Proline-rich Tyrosine Kinase Research Articles

Proline-rich tyrosine kinase 2 via enhancing signal transducer and activator of transcription 3-dependent cJun expression mediates retinal neovascularization.

Despite the involvement of proline-rich tyrosine kinase 2 (Pyk2) in endothelial cell angiogenic responses, its role in pathological retinal angiogenesis is not known. In the present study, we show that vascular endothelial growth factor A (VEGFA) induces Pyk2 activation in mediating human retinal microvascular endothelial cell (HRMVEC) migration, sprouting and tube formation. Downstream to Pyk2, VEGFA induced signal transducer and activator of transcription 3 (STAT3) activation and cJun expression in the modulation of HRMVEC migration, sprouting and tube formation. Consistent with these observations, hypoxia induced activation of Pyk2-STAT3-cJun signaling axis and siRNA-mediated downregulation of Pyk2, STAT3 or cJun levels substantially inhibited hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization. Together, these observations suggest that activation of Pyk2-mediated STAT3-cJun signaling is required for VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization in vivo.

Structural Basis for the Interaction between Pyk2-FAT Domain and Leupaxin LD Repeats.

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase and belongs to the focal adhesion kinase (FAK) family. Like FAK, the C-terminal focal adhesion-targeting (FAT) domain of Pyk2 binds to paxillin, a scaffold protein in focal adhesions; however, the interaction between the FAT domain of Pyk2 and paxillin is dynamic and unstable. Leupaxin is another member in the paxillin family and was suggested to be the native binding partner of Pyk2; Pyk2 gene expression is strongly correlated with that of leupaxin in many tissues including primary breast cancer. Here, we report that leupaxin interacts with Pyk2-FAT. Leupaxin has four leucine–aspartate (LD) motifs. The first and third LD motifs of leupaxin preferably target the two LD-binding sites on the Pyk2-FAT domain, respectively. Moreover, the full-length leupaxin binds to Pyk2-FAT as a stable one-to-one complex. Together, we propose that there is an underlying selectivity between leupaxin and paxillin for Pyk2, which may influence the differing behavior of the two proteins at focal adhesion sites.

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.

FAK and Pyk2 in disease

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are non-receptor protein tyrosine kinases that are involved in cell proliferation, migration and survival. Current research of FAK and Pyk2 is greatly focused in cancer biology and several small molecule inhibitors are being tested under clinical development. Like cancer, certain chronic diseases such as cardiovascular disease, bone disease, fibrosis, rheumatoid arthritis, and neurological disorders, share malignant characteristics of cancer. Accumulating evidence has demonstrated that FAK and Pyk2 contribute to other proliferative and degenerative diseases. Thus, the goal of this review is to briefly highlight studies that have implicated FAK and Pyk2 as players in disease progression.

Subcellular localization of proline-rich tyrosine kinase 2 during oocyte fertilization and early-embryo development in mice.

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase, is a member of the focal adhesion kinase family and is highly expressed in oocytes. Using a combination of confocal microscopy and RNAi, we localized and studied the function of both Pyk2 and tyrosine-phosphorylated Pyk2 (p-Pyk2) during mouse oocyte fertilization and early embryo development. At the onset of fertilization, Pyk2 and p-Pyk2 were detected predominantly in sperm heads and the oocyte cytoplasm. Upon formation of male and female pronuclei, Pyk2 and its activated form leave the cytoplasm and accumulate in the two pronuclei. We detected Pyk2 in blastomere nuclei and found both Pyk2 and p-Pyk2 in the pre-blastula cytoplasm. Pyk2 and its activated form then disappeared from the blastula nuclei and localized to the perinuclear regions, where blastula cells come into contact with each other. Pyk2 knockdown via microinjection of siRNA into the zygote did not inhibit early embryo development. Our results suggest that Pyk2 plays multiple functional roles in mouse oocyte fertilization as well as throughout early embryo development.

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.

Pyk2 is essential for astrocytes mobility following brain lesion.

