Pyk2 Kinase Activity Research Articles

Novel Role of Src in Priming Pyk2 Phosphorylation.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.

The Tyrosine Kinase Pyk2 Contributes to Complement-Mediated Phagocytosis in Murine Macrophages

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family and is mainly expressed in neuronal and hematopoietic cells. As FAK family members are involved in signaling connections downstream of integrins, we studied the role of Pyk2 in complement-receptor 3 (CR3, also known as Mac-1, integrin α_Mβ₂, CD11b/CD18)-mediated phagocytosis, a key process in innate immunity. Using 3 independent approaches, we observed that Pyk2 contributes to CR3-dependent phagocytosis by RAW 264.7 macrophages, but is dispensable for Fcγ receptor (FcγR)-mediated uptake. Reduction of Pyk2 expression levels via siRNA, the pharmacological inhibition of Pyk2 kinase activity as well as macrophage treatment with a cell permeable TAT fusion protein containing the C-terminus of Pyk2 (TAT-PRNK) significantly impaired CR3-mediated phagocytosis without affecting FcγR-mediated uptake. In addition, Pyk2 was strongly recruited to complement opsonized Escherichia coli and the pharmacological inhibition of Pyk2 significantly decreased uptake of the bacteria. Finally, CRISPR/Cas-mediated disruption of the pyk2 gene in RAW 264.7 macrophages confirmed the role of this protein tyrosine kinase in CR3-mediated phagocytosis. Together, our data demonstrate that Pyk2 selectively contributes to the coordination of phagocytosis-promoting signals downstream of CR3, but is dispensable for FcγR-mediated phagocytosis.

Vs-6063 (Defactinib) Targets Mesothelioma Cancer Stem Cells Directly and Through Inhibition of Tumor-Associated Macrophages and Cytokine Production

ABSTRACT Aim: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase required for survival of cancer stem cells (CSCs). PYK2, the other FAK family member, shares 60% kinase domain homology. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma. Defactinib is a potent, orally active FAK kinase inhibitor that has completed phase I and is currently in clinical development in several indications including mesothelioma. Methods: Cytokine release from human macrophages was measured in response to small molecule FAK kinase inhibitors. Serum IL-6 and F4/80-positive macrophages were assessed by ELISA and immunofluorescence respectively. Results: In addition to potent FAK inhibition, defactinib also inhibits PYK2 kinase activity with nanomolar potency. Using both FAK inhibitors and FAK siRNA, we demonstrated that FAK inhibition effectively reduces CSCs. Human monocyte-derived macrophages release IL-6 and IL-8, and these cytokines increased the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. Defactinib dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Accordingly, PYK2 siRNA decreased IL-6 and IL-8 release from human macrophages. In a xenograft model in vivo, defactinib reduced serum IL-6 while the FAK-only reference compound did not. Furthermore, defactinib substantially reduced the number of F4/80-positive macrophages in the tumors. Conclusions: These data suggest that dual targeting of FAK and PYK2 by defactinib may more effectively suppress CSCs than inhibition of FAK alone. Given the sensitivity of mesothelioma CSCs to defactinib, the use of this agent in a maintenance setting after first line chemotherapy may be an attractive strategy to extend survival of patients with mesothelioma. These data support the ongoing COMMAND study with defactinib in mesothelioma (Clinical trial NCT01870609). Disclosure: M. Keegan: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; J.E. Ring: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; V.N. Kolev: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; I.M. Shapiro: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; M.V. Padval: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; Q. Xu: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; J.A. Pachter: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.

Pyk2 Mediates Enhanced STAT3 Phosphorylation Following Collaborative Signaling Between gp130 and beta1 Integrins in Adhered Myeloma and B Cells

