PTP-PEST. LAR is a transmembrane PTPase expressed on the cell surface as two noncovalently associated subunits derived by cellular processing of a proprotein (33). LAR and an associated protein LIP.1 can be localized to focal adhesions in MCF7 cells (34). PTP1B and PTP-PEST are implicated in focal adhesion signaling because both associate with p130cas, a tyrosine-phosphorylated focal adhesion-associated protein. PTP1B directly associates with the SH3 domain of p130cas, and overexpression of PTP1B leads to decreased tyrosine phosphorylation of p130cas (35). A proline-rich sequence of PTP-PEST can also bind the p130cas SH3 domain, but in addition, the catalytic domain can directly interact with tyrosine-phosphorylated p130cas, and PTP-PEST may function to specifically dephosphorylate p130cas (36, 37). Yersinia pseudotuberculosis, which is a bacterial pathogen, encodes a PTPase that binds and dephosphorylates both FAK and p130cas upon entry into cells (38). This enzyme is essential for the virulence of a number of Yersinia species (39). Thus, three cellular PTPases and one bacterial PTPase have been implicated as potential regulators of tyrosine phosphorylation of focal adhesion proteins.
In this study, we have tested the hypothesis that a PTPase may be intimately associated with FAK. PTPase activity was found to specifically associate with FAK, both in vitro and in vivo. However, this interaction appeared to be indirect and was in part mediated by the focal adhesion-associated protein paxillin. The PTPase was identified as PTP-PEST and was found to directly bind to paxillin in vitro via its noncatalytic C-terminal domain. The finding that paxillin associates with both a PTK and a PTPase suggests novel models for the regulation of tyrosine phosphorylation by the assembly of complexes containing both enzymes.
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MATERIALS AND METHODS |
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Cell Culture Techniques-- Chicken embryo (CE) cells were prepared and maintained as described (40). Swiss 3T3 cells were maintained in Dulbecco's modified Eagle's medium-H + 10% calf serum and Cos cells in Dulbecco's modified Eagle's medium-H + 10% fetal bovine serum.
Expression of Exogenous Proteins in CE and Cos Cells-- The replication competent avian retroviral vector RCAS A (BH) (41) was used to express wild type FAK, dl853-963, dl965-1012, and C-tagged FAK as described previously (42, 43). For expression of PTP-PEST in Cos cells, the murine cDNA was engineered to encode a C-terminal epitope tag (KT3) using a PCR-based strategy. A 3' primer that created a BamHI site and destroyed the termination codon was used, and the PCR product was inserted into the plasmid pctag (43) (the BamHI site was engineered so the PTP-PEST and KT3 codons of pctag were in frame). The tagged cDNA was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA). To create a deletion mutant lacking the paxillin binding site, the sequences encoding amino acids 1-300 were amplified by PCR using a primer that created an EcoRI site at codon 300 that was in frame with the EcoRI site at codon 494 of PTP-PEST. The PCR product was substituted for the sequences encoding amino acids 1-494 of epitope-tagged PTP-PEST, then subcloned into pcDNA3. Cos cells were transiently transfected with the PTP-PEST constructs using LipofectAMINE and the protocol recommended by the manufacturer (Life Technologies, Inc.), and the cells were lysed 48 h later.
In Gel Phosphatase Assay--
Glutathione
S-transferase (GST)-FER (kindly provided by Dr. Shelton
Earp, University of North Carolina-Chapel Hill) was used as a PTK
source for phosphorylation of the substrate. One mg of poly(Glu,Tyr)
was incubated with 10 µg GST-FER in 500 µl of reaction buffer (200 µCi of [-32P]ATP, 50 mM imidizole (pH
7.2), 10 mM dithiothreitol, 30 mM
MgCl2, 1 mM MnCl2, 1 mM
vanadate, 0.05% Triton X-100, 0.2 mM cold ATP, and 0.1%
-mercaptoethanol) for 4-6 h at room temperature with constant
rotation as described (44). The labeled poly(Glu,Tyr) was purified by
trichloroacetic acid precipitation and chromatography through a
Sephadex G-50 column. [
-32P]ATP-labeled poly(Glu,Tyr)
(1 × 106 cpm) was incorporated into a 10%
SDS-polyacrylamide gel prior to polymerization. After separating the
samples by electrophoresis, the SDS was removed, and the gel was
incubated in denaturing buffer (6 M guanidine chloride and
0.3%
-mercaptoethanol) and then in renaturing buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.04% Tween 20, and 0.3%
-mercaptoethanol) as described (44). The gel was incubated in reaction buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.04% Tween 20, 0.3%
-mercaptoethanol, and 3 mM dithiothreitol) for 1 h and then in fresh buffer
overnight. After drying, the gel was exposed to x-ray film
overnight.
