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J. Biol. Chem., Vol. 280, Issue 15, 14556-14562, April 15, 2005

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Tyrosine Phosphorylation of ₃ Integrin Provides a Binding Site for Pyk2^*

Boyd Butler and Scott D. Blystone

From the Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York 13210

Received for publication, October 15, 2004 , and in revised form, December 23, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Integrins expressed on leukocytes possess the ability to maintain themselves in a non-adhesive state, thus preventing unwarranted adhesion and uncontrolled inflammation. Leukocyte adhesion is regulated through the modulation of integrin receptors such as _V3. Firm adhesion to the extracellular matrix and directed cellular motility requires the reorganization of the actin cytoskeleton. The ability of ₃ to recruit signaling and scaffolding molecules to propagate _V3 -mediated signals is regulated in part by the phosphorylation of the ₃ cytoplasmic tail. The identities of integrin-associated signaling molecules within _V3 podosomes and in particular the proximal binding partners of the ₃ cytoplasmic tail are not completely known. Here we show that _V3 ligation induces Pyk2-Tyr-402 phosphorylation and its association with the ₃ cytoplasmic tail in a ₃-Tyr-747 phosphorylation-dependent manner. Pyk2 binding to the ₃ cytoplasmic tail is direct and dependent upon Pyk2-Tyr-402 and ₃ -Tyr-747 phosphorylations. These data identify Pyk2 as a phosphorylated ₃ binding partner, providing a potential structural and signaling platform to achieve _V3 -mediated remodeling of the actin cytoskeleton.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The ability of hematopoietic cells and in particular leukocytes to self-regulate adhesion to complex extracellular matrices is an essential requirement for the maintenance of immunosurveillance. Hematopoietic cells migrate throughout the vasculature in a non-adhesive state until they encounter proinflammatory or thrombotic signals. These signals induce an arrest from the circulation by means of firm adhesion to the vascular walls, extravasation from the vascular compartment, followed by migration to the site of inflammation. The ability of leukocytes to undergo this spatially and temporally integrated multistep process is regulated in part by the integrin _V3.

The _V3 integrin plays a pivotal role in the ability of many cell types to adhere to the extracellular matrix and can also serve as a route of pathogen entry (1–3). Termed the vitronectin (Vn)¹ receptor, _V3 does in fact recognize a number of extracellular matrix ligands including fibronectin, tenascin, and osteopontin (4). Our laboratory has previously shown that, although _V3 recognizes many different ligands, there is differentially regulated _V3-mediated adhesion to individual ligands, particularly Vn and fibronectin (5). Integrin ligation by the extracellular matrix initiates conformational changes within integrin structure and, in the case of _V3, induces tyrosine phosphorylation of residues within the ₃ cytoplasmic domain. Both of these events are involved in the recruitment of cytosolic proteins in a phosphorylation- and conformation-dependent manner leading to an integrin-associated protein complex. Although phosphorylation of the ₃ cytoplasmic tail is required for _V3-mediated adhesion to Vn, the cytoplasmic tail itself exhibits no signaling capabilities. Therefore, signaling molecules must be recruited to propagate adhesion-initiated signals to downstream effector proteins.

We have previously demonstrated that hematopoietic cells expressing non-tyrosine phosphorylatable ₃ integrins fail to adhere due to an inability to form actin stress fibers (5). Similarly, in mice expressing only non-phosphorylatable ₃ integrins, a failure in the maintenance of platelet aggregation can also be attributed to defects in myosin-based contraction of actin fibrils (6–9). These findings are likely related to the requirement of ₃ phosphorylation for both Rho GTPase activation and recruitment of the Arp2/3 complex to ₃-containing podosomes (10) as both of these events are known to lead to actin reorganization within cells. Because it seems unlikely that either Rho or Arp2/3 interact directly with phosphorylated ₃, we have pursued identification of the proximal binding partner for phosphorylated ₃.

