Control of Dendritic Cell Migration,T Cell-Dependent Immunity,and Autoimmunity by Protein Tyrosine Phosphatase PTPN12 Expressed in Dendritic Cells

Dendritic cells (DCs) capture and process antigens in peripheral tissues, migrate to lymphoid tissues, and present the antigens to T cells. PTPN12, also known as PTP-PEST, is an intracellular protein tyrosine phosphatase (PTP) involved in cell-cell and cell-substratum interactions. Herein, we examined the role of PTPN12 in DCs, using a genetically engineered mouse lacking PTPN12 in DCs. Our data indicated that PTPN12 was not necessary for DC differentiation, DC maturation, or cytokine production in response to inflammatory stimuli. However, it was needed for full induction of T cell-dependent immune responses in vivo. This function largely correlated with the need of PTPN12 for DC migration from peripheral sites to secondary lymphoid tissues. Loss of PTPN12 in DCs resulted in hyperphosphorylation of the protein tyrosine kinase Pyk2 and its substrate, the adaptor paxillin. Pharmacological inhibition of Pyk2 or downregulation of Pyk2 expression also compromised DC migration, suggesting that Pyk2 deregulation played a pivotal role in the migration defect caused by PTPN12 deficiency. Together, these findings identified PTPN12 as a key regulator in the ability of DCs to induce antigen-induced T cell responses. This is due primarily to the role of PTPN12 in DC migration from peripheral sites to secondary lymphoid organs through regulation of Pyk2.     

The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion,but Not Permeability,and Controls Vascular Development and Embryonic Viability

Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability.     

Macrophage Fusion Is Controlled by the Cytoplasmic Protein Tyrosine Phosphatase PTP-PEST/PTPN12

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.     

Outside-in signaling pathway linked to CD146 engagement in human endothelial cells

CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.     

A Role for the Protein Tyrosine Phosphatase CD45 in Macrophage Adhesion through the Regulation of Paxillin Degradation

CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility. These defects are accompanied by substantially decreased levels of the cytoskeletal-associated protein paxillin, without affecting the levels of other proteins. Degradation of paxillin in CD45-deficient macrophages is calpain-mediated, as treatment with a calpain inhibitor restores paxillin levels in these cells and enhances cell spreading. Inhibition of the tyrosine kinases proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK), kinases that are capable of mediating tyrosine phosphorylation of paxillin, also restored paxillin levels, indicating a role for these kinases in the CD45-dependent regulation of paxillin. These data demonstrate that CD45 functions to regulate Pyk2/FAK activity, likely through the activity of Src family kinases, which in turn regulates the levels of paxillin to modulate macrophage adhesion and migration.     

p130Cas promotes invasiveness of three-dimensional ErbB2-transformed mammary acinar structures by enhanced activation of mTOR/p70S6K and Rac1

ErbB2 over-expression is detected in approximately 25% of invasive breast cancers and is strongly associated with poor patient survival. We have previously demonstrated that p130Cas adaptor is a crucial mediator of ErbB2 transformation. Here, we analysed the molecular mechanisms through which p130Cas controls ErbB2-dependent invasion in three-dimensional cultures of mammary epithelial cells. Concomitant p130Cas over-expression and ErbB2 activation enhance PI3K/Akt and Erk1/2 MAPK signalling pathways and promote invasion of mammary acini. By using pharmacological inhibitors, we demonstrate that both signalling cascades are required for the invasive behaviour of p130Cas over-expressing and ErbB2 activated acini. Erk1/2 MAPK and PI3K/Akt signalling triggers invasion through distinct downstream effectors involving mTOR/p70S6K and Rac1 activation, respectively. Moreover, in silico analyses indicate that p130Cas expression in ErbB2 positive human breast cancers significantly correlates with higher risk to develop distant metastasis, thus underlying the value of the p130Cas/ErbB2 synergism in regulating breast cancer invasion. In conclusion, high levels of p130Cas favour progression of ErbB2-transformed cells towards an invasive phenotype.     