Protein Analysis-- Polyclonal antiserum directed against part of the noncatalytic portion of PTP-PEST was generated in rabbits using a trpE fusion protein containing amino acids 286-471 (49). A polyclonal antiserum was used to recognize FAK (BC3; a gift of Tom Parsons, University of Virginia), and commercially available monoclonal antibodies were used to recognize paxillin and p130cas (Transduction Laboratories, Lexington, KY). Cells were lysed in ice-cold modified radioimmunoprecipation assay buffer (50 mM Tris-HCl [pH 7.3], 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.5% aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1.5 mM orthovanadate) or Triton X-100 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 10% glycerol, 0.5% aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1.5 mM orthovanadate). The protein concentration of the lysates was determined by the bicinchoninic acid assay (Pierce). For in vitro phosphatase assays, the cells were lysed in the absence of vanadate. For immunoprecipitations, antibodies were incubated with 0.2-1 mg of cell lysate for 1 h on ice. The immunecomplexes were recovered by incubation with protein A-Sepharose beads (Pharmacia Biotech Inc.) or anti-mouse IgG-coupled agarose beads (Sigma) for 1 h at 4 °C. After washing three times in lysis buffer and twice in Tris-buffered saline (50 mM Tris-HCl (pH 7.5), 150 mM NaCl), the immunecomplexes were dissociated by boiling in SDS sample buffer. The samples were analyzed using the in gel PTPase assay (44) or by SDS-PAGE and Western blotting (50). For Western blotting, primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies or protein A and ECL (Amersham Corp.).
Expression of GST Fusion Proteins--
GST fusion proteins
containing the N- and C-terminal domains of FAK have been described
(45). Additional fusion constructs were engineered using PCR to amplify
C-terminal domain fragments of FAK, and the PCR products were inserted
into pGEX2TK (Pharmacia). GST-H-FAK contains codons 765-1052,
GST-B-FAK contains codons 858-1052, and GST-S-FAK contains codons
909-1052. Similarly, fragments of PTP-PEST were amplified by PCR and
subcloned into pGEX2TK. Fusion proteins containing residues 1-301,
297-775, 441-775, and 297-494 were engineered. The fusion proteins
were expressed by incubation with 0.2 mM isopropyl
-D-thiogalactopyranoside for 4-5 h at 37 °C, the
bacteria were lysed by sonication, and the fusion proteins were
purified using glutathione agarose.
Binding Experiments Using GST Fusion Proteins-- One-half to one mg of cell lysate was precleared with 10 µg of GST bound to glutathione agarose beads for 60 min at 4 °C. The cleared supernatant was then incubated with 0.1-1 µg of GST fusion protein immobilized on glutathione agarose beads for 1 h at 4 °C. The beads were washed three times in lysis buffer and twice in Tris-buffered saline. The bound proteins were released by boiling in SDS sample buffer and analyzed by either Western blotting or the in gel PTPase assay.
Far-Western Blotting--
Six to 10 µg of purified GST fusion
protein was added to 40 µl of HMK buffer (20 mM Tris (pH
7.5), 100 mM NaCl, 12 mM MgCl2) containing 1 mM dithiothreitol, 50 units of protein kinase
A (Sigma) and 100 µCi of [-32P]ATP (6000 Ci/mmol)
(NEN Life Science Products) (46, 47). After 45 min of incubation at
room temperature, the reaction was terminated with HMK stop buffer (10 mM sodium phosphate (pH 8.0), 10 mM sodium
pyrophosphate, 10 mM EDTA). Radiolabeled GST-PTP-PEST was
separated from [
-32P]ATP by chromatography on a
Sephadex G-50 column. As described previously (46, 47), protein samples
were subjected to SDS-PAGE, transferred to nitrocellulose, and
denatured in buffer containing 6 M guanidine hydrochloride.