Protein-tyrosine kinases are a class of proteins that, once associated with the integrin complex, would be capable of relaying signaling events. Proline-rich tyrosine kinase 2 (Pyk-2), also known as cell adhesion kinase- or related adhesion focal tyrosine kinase, is a nonreceptor protein-tyrosine kinase closely related to p125 focal adhesion kinase that couples receptors, including integrins, with a variety of downstream effectors such as small G proteins belonging to the Ras and Rho families, mitogen-activated protein kinases, protein kinase C, and inositol phosphate metabolism (for review, see Refs. 11 and 12). Pyk2 has been shown to play a critical role in the adhesion and migration of many cell types (13–18). Previous studies have shown that Pyk2 binds to or colocalizes with various focal adhesion proteins including Cas, Cbl, vinculin, and paxillin (15, 19–21). Pyk2 has also been shown to associate with and be involved in the activation of various signaling proteins such as Src, Pap, and PI3K (22–26). Pyk2 has also been shown to localize to integrin contact sites as well as undergo binding to integrin cytoplasmic tails (15, 27–29). Our laboratory has recently shown that Pyk2 co-localizes selectively with tyrosine-phosphorylated ₃ in podosomes (10). The present study demonstrates that Pyk2 associates with ₃ in a Pyk2 and ₃ tyrosine phosphorylation-dependent manner. The binding of Pyk2 with a ₃ cytoplasmic tail appears to be direct as demonstrated by binding of GST-Pyk2 and ₃ peptides in a phosphorylation-dependent manner. Pyk2 phosphorylation and translocation to tyrosinephosphorylated ₃ is blocked by Src kinase inhibition, known to regulate Pyk2 activity. Pharmacological blockade of Pyk2 or ₃ phosphorylation also prevents PI3K association with ₃. The phosphorylation dependence of ₃ in adhesive processes may lie in the ability to recruit the kinase and adaptor capabilities of Pyk2, a molecule suited to supporting a proadhesive signaling complex.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells and Materials—K562 cells were stably transfected with cDNA encoding wild type ₃ (K_V3) or cDNA encoding ₃ mutated at Y747F (K_V3-Y747F), Y759 (K_V3-Y759F), or Y747F,Y759F (K_V3-Y747F,Y759F) together with _V and maintained as previously described (30). The anti-₃ monoclonal antibody 7G2 was a gift of Eric J. Brown. Vn was prepared as previously described (30). Anti-GST, anti-Pyk2, anti-Pyk2^Y402P, anti-p85 (PI3K), anti-PY (4G10), anti-AktS^473P, anti-talin, anti-Src, and anti-Src^Y416P were purchased from Upstate Biotechnology (Lake Placid, NY), and anti-₃ PSSA was purchased from BIO-SOURCE (Camarillo, CA). Synthesized GRGDSP peptide was purchased from Invitrogen. All reagents unless otherwise noted were purchased from Sigma.

Immunoprecipitation and Immunoblotting—K_V3 cells (1 x 10⁷) stably expressing ₃ constructs were incubated in suspension with 1 mM GRGDSP (RGD) in serum-free media containing 50 µM Na₃VO₄ with or without PMA (10 ng/ml), PP1 (10 nM), calphostin C (100 nM), piceatannol (5 µg/ml), wortmannin (10 µM), or Y27632 (10 µM) for 15 min at room temperature. The cells were lysed in PBS buffer containing 1% Nonidet P-40, Na₃VO₄ (1 mM), aprotinin (10 µg/ml), leupeptin (10 µg/ml), and phenylmethylsulfonyl fluoride (1 mM). Cell debris was removed by centrifugation at 12,000 x g for 10 min at 4 °C. The lysates were precleared for 1 h at 4 °C with gelatin-Sepharose and immunoprecipitated with goat anti-mouse-Sepharose beads (ICN, Costa Mesa, CA) precoated with anti-₃ 7G2, anti-Pyk2, anti-talin, or anti-PI3K monoclonal antibodies for 2 h at 4 °C. Cell lysates or immunoprecipitated proteins were separated on 7.5% SDS-PAGE gels and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The membranes were blocked in Tris-buffered saline with 0.1% Tween 20 and 3% w/v bovine serum albumin at room temperature for 1 h. Membranes were then incubated with primary antibodies followed by peroxidase-coupled secondary antibodies and developed with enhanced chemiluminescence (ECL, Amersham Biosciences). For adhesion studies, 6-well tissue culture-treated plates were coated with Vn (1 µg/ml) in PBS overnight at 4 °C. K_V3 cells were plated at 3 x 10⁶ cells/well in serum-free IMDM media containing 50 µM Na₃VO₄ with or without PMA (10 ng/ml), PP1 (50 nM), calphostin C (100 nM), piceatannol (50 µg/ml), wortmannin (10 µM), or Y27632 (10 µM) for upto 1 h at 37 °Cin5%CO₂. All cells in each well were lysed and treated as described above.