Distinct Phosphotyrosine-dependent Functions of the ShcA Adaptor Protein Are Required for Transforming Growth Factor β (TGFβ)-induced Breast Cancer Cell Migration,Invasion, and Metastasis

The ErbB2 and TGFβ signaling pathways cooperate to promote the migratory, invasive, and metastatic behavior of breast cancer cells. We previously demonstrated that ShcA is necessary for these synergistic interactions. Through a structure/function approach, we now show that the phosphotyrosine-binding, but not the Src homology 2, domain of ShcA is required for TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. We further demonstrate that the tyrosine phosphorylation sites within ShcA (Tyr²³⁹/Tyr²⁴⁰ and Tyr³¹³) transduce distinct and non-redundant signals that promote these TGFβ-mediated effects. We demonstrate that Grb2 is required specifically downstream of Tyr³¹³, whereas the Tyr²³⁹/Tyr²⁴⁰ phosphorylation sites require the Crk adaptor proteins to augment TGFβ-induced migration and invasion. Furthermore, ShcA Tyr³¹³ phosphorylation enhances tumor cell survival, and ShcA Tyr²³⁹/Tyr²⁴⁰ signaling promotes endothelial cell recruitment into ErbB2-expressing breast tumors in vivo, whereas all three ShcA tyrosine residues are required for efficient breast cancer metastasis to the lungs. Our data uncover a novel ShcA-dependent signaling axis downstream of TGFβ and ErbB2 that requires both the Grb2 and Crk adaptor proteins to increase the migratory and invasive properties of breast cancer cells. In addition, signaling downstream of specific ShcA tyrosine residues facilitates the survival, vascularization, and metastatic spread of breast tumors.     

Lipopolysaccharide induces actin reorganization and tyrosine phosphorylation of Pyk2 and paxillin in monocytes and macrophages

The bacterial endotoxin LPS is a potent stimulator of monocyte and macrophage activation and induces adhesion of monocytes. Morphological changes in response to LPS have not been characterized in detail, however, nor have the signaling pathways mediating LPS-induced adhesion been elucidated. We have found that LPS rapidly induced adhesion and spreading of peripheral blood monocytes, and that this was inhibited by the Src family kinase inhibitor PP1 and the phosphatidylinositide 3-kinase inhibitor LY294002. LPS also stimulated actin reorganization, leading to the formation of filopodia, lamellipodia, and membrane ruffles in Bac1 mouse macrophages. Proline-rich tyrosine kinase 2 (Pyk2), a tyrosine kinase related to focal adhesion kinase, and paxillin, a cytoskeletal protein that interacts with Pyk2, were both tyrosine phosphorylated in response to LPS in monocytes and macrophages. Both tyrosine phosphorylation events were inhibited by PP1 and LY294002. Adhesion also stimulated tyrosine phosphorylation of Pyk2 and paxillin in monocytes, and this was further enhanced by LPS. Finally, Pyk2 and paxillin colocalized within membrane ruffles in LPS-stimulated cells. These results indicate that LPS stimulation of monocytes and macrophages results in rapid morphological changes and suggest that Pyk2 and/or paxillin play a role in this response.     

ErbB2, EphrinB1, Src kinase and PTPN13 signaling complex regulates MAP kinase signaling in human cancers

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression.     

Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades.

The protein tyrosine kinase Pyk2 acts as an upstream regulator of mitogen-activated protein (MAP) kinase cascades in response to numerous extracellular signals. The precise molecular mechanisms by which Pyk2 activates distinct MAP kinase pathways are not yet fully understood. In this report, we provide evidence that the protein tyrosine kinase Src and adaptor proteins Grb2, Crk, and p130Cas act as downstream mediators of Pyk2 leading to the activation of extracellular signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK). Pyk2-induced activation of Src is necessary for phosphorylation of Shc and p130Cas and their association with Grb2 and Crk, respectively, and for the activation of ERK and JNK cascades. Expression of a Grb2 mutant with a deletion of the amino-terminal Src homology 3 domain or the carboxyl-terminal tail of Sos strongly reduced Pyk2-induced ERK activation, with no apparent effect on JNK activity. Grb2 with a deleted carboxyl-terminal Src homology 3 domain partially blocked Pyk2-induced ERK and JNK pathways, whereas expression of dominant interfering mutants of p130Cas or Crk specifically inhibited JNK but not ERK activation by Pyk2. Taken together, our data reveal specific pathways that couple Pyk2 with MAP kinases: the Grb2/Sos complex connects Pyk2 to the activation of ERK, whereas adaptor proteins p130Cas and Crk link Pyk2 with the JNK pathway.     