The samples were renatured by incubation in serial dilutions of
denaturation buffer. Filters were washed and then blocked in 5% milk
as described (46, 47). Radioactive probe was added (to 6 × 106 cpm/ml) and incubated for 8 h at 4 °C, and then
the filter washed three times for 15 min and exposed to film.
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RESULTS |
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Association of PTPase Activity with FAK in Vitro-- Overexpression of FAK in CE cells does not induce tyrosine phosphorylation of cellular proteins unless the cells are treated with vanadate, a PTPase inhibitor (22). These results suggest that tyrosine phosphorylation of focal adhesion proteins may be controlled by both FAK and an antagonistic, perhaps intimately associated PTPase. To test whether FAK could bind a PTPase, GST fusion proteins containing the N-terminal and C-terminal noncatalytic domains of FAK were used to pull down FAK-binding proteins from lysates of CE cells. The PTPase activity associated with these fusion proteins was examined using an in gel PTPase assay. The GST-C-terminal domain of FAK associated with four major bands of PTPase activity with molecular masses of 97, 75-80 (a doublet), and 50 kDa (Fig. 1). It is not clear whether these are different PTPases or represent breakdown products of the largest, 97-kDa PTPase. The PTPase activities specifically bound to the C-terminal domain of FAK because GST alone and GST-N-FAK failed to associate with any detectable PTPase activity. A series of GST-fusion proteins containing smaller fragments of the C-terminal domain of FAK was used to further map the FAK sequences required for association with PTPase activity. The GST-S-FAK construct contains only the focal adhesion targeting sequence, a 150-amino acid sequence that has been shown to localize FAK to focal adhesions (42). Each of these constructs associated with PTPase activity in vitro (Fig. 1). Analysis of the fusion proteins by SDS-PAGE and Coomassie Blue staining demonstrated that equivalent amounts of each fusion protein were used in the analysis (Fig. 1B). These results indicate that FAK can associate with PTPase activity in vitro and that the focal adhesion targeting sequence is sufficient for association with the PTPase.
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Association of PTPase Activity with Paxillin-- The FAK sequences required for association with PTPase activity in vitro contain a binding site for paxillin, raising the possibility that FAK associates with a PTPase(s) via its interaction with paxillin. To determine whether paxillin associates with PTPase activity, paxillin immunecomplexes were analyzed using the in gel PTPase assay. No PTPase activity was detected in control immunoprecipitations lacking the primary antibody (Fig. 2). The paxillin immunecomplex, however, exhibited PTPase activity, and the molecular masses of the associated PTPase(s) were 97, 75-80, and 50 kDa (Fig. 2). The pattern of PTPase activities associated with GST-C-FAK was compared with the PTPase pattern coimmunoprecipitated with paxillin and found to be virtually identical (Fig. 2). Thus, paxillin associates with PTPase activities in vivo, and the molecular masses of the PTPase(s) are similar to those that associate with the C-terminal domain of FAK in vitro.
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Paxillin Mediates the Association between PTPase and FAK-- To determine whether paxillin was required for the association of recombinant FAK with PTPase activity in vitro, a clearing experiment was performed. Paxillin was cleared from cell lysates by repeated immunoprecipitation. After three rounds of immunodepletion using a paxillin monoclonal antibody, the amount of paxillin remaining in the lysate was undetectable by immunoprecipitation and immunoblotting (Fig. 3C). After each immunoprecipitation, the lysate was incubated with GST-C-FAK, and the PTPase activity that associated with FAK in vitro was measured using the in gel PTPase assay. GST-C-FAK could no longer associate with PTPase activity after paxillin was cleared from the lysate, whereas GST-C-FAK bound PTPase activity in uncleared lysate (Fig. 3A). Following four rounds of control immunoprecipitation using anti-mouse IgG, the amount of PTPase that associated with GST-FAK was undiminished (Fig. 3B). These results indicate that paxillin is required for the association of FAK with PTPase activity in vitro.