Pyk2 Overlays—The N terminus of Pyk2 (amino acids 1–432) was cloned into pGex4T-1 by the creation of flanking EcoR1 sites. Additionally, Pyk2-Y402F was created by PCR mutagenesis. GST-Pyk2 constructs were transfected into XL1 Blue cells, and protein production was induced by 5 mM isopropyl 1-thio--D-galactopyranoside for 2 h at 37 °C. Bacterial cells were pulse-sonicated in 10 mM Tris-HCl, pH 7.0, 1% Triton X-100, aprotinin (10 µg/ml), leupeptin (10 µg/ml), and phenylmethylsulfonyl fluoride (1 mM). Cell debris was removed by centrifugation at 12,000 x g for 10 min at 4 °C, and cell lysate was incubated with glutathione-agarose to purify GST fusion proteins. SDS-PAGE gels were performed to ensure the purity of the constructs. Phosphorylation of GST-Pyk2 constructs by recombinant c-Src was performed as described by the manufacturer (Upstate Biotechnology, Saranac Lake, NY). Synthetic integrin peptides mimicking the ₃ cytoplasmic tail, as employed in a previous study (10), as well as the corresponding cytoplasmic tail regions from other integrins with indicated modifications (10 µM) (₃ wild type (WT), ₃-Y747F, ₃-Tyr(P)-747, ₁-Tyr(P)-788, ₅-Tyr(P)-752, ₇-Tyr(P)-778) (Invitrogen) were spotted onto nitrocellulose membranes. Membranes were blocked in Tris-buffered saline with 0.1% Tween 20 and 3% w/v bovine serum albumin at room temperature for 1 h. Membranes were then incubated with recombinant GST, GST-Pyk2, or GST-Pyk2-Y402F (1 µM) after incubation with (+) or without (–) recombinant Src as described above. Membranes were then probed with anti-₃ or anti-GST and developed by ECL.

GST-Pyk2 Pull-downs—Cell lysates were prepared from K_V3, K_V3-Y747F, K_V3-Y759F, or K_V3-Y747F,Y759F cells that had been incubated with (+) or without (–) PMA and RGD. Cell lysates were incubated with Src treated or untreated GST-Pyk2, GST-Pyk2-Y402F, or GST-coated agarose beads and incubated at 4 °C for 12 h. Precipitated proteins were separated on 7.5% SDS-PAGE gels and transferred to Immobilon-P membrane (Millipore, Bedford, MA). Membranes were blocked in Tris-buffered saline with 0.1% Tween 20 and 3% w/v bovine serum albumin at room temperature for 1 h. Membranes were then incubated with mAb 7G2 followed by peroxidase-coupled secondary antibodies and developed by ECL.