CD28 ligation induces tyrosine phosphorylation of Pyk2 but not Fak in Jurkat T cells

Protein tyrosine kinases are critical for the function of CD28 in T cells. We examined whether the tyrosine kinases Pyk2 and Fak (members of the focal adhesion kinase family) are involved in CD28 signaling. We found that ligating CD28 in Jurkat T cells rapidly increases the tyrosine phosphorylation of Pyk2 but not of Fak. Paxillin, a substrate for Pyk2 and Fak, was not tyrosine-phosphorylated after CD28 ligation. CD28-induced tyrosine phosphorylation of Pyk2 was markedly reduced in the absence of external Ca2+. Previous studies have shown that the T cell antigen receptor (TCR) induces tyrosine phosphorylation of Pyk2. In this report, the concurrent ligation of CD28 and TCR increased tyrosine phosphorylation of Pyk2; however, the extent of phosphorylation by both receptors was equivalent to the sum of that induced by each receptor alone. The Syk/Zap inhibitor piceatannol blocked CD28, and TCR induced tyrosine phosphorylation of Pyk2, suggesting that Syk/Zap is involved in Pyk2 phosphorylation. In contrast, the phosphatidylinositol 3-kinase inhibitor wortmannin blocked TCR- but not CD28-induced phosphorylation of Pyk2, suggesting that CD28 and TCR activate distinct pathways to induce tyrosine phosphorylation of Pyk2. Notably, depleting phorbol 12-myristate 13-acetate-sensitive protein kinase C did not block CD28- and CD3-induced tyrosine phosphorylation of Pyk2. These data provide evidence for the involvement of Pyk2 in the CD28 signaling cascade and suggest that neither Fak nor paxillin is involved in the signaling pathways of CD28.     

CD36 signals to the actin cytoskeleton and regulates microglial migration via a p130Cas complex

The pattern recognition receptor CD36 initiates a signaling cascade that promotes microglial activation and recruitment to beta-amyloid deposits in the brain. In the present study we identify the focal adhesion-associated proteins p130Cas, Pyk2, and paxillin as novel members of the tyrosine kinase signaling pathway downstream of CD36 and show that assembly of this complex is essential for microglial migration. In primary microglia and macrophages exposed to beta-amyloid, the scaffolding protein p130Cas is rapidly tyrosine-phosphorylated and co-localizes with CD36 to membrane ruffles contemporaneous with F-actin polymerization. These beta-amyloid-stimulated events are not detected in CD36 null cells and are dependent on CD36 activation of Src family tyrosine kinases. Fyn, a Src kinase known to interact with CD36, co-precipitates with p130Cas and is an essential upstream intermediate in the signaling pathways leading to phosphorylation of the p130Cas substrate domain. Furthermore, the p130Cas-interacting kinase Pyk2 and the cytoskeletal adapter protein paxillin also demonstrate CD36-dependent phosphorylation, identifying these focal adhesion molecules as additional members of this beta-amyloid signaling cascade. Disruption of this p130Cas complex by small interfering RNA silencing inhibits p44/42 mitogen-activated protein kinase phosphorylation and microglial migration, illustrating the importance of this pathway in microglial activation and recruitment. Together, these data are the first to identify the signaling cascade that directly links CD36 to the actin cytoskeleton and, thus, implicates it in diverse processes such as cellular migration, adhesion, and phagocytosis.     