Abstract 2895Multiple myeloma is the second most common hematologic malignancy and remains incurable due to the eventual development of drug resistance. Therapy resistance has been linked to both acquired genetic changes as well as the dynamic influences of soluble and physical factors present in the microenvironment. We and others have shown that adhesion of tumor cells to the extracellular matrix component fibronectin (FN) via integrins leads to cell cycle arrest and protection from chemotherapy or Cell Adhesion-Mediated Drug Resistance (CAM-DR). The pleiotropic cytokine IL-6 has also been shown to mediate drug resistance in both solid and hematopoietic tumors [1]. To date, the influence of these two effectors on drug resistance has been studied separately; however, within the context of the bone marrow myeloma cells are influenced by both soluble and physical effectors of the microenvironment simultaneously. Using a reductionist model of the multivariant microenvironment, we recently demonstrated that unique collaborative signaling between FN-adhered cells and IL-6 leads to increased proliferation of protected tumor cells and a more malignant phenotype[2]. Our results demonstrate the FN-adhesion mediated cell-cycle arrest of myeloma cells was reversed following stimulation of adhered cells with IL-6. This phenotype was associated with a novel amplification of IL-6-induced STAT3 activation in adhered cells [2]. To further characterized the molecular events mediating this unique event we focused on the focal adhesion kinase, Proline-rich tYrosine Kinase 2 (Pyk2/FAK2). Pyk2 is a downstream intermediate of integrin signaling and has been demonstrated to amplify EGFR and cSrc-induced STAT3 activation. As such, we hypothesized that Pyk2 may be an important modulator of the enhanced STAT3 activation following multivariant signaling between beta1 integrins and gp130[3]. Our results link this amplification to Pyk2 in myeloma and murine pro-B cell lines. Western blot analysis demonstrated that Pyk2 autophophorylation on tyrosine 402 is induced following myeloma cell adhesion to FN correlating with the amplification of IL-6-induced STAT3, JAK1, and gp130 phosphorylation in both myeloma and pro B cell line models. Targeting Pyk2 with RNA interference attenuated the adhesion-associated amplification of STAT3 signaling as well as JAK1 and gp130 phosphorylation, but did not influence the limited STAT3 activation in cells grown in suspension. Further we demonstrated that Pyk2 kinase activity was similarly required for the activation of JAK1/STAT3 signaling under collaborative conditions, but not by IL-6 alone. Consistent with previous results ERK1/2 and Akt signaling were not affected by Pyk2 siRNA or kinase activity. Importantly, Pyk2 siRNA did not inhibit myeloma cell adhesion (n=4, p-value >0.05). Our previous results demonstrated that that the enhanced STAT3 signaling involved a FN-adhesion specific binding of unphosphorylated STAT3 with gp130 (independent of IL-6 stimulation). To determine if Pyk2 was similarly recruited to gp130 under co-stimulatory conditions we have initially used confocal microscopy. Confocal imaging with antisera to Pyk2 and gp130 demonstrated colocalization of the two effectors upon adhesion of myeloma cell to FN, but not in those grown in suspension. These data demonstrate that within the context of multivariant stimulation (IL-6 and FN-adhesion) a unique Pyk2-mediated JAK1/STAT3 signaling cascade is associated with unique biologic sequelae. We are currently exploring the biological sequelae of Pyk2-mediated collaborative signaling between integrins and IL-6. Key endpoints currently under investigation include proliferation and drug-response. These findings are significant because they suggests strategies targeting Pyk2 could be used to block cooperative signaling between integrins and IL-6, and inhibit the proliferation and/or therapy resistance conferred to tumor cells by the multivariant bone marrow niche. Disclosures:No relevant conflicts of interest to declare.

Phosphoproteomic characterization of PYK2 signaling pathways involved in osteogenesis

The PYK2 tyrosine kinase is a negative regulator of bone formation, but aside from the requirement for PYK2 kinase activity there has been little progress toward understanding of the molecular mechanism involved in this function. To gain insight into the signaling pathways modulated by PYK2 we sought to identify PYK2 substrates. Challenges inherent to a quantitative phosphoproteomic analysis for non-receptor tyrosine kinases were overcome by employing an inducible PYK2 overexpression system in NIH3T3 cells in combination with a selective PYK2 inhibitor. The identification of a number of known PYK2 substrates and interacting partners validated the methodology. Results of the inducible cell system were extended to a cell model of osteogenesis, examining the effect of the PYK2 inhibitor on the phosphorylation state of targets identified in the phosphoproteomic study. Consistent with phosphoproteomic analysis, increased osteogenesis associated with a selective PYK2 inhibitor was accompanied by reduced phosphorylation of paxillin, Gab1 and p130(Cas), along with reduction of phosphorylation levels of the Met activation loop. These results further confirmed the utility of the methodology and point to a previously unknown bi-directional activation pathway between PYK2 and Met.