Cell Adhesion Assays—96-Well microtiter plates (Immulon II, Dynatech, Chantilly, VA) were coated with Vn (1 µg/ml) in PBS overnight at 4 °C. Wells were washed twice in PBS and post-coated with 1% casein in PBS for 1 h at room temperature. K_V3 cells were added at 1 x 10⁵ cells/well in Hanks' buffered saline solution containing 1.0 mM Ca²⁺ and Mg²⁺ with or without PMA (10 ng/ml), PP1 (50 nM), calphostin C (100 nM), piceatannol (50 µg/ml), wortmannin (10 µM), or Y27632 (10 µM) and allowed to adhere for 1 h at 37 °C. Wells were rinsed three times in Hanks' buffered saline solution without Ca²⁺ or Mg²⁺, and adherent cells were fixed in 3.7% formaldehyde for 1 h at 4 °C and stained with 0.05% crystal violet for 30 min at room temperature. Crystal violet was dispersed in methanol and quantified by absorbance at 570 nm using an Emax microplate reader (Amersham Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pyk2 Phosphorylation and Activity Are Induced by _v3 Ligation—Our previous reports demonstrate a requirement for phosphorylation at Tyr-747 of the ₃ cytoplasmic tail for both hematopoietic cell adhesion and Rho association with ₃ (5). To determine whether or not Pyk2 may mediate these effects, we first evaluated whether or not Pyk2 association with ₃ was regulated by the phosphorylation of one or both partners. We evaluated Pyk2-Tyr-402 phosphorylation in the presence of known soluble inducers of ₃ tyrosine phosphorylation as well as when K562 cells expressing _V3 were adherent to Vn. Although Mn²⁺ (5 mM) and Na₃VO₄ (100 µM) induced ₃-Tyr-747 phosphorylation, there was no significant phosphorylation of Pyk2-Tyr-402 at 15 min with these same stimuli (Fig. 1A). However, when _V3 was ligated by incubation with RGD (1 mM) peptide or by adhesion to Vn, there was a significant increase in Pyk2-Tyr-402 phosphorylation, and phosphorylation of Pyk2 was augmented with the addition of PMA (Fig. 1A). Previous studies have shown that in many cell types including islet cells and muscle cells, PMA induces Pyk2-Tyr-402 phosphorylation (31, 32). Conversely it has also been shown in T cells that PMA ablates Pyk2-Tyr-402 phosphorylation (33). Cells of hematopoietic lineage often require additional stimuli, such as thrombin or cytokines, mimicked in vitro by PMA to achieve integrin-mediated firm adhesion (23, 34–36). In K562 cells expressing _V3 we see an immediate induction of Pyk2 phosphorylation by PMA within 5 min that dissipates by 10 min (Fig. 1B). With the addition of Na₃VO₄ (100 µM) we see that, whereas ₃ phosphorylation is robust at 15 min, Pyk2-Tyr-402 phosphorylation is prolonged only slightly to 10 min. However, with _V3 ligation, by the addition of RGD peptide we find that ₃-Tyr-747 and Pyk2-Tyr-402 phosphorylations remain robust at 15 min (Fig. 1B). The exact role that PMA plays in _V3-mediated adhesion remains unclear; however, PMA activates protein kinase Cs, PI3K, and Src family kinases, and it has been demonstrated that Src family kinases and Pyk2 associate in an activation-dependent manner, leading to Pyk2 phosphorylation and Pyk2-mediated signaling (13, 22, 24, 37, 38). We have previously shown in K562 cells expressing _V3 that in the presence of PMA, PI3K and Rho are activated when these cells are adherent to Vn (5). We examined the role of cellular activation by PMA in Pyk2 and PI3K activation. In cells adherent to Vn in the presence of PMA, Src inhibition by PP1 causes a complete inhibition of Pyk2-Tyr-402 phosphorylation as well as PI3K phosphorylation and activity as indicated by Akt Ser-473 phosphorylation (Fig. 1C) with no effect on ₃ phosphorylation (see Fig. 3A). Although wortmannin inhibits PI3K phosphorylation and activity, there was no effect on Pyk2-Tyr-402 phosphorylation (Fig. 1C), suggesting that Pyk2 phosphorylation occurs independently of PI3K activity during _V3-mediated adhesion. These data indicate that Pyk2 activation via phosphorylation at Tyr-402 requires _V3 ligation and Src activity. These data also suggest that Pyk2 activation during _V3-mediated adhesion may contribute to PI3K activation in hematopoietic cells.