Proline-rich tyrosine kinase 2 (Pyk2) is a calcium-dependent, non-receptor protein-tyrosine kinase of the focal adhesion kinase (FAK) family. Pyk2 is enriched in the brain, especially the forebrain. Pyk2 is highly expressed in neurons but is also present in astrocytes, where its role is not known. We used Pyk2 knockout mice (Pyk2(-/-) ) developed in our laboratory to investigate the function of Pyk2 in astrocytes. Morphology and basic properties of astrocytes in vivo and in culture were not altered in the absence of Pyk2. However, following stab lesions in the motor cortex, astrocytes-mediated wound filling was slower in Pyk2(-/-) than in wild-type littermates. In an in vitro wound healing model, Pyk2(-/-) astrocytes migrated slower than Pyk2(+/+) astrocytes. The role of Pyk2 in actin dynamics was investigated by treating astrocytic cultures with the actin-depolymerizing drug latrunculin B. Actin filaments re-polymerization after latrunculin B treatment was delayed in Pyk2(-/-) astrocytes as compared with wild-type astrocytes. We mimicked wound-induced activation by treating astrocytes in culture with tumor-necrosis factor alpha (TNFα), which increased Pyk2 phosphorylation at Tyr402. TNFα increased PKC activity, and Rac1 phosphorylation at Ser71 similarly in wild-type and Pyk2-deficient astrocytes. Conversely, we found that gelsolin, an actin-capping protein known to interact with Pyk2 in other cell types, was less enriched at the leading edge of migrating Pyk2(-/-) astrocytes, suggesting that its lack of recruitment mediated in part the effects of the mutation. This work shows the critical role of Pyk2 in astrocytes migration during wound healing.

Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.

Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases.

Abstract B202: Combined therapy of temozolomide and PF-562271, a PYK2 inhibitor, reduces glioma tumor growth and dispersal compare to temozolomide monotherapy

Abstract Microglia infiltrate majority of gliomas and release factors, which favor tumor growth and dispersal. Previously we demonstrated that microglia residing within the tumor stimulate glioma cell dispersal through the proline rich tyrosine kinase 2 (Pyk2) signaling cascade (Rolon-Reyes et al. 2015). We hypothesize that the use of pharmacological inhibitors of Pyk2 in applied therapy can significantly reduce the dispersal of tumor cells and improve the outcome of treatment. In the present study, we investigated both in vitro and in vivo, the effect of the combined treatment of glioblastoma with temozolomide (TMZ) together with PF-562271 (a Pyk2 and FAK inhibitor), vs. TMZ monotherapy. Proliferation and migration assays were performed for a number of primary human glioma cell lines, as well as U87 and HS683cell lines in the presence and absence of microglia. Tumor size and invasion area were evaluated in brains of GL261 tumor bearing C57BL/6 mice for the assessment of the effectiveness of treatment. Our data demonstrate that PF-562271 (16nM) and TMZ (100μM) given alone reduced in vitro migration and proliferation of glioma cells in the presence of microglia. PF-562271 (16nM) given in combination with TMZ at a concentration of just 10μM provided the same effect as TMZ 100 μM along, without reduction of efficacy. In vivo experiments demonstrated that oral administration of PF-562271 (twice/daily, 25 mg/kg, orally) reduced invasion of glioma cells at the tumor edge, while TMZ (50 mg/kg, once/day, orally) reduced the tumor growth. Combined treatment revealed significantly more prominent effect on both tumor growth and invasion compare to monotherapy. These data indicate that the combination of TMZ and PF-562271 reduces both tumor growth and dispersal of tumor cells to surrounding areas. Moreover, in vitro experiments revealed a complex effect of these drugs on glioma cell proliferation and migration, allowing the reduction of the applied concentration of TMZ without the loss of its therapeutic effect. This research was made possible by NIH grant numbers: 1SC2GM102040-01A1, 8G12MD007583-27, Title V PPOHA grant number P031M105050, NIH NIMH R25 MH055929 and RO1-CA18382701A1. Citation Format: Kimberleve Rolon-Reyes, Luis A. Cubano, Alfredo Quiñones-Hinojosa, Lilia Kucheryavykh. Combined therapy of temozolomide and PF-562271, a PYK2 inhibitor, reduces glioma tumor growth and dispersal compare to temozolomide monotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B202.

The role of non-receptor protein tyrosine kinases in the excitotoxicity induced by the overactivation of NMDA receptors.

Protein tyrosine phosphorylation is one of the primary modes of regulation of N-methyl-d-aspartate (NMDA) receptors. The non-receptor tyrosine kinases are one of the two types of protein tyrosine kinases that are involved in this process. The overactivation of NMDA receptors is a primary reason for neuron death following cerebral ischemia. Many studies have illustrated the important role of non-receptor tyrosine kinases in ischemia insults. This review introduces the roles of Src, Fyn, focal adhesion kinase, and proline-rich tyrosine kinase 2 in the excitotoxicity induced by the overactivation of NMDA receptors following cerebral ischemia.