The Rap GTPases mediate CXCL13‐ and sphingosine1‐phosphate‐induced chemotaxis, adhesion, and Pyk2 tyrosine phosphorylation in B lymphocytes

The localization of B cells to lymphoid organs where they can become activated and differentiate into antibody-secreting plasma cells is controlled by multiple chemoattractants that promote cell migration and integrin-mediated adhesion. CXCL13 and sphingosine 1-phosphate (S1P) are two important chemoattractants that control the trafficking of B cells. CXCL13 directs B lymphocytes to lymphoid follicles where they receive survival signals and, if activated, undergo a germinal center response. In contrast, S1P allows B cells and plasma cells to exit lymphoid organs and re-enter the circulation. The Rap1 GTPase is a key regulator of cell adhesion and cell migration in a number of systems. We now show that Rap activation is required for CXCL13 and S1P to induce B cell migration as well as adhesion to ICAM-1 and VCAM-1. We also show that Pyk2, a tyrosine kinase involved in cytoskeleton rearrangements and B cell migration, is a downstream target of both CXCL13 and S1P signaling and that Rap activation is important for CXCL13 and S1P to stimulate tyrosine phosphorylation of Pyk2, a modification that increases Pyk2 kinase activity. This suggests that the ability of CXCL13 and S1P to direct the trafficking and localization of B cells in vivo may be dependent on Rap activation.

Loss of proline‐rich tyrosine kinase 2 function induces spreading and motility of epithelial prostate cells

Although prostate carcinoma is an aggressive cancer preferentially metastasizing to the bones, many prostate tumors remain localized and confined to the prostate indefinitely. Prediction of the behavior of anatomically localized and moderately differentiated prostate tumors remains difficult because of lack of prognostic markers. Cell motility is an important step in the progression of epithelial tumor toward invasive metastatic carcinomas and changes in the expression and function of adhesion molecules contribute to the acquisition of a more malignant phenotype. Proline-rich tyrosine kinase 2 (Pyk2) is implicated in regulating the organization of actin cytoskeleton, a process critical for cell migration, mitosis, and tumor metastasis. In this report, we investigated whether Pyk2 played a role in the acquisition of an aggressive phenotype in prostate cell. Data reported here demonstrate that loss of Pyk2 kinase function results in induction of cell motility and migration in EPN cells, a line of non-transformed epithelial cells derived from human normal prostate tissue. Changes in motility and migration of prostate cells were associated with changes in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. Ablation of Pyk2 kinase activity caused a dramatic decrease of the expression of E-cadherin and IRS1 and an increase of the expression of alpha5-integrin. In addition, a massive reorganization of actin cytoskeleton was observed. Our data indicate that Pyk2 plays a central role in the mechanism that regulate cell-cell and cell-substrate interaction and lack of its kinase activity induces prostate cells to acquire a malignant, migrating phenotype.

Pyk2/CAKβ Tyrosine Kinase Activity-mediated Angiogenesis of Pulmonary Vascular Endothelial Cells

Endothelial cell spreading, migration, and morphogenesis are essential for angiogenesis, the formation of new blood vessels. In the present study, we explored roles of tyrosine kinase Pyk2 in angiogenesis of pulmonary endothelial cells. We found that tyrosine kinase Pyk2 was particularly enriched in pulmonary vascular endothelial cells and lung, a major organ site for tumor metastasis. By using adenovirus-mediated expression of various Pyk2 mutants, we demonstrated that Pyk2 tyrosine kinase activity was essential for the pulmonary vascular endothelial cell spreading, migration, morphogenesis, as well as pulmonary vein and artery angiogenesis ex vivo. We further showed that Pyk2 kinase activity was required for the expression of focal adhesion kinase, p130Crk-associated substrate, and its homologue human enhancer of filamentation 1, thus regulating formation of focal adhesions and cytoskeletal reorganization. These results indicate that Pyk2 plays a crucial role in the pulmonary endothelial cell motility such as spreading and migration necessary for angiogenesis.