View larger version (70K): [in this window] [in a new window]   FIG. 1. Ligation-dependent phosphorylation of Pyk2 in an Src-dependent manner. A, for suspension experiments KV3 with cells were incubated (+) or without (–) PMA (10 ng/ml), Mn2+ (5 mM), Na3VO4 (100 µM), or RGD peptide (1 mM) for 15 min. For adhesion lanes KV3 plates cells were plates on 6-well coated with Vn (1 µg/ml) in the presence or absence of PMA and allowed to adhere for 1 h at 37°C. Cell lysates were immunoprecipitated (IP) with either anti-Pyk2 or anti-3 bound to Sepharose. Western blotting was performed using PSSA antibodies to Pyk2-Tyr-402 and 3 -Tyr-747. Total Pyk2 was determined from lysate blots using of the preimmunoprecipitate lysate. Total 3 was determined by Western blot of of the 3 immunoprecipitation probed with mAb 7G2. B, KV3 cells in suspension were incubated with or without PMA, Na3VO4, or RGD peptide for 0, 5, 10, or 15 min. Cell lysates were immunoprecipitated with either anti-Pyk2 or anti-3 bound to Sepharose. Western blotting was performed using PSSA antibodies to Pyk2-Tyr-402 and 3 -Tyr-747. Total Pyk2 was determined from lysate blots using of the preimmunoprecipitate lysate. Total 3 was determined by Western blot of of the 3 immunoprecipitation probed with mAb 7G2. C, KV3 presence cells in suspension in the of PMA and RGD were incubated with or without specific inhibitors for Src kinases (10 nM PP1) or PI3K (10 µM wortmannin (Wn)). Cell lysates were immunoprecipitated with either anti-Pyk2 or anti-PI3K bound to Sepharose. Western blotting for Akt phospho-Ser-473 was performed on whole cell lysate. Western blotting for Pyk2, Src, or PI3K was performed using PSSA antibody to Pyk2-Tyr-402, Src Tyr-416, or anti-PY (4G10) for PI3K. Total Pyk2, PI3K, and Akt were determined from lysate blots using of the preimmunoprecipitate lysate. Shown are representative blots for more than three individual experiments.

View larger version (56K): [in this window] [in a new window]   FIG. 3. Selective inhibition of Pyk2 association with 3. A, KV3 cells were either kept in suspension in the presence or absence (–) of PMA (10 ng/ml) and RGD (1 mM) or allowed to adhere to Vn in the presence or absence (–) of PMA. Suspension and adhesion groups were incubated with or without known inhibitors to Src kinases (10 nM PP1), PI3K (10 µM wortmannin), protein kinase C (10 µM calphostin C (CC)), Rho-associated coiled kinase (10 µM Y27632), or Syk (50 µg/ml piceatannol (Pic)). Cell lysates were immunoprecipitated with anti-3 or anti-Pyk2 bound to Sepharose. Western blotting was performed using PSSA antibodies to Pyk2-Tyr-402 or 3-Tyr-747 or mAb to the p85 subunit of PI3K. Total Pyk2 and PI3K were determined from lysate blots using of the preimmunoprecipitate lysate. Total 3 was determined by a Western blot of of the 3 immunoprecipitation probed with mAb 7G2. Shown are representative blots for more than three individual experiments. B, selective inhibition of V3-mediated adhesion to Vn. KV3 cells were allowed to adhere to Vn in microtiter plates (96 wells) in the presence or absence of PMA, PP1, wortmannin, calphostin C, Y27632, or piceatannol, and quantified as described under "Experimental Procedures." Data bars represent the mean absorbance ± S.D. from triplicate wells from three separate experiments. Ctrl, control.

View larger version (71K): [in this window] [in a new window]   FIG. 2. Pyk2 associates with the 3 cytoplasmic tail in a phosphorylation-specific manner. A, Pyk2 is only associated with phosphorylated Tyr-747. KV3, KV3-Y747F, KV3-Y759F, or KV3 -Y747F,Y759F cells were incubated with (+) or without (–) PMA (10 ng/ml) and RGD peptide (1 mM) in suspension or allowed to adhere to Vn in the presence of PMA, and cell lysates were immunoprecipitated (IP) with anti-3 or anti-Pyk2 bound to Sepharose. Western blotting was performed using PSSA antibodies to Pyk2-Tyr-402 or 3-Tyr-747 anti-Pyk2 mAb or anti-talin mAb. Total Pyk2 and talin were determined from lysate blots using of the preimmunoprecipitate lysates. Total 3 was determined by Western blot of of the 3 immunoprecipitation probed with mAb 7G2. B, decreasing 3 phosphorylation leads to decreasing Pyk2 association. KV3 cells were incubated with PMA and RGD in the presence of indicated concentrations of Na3VO4, and cell lysates were immunoprecipitated with anti-bound to 3 Sepharose. Western blotting was performed using PSSA antibodies to Pyk2-Tyr-402 or 3-Tyr-747. Total Pyk2 was determined from lysate blots using of the preimmunoprecipitate lysate. Total 3 was determined by Western blot of of the 3 immunoprecipitation probed with mAb 7G2. C, KV3 cells were allowed to adhere to Vn (1 µg/ml) in the presence (+) or absence (–) of PMA. Cells were lysed at indicated times, and cell lysates were immunoprecipitated with anti-3 or anti-Pyk2 bound to Sepharose. Western blotting was performed using PSSA antibodies to Pyk2-Tyr-402 or 3-Tyr-747. Shown are representative blots for more than three individual experiments.