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The 97-kDa PTPase Is PTP-PEST-- To determine whether PTP-PEST was the paxillin-associated PTPase, paxillin was immunoprecipitated from CE cells or Swiss 3T3 cells, and the immunecomplexes were probed with an anti-PTP-PEST antibody in a Western blot (Fig. 5A). PTP-PEST was detected in paxillin immunecomplexes formed from each cell type. Control immunecomplexes lacking the primary paxillin monoclonal antibody did not contain PTP-PEST. Interestingly, the molecular mass of PTP-PEST differed between species. Murine PTP-PEST exhibited a molecular mass of 120 kDa, whereas avian PTP-PEST was 97 kDa. Thus, the molecular mass of avian PTP-PEST coincides with that of the major PTPase activity that associates with paxillin. In addition, PTP-PEST was immunoprecipitated from CE cells and Swiss 3T3 cells, and the immunecomplexes were Western blotted for paxillin. Control immunoprecipitations using nonimmune serum contained no paxillin, whereas PTP-PEST immunecomplexes contained detectable paxillin (Fig. 5B). To confirm that the PTPase activity originally detected by its association with recombinant FAK was indeed PTP-PEST, CE and Swiss 3T3 cell lysates were incubated with GST-C-FAK, and the bound proteins were blotted for PTP-PEST. Indeed, PTP-PEST was found to be associated with GST-C-FAK (Fig. 5C). Despite the presence of PTPase activity in FAK immunecomplexes (Fig. 4), PTP-PEST could not be detected in FAK immunecomplexes by Western blotting. This presumably reflects the low stoichiometry of association with FAK. These results identify PTP-PEST as the PTPase that coimmunoprecipitates with paxillin and associates with the C-terminal domain of FAK in vitro.
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The Noncatalytic Domain of PTP-PEST Binds Paxillin-- To map the paxillin binding site within PTP-PEST, fragments of the PTPase were expressed as GST fusion proteins. The fusion proteins were immobilized on glutathione beads and incubated with CE cell lysates, and the cellular proteins that bound to PTP-PEST were probed for paxillin by Western blotting. The PTP-PEST 1-305 construct (containing the catalytic domain) did not associate with paxillin (Fig. 7), whereas the PTP-PEST 297-775 construct did. Thus, the paxillin binding site resides in the C-terminal noncatalytic domain of PTP-PEST. To further narrow the binding site, two additional constructs were engineered. PTP-PEST 441-775 exhibited no paxillin binding activity, whereas PTP-PEST 297-494 bound paxillin as well as the construct containing the entire C-terminal domain (Fig. 7). Therefore, the paxillin binding site on PTP-PEST resides between residues 297 and 494. To determine whether this region of PTP-PEST also mediated association with paxillin in vivo, a deletion mutant lacking residues 300-494 was engineered. Both wild type PTP-PEST and the mutant were epitope-tagged using the KT3 tag and subcloned into pcDNA3. The constructs were transiently transfected into Cos cells. Cells were lysed, and exogenously expressed PTP-PEST was immunoprecipitated with the antibody recognizing the tag. Each immunecomplex was probed for paxillin by Western blotting. Wild type PTP-PEST coimmunoprecipitated paxillin from Cos cells, whereas control immunoprecipitations lacking the primary antibody did not (Fig. 8). Cos cells express SV40 large T antigen, which also contains the KT3 tag. Paxillin was not coimmunoprecipitated with large T antigen, because KT3 immunoprecipitations from untransfected Cos cells did not contain detectable paxillin (Fig. 8). Thus, in transiently transfected Cos cells, the paxillin/PTP-PEST interaction can be reconstituted. The mutant PTP-PEST protein, dl300-494, was successfully expressed in Cos cells but failed to coimmunoprecipitate paxillin (Fig. 8). Therefore, the region of PTP-PEST extending from residue 297 to residue 494 is required for association with paxillin both in vitro and in vivo.
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PTP-PEST Binds Directly with Paxillin--
A far-Western blotting
(also known as gel overlay) strategy was applied to determine whether
PTP-PEST might directly bind paxillin. Paxillin immunecomplexes were
subjected to SDS-PAGE and transferred to nitrocellulose. The GST fusion
protein containing PTP-PEST sequences 297-494 was radiolabeled using
[-32P]ATP and protein kinase A, which phosphorylates a
site at the junction of the GST and PTP-PEST polypeptides. This fusion
protein was used as the probe for the proteins immobilized on the
membrane. The PTP-PEST probe bound to two proteins in the lane
containing the paxillin immunecomplex (Fig.