Unique Regulation of c-Jun N-Terminal Kinase by PYK2/CAK-β in Angiotensin II-Stimulated Vascular Smooth Muscle Cells

Activation of tyrosine kinases is believed to play a central role in angiotensin II (AngII) signaling. Here, we have investigated whether a tyrosine kinase, PYK2, is functionally involved in AngII-induced c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells (VSMCs). Adenovirus expressing PYK2 kinase-inactive mutant K457A or a tyrosine phosphorylation site mutant Y402F was transfected in VSMCs. AngII-induced JNK phosphorylation was markedly enhanced by K457A, whereas it was suppressed by Y402F. Protein synthesis induced by AngII was also enhanced by K457A and inhibited by Y402F. In this regard, K457A suppressed PYK2 kinase activation by AngII, whereas it enhanced AngII-induced PYK2 Tyr402 phosphorylation. By contrast, Y402F inhibited PYK2 Tyr402 phosphorylation, whereas it markedly enhanced AngII-induced PYK2 kinase activation. Thus, we conclude that PYK2 kinase activity negatively regulates JNK activation and protein synthesis, whereas Tyr402 phosphorylation positively regulates these events in AngII-stimulated VSMCs, suggesting a unique role of PYK2 in mediating vascular remodeling.

G13α-mediated PYK2 Activation: PYK2 IS A MEDIATOR OF G13α-INDUCED SERUM RESPONSE ELEMENT-DEPENDENT TRANSCRIPTION

G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.

Thrombin-stimulated phosphatidylinositol 3-kinase activity in platelets is associated with activation of PYK2 tyrosine kinase: activation of both enzymes is aggregation independent.

In this study, we investigated the activation of a new member of the focal adhesion kinase family of tyrosine kinases, the proline-rich tyrosine kinase, or PYK2, in platelets. We show that PYK2 is tyrosine phosphorylated and its activity is increased during early stages of platelet aggregation. This activation coincided with increased association of phosphatidylinositol (PI) 3-kinase and PYK2, as determined by both anti-PI 3-kinase and anti-PYK2 immunoprecipitates. However, under basal conditions, some association of PYK2 and PI 3-kinase was consistently observed, even though little or no tyrosine phosphorylated PYK2 could be detected. In addition, both increased PI 3-kinase activity and increased PYK2 activity could be detected in immunoprecipitates following thrombin stimulation. All of these events were unaffected by blocking platelet aggregation with arginine-glycine-aspartate-serine (RGDS) peptide, which interferes with binding of the platelet integrin alpha(IIb)beta(3) to fibrinogen. Neither was the activation of the PYK2 kinase activity affected by blocking PI 3-kinase activity. These results support a model in which PYK2 is associated with PI 3-kinase in unstimulated platelets and following activation of platelets, there is an increase in tyrosine phosphorylation of PYK2, increased PYK2 activity, and increased association of PYK2 with PI 3-kinase, which may contribute to the increase in PI 3-kinase activity. All of these were found to be early events independent of subsequent platelet aggregation.

Tyrosine kinase Pyk2 mediates G-protein-coupled receptor regulation of the Ewing sarcoma RNA-binding protein EWS

Ewing family tumors result from the effects of chromosomal translocations that fuse the Ewing sarcoma (EWS) gene to various genes encoding transcription factors [1]. The resulting chimeric EWS fusion proteins are transcriptional activators with transforming potential that have received much study [2]. By contrast, the cellular function of somatic EWS remains obscure. EWS belongs to a family of RNA-binding proteins thought to play role in RNA synthesis or processing [3,4]. Here, we show that EWS interacts with Pyk2, a protein tyrosine kinase implicated in a variety of signal transduction processes [5–7][8]. G-protein-coupled receptor signaling and other stimuli of Pyk2 kinase activity significantly block the interaction between EWS and Pyk2. Furthermore, as assessed by sucrose gradient centrifugation, EWS partitions with dense ribosome-containing fractions in a manner that is enhanced by signaling from the G-protein-coupled m1 muscarinic acetylcholine receptor (mAChR). We conclude that extranuclear EWS is a previously unrecognized target of G-protein-coupled receptor regulation.