In a previous report from our laboratory we show that Tyr-747 phosphorylation of ₃ in adherent K_V3 cells decreases as cells adhere and spread (10). We concluded in this study that this is due to masking of the phospho-epitope recognized by the ₃ PSSA antibody by associated protein complexes. In this same study we showed that Rho-associated coiled kinase inhibition by Y27632 delays the loss of ₃ phosphorylation presumably by preventing masking proteins from translocating to the ₃ cytoplasmic tail. In support of this hypothesis we show here see that Rho-associated coiled kinase inhibition with Y27632 decreases Pyk2 and PI3K association with ₃ even though Pyk2-Tyr-402 and ₃-Tyr-747 phosphorylations are maintained (Fig. 3A). In suspension cells, piceatannol, a syk inhibitor that prevents ₃ phosphorylation, does not inhibit Pyk2-Tyr-402 phosphorylation, suggesting that ₃-Tyr-747 and Pyk2-Tyr-402 phosphorylation events are independent of each other. These data suggest that Pyk2-Tyr-402 and ₃-Tyr-747 phosphorylations are required for their association.

Pyk2 Binds Directly to the ₃ Cytoplasmic Tail in a Phosphorylation-dependent Manner—To determine whether or not the Pyk2 and ₃ association was direct, we employed ₃ overlay assays and GST-Pyk2 pull-downs. As a control we show that we are able to phosphorylate GST-Pyk2-Tyr-402 by recombinant Src (Fig. 4A). When we spot peptides corresponding to various integrin cytoplasmic tails onto membranes and overlay them with phosphorylated or non-phosphorylated GST-Pyk2, we observe that the Pyk2 N terminus binds robustly to ₃ cytoplasmic tail peptides in a Pyk2-Tyr-402 and ₃-Tyr-747 phosphorylation-specific manner (Fig. 4A). Only Pyk2 that has been phosphorylated by recombinant Src is capable of binding to ₃, and only ₃ phosphorylated at Tyr-747 supported GST-Pyk2 binding. Although to a much lesser degree than ₃, GST-Pyk2 peptide was also found to bind to the phospho-₁ peptide (Fig. 4A) but not to the phospho-₅ peptide. Because it has been previously shown that Src associates with the ₃ cytoplasmic tail (42), we wanted to ensure that Src did not bridge Pyk2 and ₃ or that there was any residual Src from the kinase assays; therefore, we probed for Src on the overlays and were unable to detect Src (Fig. 4A). Additionally, using GST-Pyk2, phospho-GST-Pyk2, or GST alone, we show that only phospho-GST-Pyk2 is able to precipitate phosphorylated ₃ from cell lysates and only under conditions where ₃ is tyrosine-phosphorylated (Fig. 4B). To further determine the role of phosphorylation of the ₃ cytoplasmic tail in the recruitment of Pyk2, we addressed whether or not competitive inhibition of ₃ integrin cytoplasmic tail tyrosines would result in a decrease in ₃/Pyk2 association. K562 cells expressing WT ₃ were loaded with cell-permeant human immunodeficiency virus TAT pep-tides corresponding to the cytoplasmic tails of WT _{3, 3}-Y747P, ₃-Y747F, ₁-Y788P, ₅-Y752P, or ₇-Y778P and adhered to Vn in the presence of PMA. Human immunodeficiency virus TATphospho-₃ completely inhibited association of Tyr-402-phosphorylated Pyk2 with endogenous ₃ cytoplasmic tail (Fig. 5). There was partial inhibition with the WT ₃ peptide that may be explained by the ability of this peptide to become phosphorylated once inside cells (data not shown). As shown in Fig. 4A, phospho-₁ also associates with ₃, and indeed we see a decrease in Pyk2 association with ₃ in the presence of phospho-₁. Interestingly, only ₃ and ₁ peptides inhibit the interaction between Pyk2 and ₃, and they are the only subunits reported to be tyrosine-phosphorylated (43). Furthermore, these peptides encompass a very short and very well conserved region of cytoplasmic tails proposed to be a phosphotyrosine binding domain (10). As such, it is surprising that there was no inhibition of the ₃ and Pyk2 association in the presence of ₅ or ₇ peptides, perhaps indicating a selective mechanism. Although we cannot ascertain the phosphorylation state of the TAT peptides within the cell, the peptides do remain intact for greater than 4 h (data not shown). Our laboratory has demonstrated that the Tyr-747 phosphopeptides, but not Y747F peptides, also block Arp2/3 localization with ₃, a phosphorylation-dependent event (10). These data suggest that Pyk2 associates directly with the ₃ cytoplasmic tail and that this binding event is controlled by the phosphorylation of Tyr-747 of the ₃ cytoplasmic tail and Tyr-402 of Pyk2.