9). The 68-kDa protein is paxillin, and
the 45-kDa protein may be a paxillin-related species that can be
immunoprecipitated and detected in Western blots using paxillin
monoclonal antibodies (65). As controls, p130cas
and FAK immunecomplexes were also analyzed. The PTP-PEST probe did not
bind any protein in the lane containing the FAK immunecomplex. It did,
however, bind to p130cas (Fig. 9). These results
demonstrate that a fragment of the noncatalytic domain of PTP-PEST can
directly bind to paxillin and p130cas in
vitro.
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DISCUSSION |
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In this study, we set out to test the hypothesis that FAK intimately associated with a PTPase. Indeed, PTPase activity did associate with FAK, both in vitro and in vivo. PTPase activity was also detected in paxillin immunecomplexes. Two lines of evidence suggest that the observed association of PTPase activity with FAK was indirect. First, following immunodepletion of paxillin from cell lysates, recombinant FAK was unable to associate with PTPase activity in vitro. Second, mutants of FAK that are defective for paxillin binding fail to coimmunoprecipitate PTPase activity. PTP-PEST, a cytoplasmic member of a family of PTPases, was identified as the paxillin binding PTPase. A region of the noncatalytic domain of PTP-PEST contains the paxillin binding site and can directly bind paxillin in vitro. Therefore, we favor a model in which PTP-PEST can directly associate with paxillin and the small amount of PTP-PEST associated with FAK does so indirectly, via paxillin (Fig. 10).
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PTP-PEST is a widely expressed PTPase belonging to the PEST family of PTPases, which are characterized by proline, glutamic acid, serine, and threonine rich sequences (48, 51). Immunofluorescence and subcellular fractionation studies indicate that PTP-PEST is localized to the cytoplasm of the cell (48). The enzymatic activity of PTP-PEST can be repressed by phosphorylation of serine residue 39 by either protein kinase A or protein kinase C (52). The adaptor molecule Shc binds via its phosphotyrosine binding (PTB) domain to a sequence in the noncatalytic domain of PTP-PEST (NPLH) (53, 54). An abundance of proline rich sequences in the noncatalytic domain of PTP-PEST serve as docking sites for other signaling molecules. Grb2 binds via its SH3 domain to the noncatalytic domain of PTP-PEST, and through this interaction PTP-PEST can associate with tyrosine-phosphorylated EGF receptor (48). The SH3 domain of the Csk PTK can also associate with a proline rich sequence located distally, in the noncatalytic domain of PTP-PEST (49). A distinct proline rich sequence in the noncatalytic domain of PTP-PEST serves as a docking site for the SH3 domain of p130cas (37). Tyrosine-phosphorylated p130cas associates tightly with the catalytic domain of "substrate trapping" mutants of PTP-PEST and can be dephosphorylated by PTP-PEST, suggesting that p130cas may be a specific substrate (36). The SH3 domain mediated interaction between p130cas and PTP-PEST may facilitate dephosphorylation of p130cas in vivo (37).
In addition to these associated proteins, we have found that paxillin directly binds to the noncatalytic domain of PTP-PEST. Residues 297-494 of PTP-PEST contain both paxillin and p130cas binding sites because this fragment of PTP-PEST recognizes both proteins on a far-Western blot. A sequence within this region (PPPKPPR) has been identified as the binding site for the SH3 domain of p130cas (37). The paxillin binding site has not been further delineated; thus, the proximity of the paxillin and p130cas binding sites is not known. Likewise, it remains to be determined whether paxillin and p130cas can simultaneously dock to PTP-PEST. It may be noteworthy that paxillin and p130cas can be coimmunoprecipitated (55).2 Whether this observation is due to a direct interaction or is mediated via an intermediate protein, e.g. PTP-PEST or FAK, remains to be established. Coimmunoprecipitation and Western blotting experiments suggest that more than 15% of the cellular PTP-PEST is in complex with paxillin. This relatively high stoichiometry of association between endogenous PTP-PEST and endogenous paxillin presumably reflects the functional importance of the interaction. The finding that PTP-PEST associates with paxillin lends further credence to the hypothesis that PTP-PEST may control signaling by dephosphorylation of focal adhesion-associated proteins.