Paxillin phosphorylation and complexing with Erk and FAK are regulated by PLD activity in MDA-MB-231 cells

MDA-MB-231 cells are highly aggressive human breast adenocarcinoma cells that depend on PLD activity for survival. In response to the stress of serum withdrawal, there is increased motility and invasiveness of these cells that is associated with a rapid increase in PLD activity. In addition, PLD activity is elevated in response to most mitogenic signals. Similar to PLD, paxillin, a focal adhesion adaptor protein, and Erk, mitogen-activated protein kinase, play vital roles in cell motility through regulation of focal adhesion dynamics. Here, we addressed whether there is a functional correlation between paxillin and PLD that may influence cancer cell motility. We investigated the role of PLD activity on paxillin regulation, Erk activation and formation of a paxillin-Erk and paxillin-FAK association. Inhibition of PLD activity led to an increase in paxillin tyrosine phosphorylation, a decrease in Erk activation, as measured by phosphorylation, and enhanced association of paxillin with Erk. In addition, we found that paxillin tyrosine phosphorylation depends upon Erk activity and may be a consequence of an increased association with FAK. Taken together, these results suggest that Erk activity is governed by PLD activity and regulates the tyrosine phosphorylation of paxillin, potentially explaining its role in cell motility. This study indicated that PLD, Erk, paxillin and FAK participate in the same signaling pathway in this breast cancer cell line.     

Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC-12 cells

Cell adhesions play an important role in neurite extension. Paxillin, a focal adhesion adaptor protein involved in focal adhesion dynamics, has been demonstrated to be required for neurite outgrowth. However, the molecular mechanism by which paxillin regulates neurite outgrowth is unknown. Here, we show that paxillin is phosphorylated by p38MAPK in vitro and in nerve growth factor (NGF)-induced PC-12 cells. Ser 85 (Ser 83 for endogenous paxillin) is identified as one of major phosphorylation sites by phosphopeptide mapping and mass spectrometry. Moreover, expression of the Ser 85 --> Ala mutant of paxillin (paxS85A) significantly inhibits NGF-induced neurite extension of PC-12 cells, whereas expression of wild-type (wt) paxillin does not influence neurite outgrowth. Further experiments indicate that cells expressing paxS85A exhibit small, clustered focal adhesions which are not normally seen in cells expressing wt paxillin. Although wt paxillin and paxS85A have the same ability to bind vinculin and focal adhesion kinase, wt paxillin more efficiently associates with Pyk2 than paxS85A. Thus, phosphorylation of paxillin is involved in NGF-induced neurite extension of PC-12 cells, probably through regulating focal adhesion organization.     

PKC-regulated myogenesis is associated with increased tyrosine phosphorylation of FAK,Cas, and paxillin,formation of Cas-CRK complex,and JNK activation

Previous reports suggest that PKC plays an important role in regulating myogenesis. However, the regulatory signaling pathways are not fully understood. We examined the effects of PKC downregulation on signaling events during skeletal muscle differentiation. We found that downregulation of PKC results in increased myogenesis in C2C12 cells as measured by creatine kinase activity and myogenin expression. We showed that, during differentiation, downregulation of PKC expression results in increased tyrosine phosphorylation of FAK, Cas, and paxillin, concomitant with enhanced Cas-CrkII complex formation, which leads to activation of JNK2. But in proliferated muscle cells, PKC inhibition results in FAK and Cas tyrosine dephosphorylation. Further, disruption of actin cytoskeleton by cytochalasin D prevents the activation of FAK and Cas as well as the formation of Cas-CrkII complex stimulated by PKC downregulation during muscle cell differentiation. Finally, we observed that PKC downregulation increases the tyrosine phosphorylation of focal adhesion associated proteins. Based on the above data, we propose that PKC downregulation results in enhanced tyrosine phosphorylation of FAK, Cas, and paxillin, thus promoting the establishment of Cas-CrkII complex, leading to activation of JNK and that these interactions are dependent upon the integrity of actin cytoskeleton during muscle cell differentiation. Data presented here significantly contribute to elucidating the regulatory role of PKC in myogenesis possibly through integrin signaling pathway.     