View larger version (58K): [in this window] [in a new window]   FIG. 4. Pyk2 binds directly to the 3 cytoplasmic tail in a phosphorylation-dependent manner. A, Pyk2 is phosphorylated in vitro by Src, and phosphorylated Pyk2 binds to phosphorylated 3 GST-Pyk2 constructs were created as described under "Experimental Procedures." GST-Pyk2 WT, GST-Pyk2-Y402F, or GST alone were incubated with recombinant Src kinase. The phosphorylation state was determined by Western blot using PSSA to Pyk2-Tyr-402. The N terminus of Pyk2 binds directly to the 3 cytoplasmic tail in a phosphorylation-dependent manner. Recombinant 3 wild type (WT), phospho-3-Y747P, phospho-1-Y788P, phospho-5-Y752P, or 3-Y747F were spotted onto lulose membrane and overlaid nitrocelwith recombinant GST, GST-Pyk2 WT, or GST-Pyk2-Y402F that was previously incubated with (+) or without (–) recombinant Src kinase. Blotting was performed using anti-Pyk2 mAb, anti-Src or anti-GST mAb. Shown are representative blots for more than three individual experiments. B,KV3 cells were incubated with (+) or without (–) PMA (10 ng/ml) and RGD peptide (1 mM). Cell lysates were then precipitated with GST, GST-Pyk2 WT, or GST-Pyk2-Y402F bound to glutathione-agarose that had previously been incubated with (+) or without (–) recombinant Src kinase. Western blotting was performed using mAb 7G2. Shown are representative blots for more than three individual experiments.

View larger version (52K): [in this window] [in a new window]   FIG. 5. Pyk2 association with 3 is ablated by exogenous phosphorylated 3 peptides. KV3 cells were incubated with human immunodeficiency virus TAT peptides corresponding to the cytoplasmic tails of 3, 3-Y747F, phospho-3-Y747P, phospho-5-Y752P, phospho-1-Y788P, or phospho-7-Y778P for 15 min before adhesion to Vn in the presence of PMA (10 ng/ml). Cell lysates were immunoprecipitated (IP) with anti-3 bound to Sepharose. Western blotting was performed using PSSA antibodies to Pyk2-Tyr-402 or 3-Tyr-747. Total Pyk2 was determined from lysate blots using of the preimmunoprecipitate lysate. Total 3 was determined by Western blot of of the 3 immunoprecipitation probed with mAb 7G2. Shown are representative blots for more than three individual experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Using direct peptide association we show that Pyk2 phosphorylated at Tyr-402 binds directly to Tyr-747-phosphorylated ₃ cytoplasmic tails and that both of these phosphorylation events are required for direct binding (Fig. 4, A and B). We also demonstrate that Pyk2 associates in cells with the ₃ cytoplasmic tail in a phosphorylation-dependent manner consistent with previously demonstrated colocalization visualized by microscopy. We do additionally see that Pyk2 binds to the ₁ cytoplasmic tail and that Pyk2 association with the ₃ cytoplasmic tail is diminished in the presence of exogenous ₁ (Fig. 4A and 5). It has been shown previously that Pyk2 localizes to ₁-mediated focal contacts and that ₁ can become tyrosinephosphorylated (28, 43).