Presumably, the function of the paxillin/PTP-PEST-containing complex is to regulate the PTPase. Conceivably, paxillin binding could regulate the enzymatic activity of the PTPase, in either a stimulatory or inhibitory manner. A more likely scenario is that paxillin regulates the accessibility of PTP-PEST to its substrates. PTP-PEST may be targeted to focal adhesions, the site of some of its substrates, e.g. p130cas, via its association with paxillin. However, immunofluorescence experiments have failed to reveal PTP-PEST in focal adhesions (48).2 This observation is somewhat surprising, given the relatively high stoichiometry of association of PTP-PEST with paxillin. Perhaps only a fraction of the cellular PTP-PEST resides in focal adhesions or retargeting of PTP-PEST to focal adhesions may only occur under certain conditions, e.g. immediately following cell adhesion. Alternatively, paxillin may control the access of PTP-PEST to its substrates by serving as a scaffold to recruit both enzyme and its substrates into a complex.
The discovery that PTP-PEST binds paxillin suggests that paxillin itself or other paxillin-binding partners might also serve as PTP-PEST substrates. FAK is a paxillin-binding protein and therefore could also serve as a PTP-PEST substrate (47, 56). An epitope-tagged FAK variant, which fails to bind paxillin (47) and PTP-PEST, is remarkably normal in the regulation of its tyrosine phosphorylation.2 Thus, the recruitment of PTP-PEST via paxillin may not be the exclusive mechanism for the regulation of dephosphorylation of FAK. It is particularly intriguing to speculate that PTP-PEST might be able to dephosphorylate only a subset of the phosphorylation sites on FAK or paxillin. One other interesting set of candidate substrates are the Src family PTKs. Src can bind via its SH3 domain to paxillin (57), and both Src and Fyn can bind via their SH2 domains to FAK (12, 58, 59). If PTP-PEST were brought into the proximity of Src (or the FAK/Src complex) via paxillin, the active conformation of Src might be stabilized by dephosphorylation of the negative site of regulation (60). This mechanism of activation is somewhat analogous to the activation of the Src-like PTK, lck, in T cells by the CD45 PTPase (61, 62). Alternatively, PTP-PEST might dephosphorylate tyrosine 416 of Src, which is the major site of autophosphorylation and a positive regulatory site (60). Thus, models evoking PTP-PEST as both a positive and negative regulator of Src can be proposed.
The findings that PTP-PEST dephosphorylates p130cas and associates with paxillin provides clues into how the enzyme might be regulated. Stimuli that induce the dephosphorylation of focal adhesion-associated proteins might enhance the activity of PTP-PEST. Alternatively, stimuli that induce tyrosine phosphorylation of focal adhesion-associated proteins might inhibit the activity of PTP-PEST. In this regard, it may be noteworthy that integrin-dependent cell adhesion induces tyrosine phosphorylation of focal adhesion proteins and stimulates the activity of protein kinase C (63, 64). Because serine phosphorylation of PTP-PEST by either protein kinase C or protein kinase A inhibits its activity (52), integrin-induced serine phosphorylation of PTP-PEST by protein kinase C could contribute to the elevation of phosphotyrosine levels following cell adhesion.
Finally, it is intriguing that multimolecular complexes containing a PTK (FAK), a PTPase (PTP-PEST), and paxillin can be immunoprecipitated from cell lysates. We speculate that paxillin may play a role in coordinating the tyrosine phosphorylation of focal adhesion associated proteins by recruiting PTP-PEST and or FAK to their substrates. Thus, paxillin may play a novel role in regulating tyrosine phosphorylation of cellular proteins in both a spatial and a temporal fashion.
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ACKNOWLEDGEMENTS |
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We thank Dr. Keith Burridge for his critical comments during the course of this investigation. Thanks to Drs. Shelton Earp and Tom Parsons for the provision of antibodies.
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FOOTNOTES |
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* This project was supported by Grant CB-173 from the American Cancer Society and Grant GM53666 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Department of Cell Biology and Anatomy, 534 Taylor Hall, CB 7090, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Tel.: 919-966-0391; Fax: 919-966-1856; E-mail: crispy4{at}med.unc.edu.
1 The abbreviations used are: FAK, focal adhesion kinase; PTK, protein-tyrosine kinase; cas, crk-associated substrate; PTP or PTPase, protein-tyrosine phosphatase; SH, Src homology; CE, chicken embryo; PCR, polymerase chain reaction; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
2 Y. Shen and M. D. Schaller, unpublished observations.
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REFERENCES |
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