Interaction of Pyk2 and PTP-PEST with leupaxin in prostate cancer cells

We have identified the presence of leupaxin (LPXN), which belongs to the paxillin extended family of focal adhesion-associated adaptor proteins, in prostate cancer cells. Previous studies have demonstrated that LPXN is a component of the podosomal signaling complex found in osteoclasts, where LPXN was found to associate with the protein tyrosine kinases Pyk2 and c-Src and the cytosolic protein tyrosine phosphatase-proline-, glutamate-, serine-, and threonine-rich sequence (PTP-PEST). In the current study, LPXN was detectable as a 50-kDa protein in PC-3 cells, a bone-derived metastatic prostate cancer cell line. In PC-3 cells, LPXN was also found to associate with Pyk2, c-Src, and PTP-PEST. A siRNA-mediated inhibition of LPXN resulted in decreased in vitro PC-3 cell migration. A recombinant adenoviral-mediated overexpression of LPXN resulted in an increased association of Pyk2 with LPXN, whereas a similar adenoviral-mediated overexpression of PTP-PEST resulted in decreased association of Pyk2 and c-Src with LPXN. The overexpression of LPXN in PC-3 cells resulted in increased migration, as assessed by in vitro Transwell migration assays. On the contrary, the overexpression of PTP-PEST in PC-3 cells resulted in decreased migration. The overexpression of LPXN resulted in increased activity of Rho GTPase, which was decreased in PTP-PEST-overexpressing cells. The increase in Rho GTPase activity following overexpression of LPXN was inhibited in the presence of Y27632, a selective inhibitor of Rho GTPase. In conclusion, our data demonstrate that LPXN forms a signaling complex with Pyk2, c-Src, and PTP-PEST to regulate migration of prostate cancer cells. PC-3; protein tyrosine phosphatase-proline-, glutamate-, serine-, and threonine-rich sequence; c-Src; migration     

A phosphoproteomic analysis of the ErbB2 receptor tyrosine kinase signaling pathways

Overexpression of the ErbB2 receptor tyrosine kinase is common in human cancers and is associated with an increased level of metastasis. To better understand the cellular signaling networks activated by ErbB2, a phosphoproteomic analysis of tyrosine-phosphorylated proteins was carried out in ErbB2-overexpressing breast and ovarian cancer cell lines. A total of 153 phosphorylation sites were assigned on 78 proteins. Treatment of cells with Herceptin, a monoclonal antibody that inhibits ErbB2 activity, significantly reduced the number of detectable protein phosphorylation sites, suggesting that many of these proteins participate in ErbB2-driven cell signaling. Of the 71 proteins that were differentially phosphorylated, only 13 were previously reported to directly associate with ErbB2. The differentially phosphorylated proteins included kinases, adaptor/docking proteins, proteins involved in cell proliferation and migration, and several uncharacterized RNA binding proteins. Selective depletion of some of these proteins, including RNA binding proteins SRRM2, SFRS1, SFRS9, and SFRS10, by siRNAs reduced the rate of migration of ErbB2-overexpressing ovarian cancer cells.     

Hypophosphorylated and inactive Pyk2 associates with paxillin at the microtubule organizing center in hematopoietic cells

Pyk2 is a non-receptor tyrosine kinase that regulates cellular adhesion. We generated antibodies to a peptide corresponding to the N-terminus (NT) of Pyk2 and another to a portion of the C-terminal (CT) domain. Only the CT antiserum recovered paxillin-associated Pyk2. These antibodies recognized overlapping but biochemically distinct molecular species of Pyk2 since the CT antiserum recovered Pyk2 after NT antibody immunodepletion. Furthermore, the CT antibody could not immunoblot NT antibody-captured Pyk2. Phosphorylation partially accounts for the differential binding of these antibodies as dephosphorylation of Pyk2 recovered with the NT antibodies allows for recognition by the CT antibody. Additionally, Pyk2 recovered with the NT antibody displays increased serine/threonine phosphorylation. We suggest that the NT epitope is inaccessible to the antibody because Pyk2 is in a closed confirmation in association with paxillin. Upon induction of serine and/or threonine phosphorylation of Pyk2, it opens to a confirmation that allows for antibody binding to the NT epitope but at the same time no longer binds paxillin or the CT antiserum. These antibodies also display differential staining of Pyk2 in both T cells and macrophages. Pyk2 recognized by the CT antibody, but not the NT antibody, colocalized with paxillin at the microtubule-organizing center (MTOC). The MTOC-bound Pyk2 was not tyrosine phosphorylated upon T cell activation. We hypothesize that a reservoir of primarily inactive Pyk2 associates with paxillin at the MTOC, which may allow for rapid delivery of Pyk2 to specific sites of adhesion.