We have previously shown that PI3K associates with ₃ only when ₃ is tyrosine-phosphorylated, and it has also been shown that Pyk2 and PI3K are associated in activated platelets (23, 25). Here we show that PI3K activation and association with ₃ are dependent upon ₃ tyrosine phosphorylation and do not occur when Pyk2 activation or association with ₃ is disrupted (Figs. 1 and 3).

Previous work has demonstrated that adhesion to the extracellular matrix or other cells as well as ligation of integrins with mAb induces Pyk2-Tyr-402 phosphorylation. ₃ ligation in osteoclasts induces Pyk2-Tyr-402 phosphorylation and Pyk2 localization to the sealing zone as well as an association between Src and Pyk2 (14, 15, 29). In primary neutrophils and macrophages Pyk2-Tyr-402 phosphorylation is induced by integrin ligation, and Pyk2 is localized to podosomes (15, 50). We have shown that localization of Pyk2-Tyr-402 phosphorylation to _V3 podosomes requires ₃ phosphorylation (10). In this study we also show that _V3 ligation with RGD or by adhesion to Vn induces Pyk2-Tyr-402 phosphorylation (Figs. 1, 2, 3, 4). We further show that phosphorylation of Pyk2-Tyr-402 most likely precedes its association with Tyr-phosphorylated ₃ and that Pyk2-Tyr-402 and ₃-Tyr-747 phosphorylations are required for their association (Figs. 2, 3, 4, 5). Although Pyk2 and ₃ association requires both Pyk2-Tyr-402 and ₃-Tyr-747 phosphorylations, these phosphorylation events appear to occur independently of each other as we show that even with the inhibition of ₃ phosphorylation by either mutation or with piceatannol, we see Pyk2 phosphorylation upon integrin ligation. Additionally, in the presence of PP1, which inhibits Pyk2 phosphorylation, we observe ₃-Tyr-747 phosphorylation (Figs. 1, 2, 3). Recent evidence has shown that Src plays a role in Pyk2-Tyr-402 phosphorylation; however, Pyk2 also undergoes autophosphorylation in a trans-dependent manner (51), also suggesting that Pyk2-Tyr-402 phosphorylation may precede its association with ₃. Together these data indicate that the protein-tyrosine kinase Pyk2 may function to link ₃ integrins with downstream effectors in a manner regulated by ₃ phosphorylation induced by ligand binding.

₃ ligation has been shown to induce Src activity, and Src was shown to bind constitutively to the ₃ cytoplasmic tail (42). Taken together with the PP1 inhibition of Pyk2-Tyr-402 phosphorylation, it could be postulated that Src activation by PMA or ₃ clustering or synergistically by both initiates Pyk2 activation and facilitates the translocation to the phosphorylated ₃ cytoplasmic tail. Therefore, we believe that Src plays a pivotal role in the activation and translocation of Pyk2 to and association with the ₃ cytoplasmic tail and that this association is direct and requires the independent phosphorylations of Tyr-747 of ₃ and Tyr-402 of Pyk2.

	FOOTNOTES

 
^* This work was supported by NIAID, National Institutes of Health Grants R01AI40602 and K02AI57384 (to S. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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: Dept. of Cell and Developmental Biology, SUNY Upstate Medical University, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464-8512; Fax: 315-464-8535; E-mail: Blystons{at}upstate.edu.

¹ The abbreviations used are: Vn, vitronectin; PI3K, phosphatidylinositol 3-kinase; GST, glutathione S-transferase; PSSA, phospho-specific antibody; RGD, GRGDSP; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; WT, wild type; mAb, monoclonal antibody.

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