CD19 controls Toll-like receptor 9 responses in human B cells

CD19 is a B cell-specific molecule that serves as a major costimulatory molecule for amplifying B-cell receptor (BCR) responses. Biallelic CD19 gene mutations cause common variable immunodeficiency in human subjects. BCR- and Toll-like receptor (TLR) 9-induced B-cell responses are impaired in most patients with common variable immunodeficiency. We sought to analyze whether CD19 is required for TLR9 function in human B cells. Expression of surface activation markers was assessed after anti-IgM or CpG stimulation by using flow cytometry on B cells from patients with 1 or 2 defective CD19 alleles, which decrease or abrogate CD19 expression, respectively. The phosphorylation or interaction of signaling molecules was analyzed by using phospho flow cytometry, immunoblotting, or co-immunoprecipitation in CD19-deficient or control B cells and in a B-cell line in which CD19 has been knocked down with lentivirus-transduced short hairpin RNA. B cells from subjects with 1 or 2 defective CD19 alleles showed defective upregulation in vitro of CD86, transmembrane activator and CAML interactor (TACI), and CD23 activation markers after TLR9 stimulation. TLR9 ligands normally induce phosphorylation of CD19 through myeloid differentiation primary response gene-88 (MYD88)/proline-rich tyrosine kinase 2 (PYK2)/LYN complexes, which allows recruitment of phosphoinositide 3-kinase (PI3K) and phosphorylation of Bruton tyrosine kinase (BTK) and AKT in human B cells with a different kinetic than that of BCRs. In addition, inhibition of PI3K, AKT, or BTK, as well as BTK deficiency, also resulted in TLR9 activation defects in B cells similar to those in patients with CD19 deficiency. CD19 is required for TLR9-induced B-cell activation. Hence CD19/PI3K/AKT/BTK is an essential axis integrating BCRs and TLR9 signaling in human B cells.

Targeting PYK2 mediates microenvironment-specific cell death in multiple myeloma.

Multiple myeloma (MM) remains an incurable malignancy due, in part, to the influence of the bone marrow microenvironment on survival and drug response. Identification of microenvironment-specific survival signaling determinants is critical for the rational design of therapy and elimination of MM. Previously, we have shown that collaborative signaling between β1 integrin-mediated adhesion to fibronectin and interleukin-6 confers a more malignant phenotype via amplification of signal transducer and activator of transcription 3 (STAT3) activation. Further characterization of the events modulated under these conditions with quantitative phosphotyrosine profiling identified 193 differentially phosphorylated peptides. Seventy-seven phosphorylations were upregulated upon adhesion, including PYK2/FAK2, Paxillin, CASL and p130CAS consistent with focal adhesion (FA) formation. We hypothesized that the collaborative signaling between β1 integrin and gp130 (IL-6 beta receptor, IL-6 signal transducer) was mediated by FA formation and proline-rich tyrosine kinase 2 (PYK2) activity. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation under co-stimulatory conditions. Co-culture of MM cells with patient bone marrow stromal cells (BMSC) showed similar β1 integrin-specific enhancement of PYK2 and STAT3 signaling. Molecular and pharmacological targeting of PYK2 specifically induced cell death and reduced clonogenic growth in BMSC-adherent myeloma cell lines, aldehyde dehydrogenase-positive MM cancer stem cells and patient specimens. Finally, PYK2 inhibition similarly attenuated MM progression in vivo. These data identify a novel PYK2-mediated survival pathway in MM cells and MM cancer stem cells within the context of microenvironmental cues, providing preclinical support for the use of the clinical stage FAK/PYK2 inhibitors for treatment of MM, especially in a minimal residual disease setting.

Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

Retraction Note To: Functional activation of Proline-rich Tyrosine Kinase2 (PYK2) in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a representative systemic autoimmune disease characterized by activated T cells and polyclonally activated B cells that produce autoantibodies. Activation of autoreactive T and B cells plays a pivotal role in the pathogenesis of this disease. A role of focal adhesion kinase (FAK) in the pathogenesis has been suggested. Proline-rich tyrosine kinase2 (PYK2) is structurally related to FAK, however, the functional activation of PYK2 in SLE remains unclear. In the present study, we showed that PYK2 is significantly increased and activated in peripheral blood mononuclear cells (PBMCs) of patients with SLE. In addition, we showed the involvement of PYK2 proteins in the up-regulation of CD40L and CTLA4 expression and PBMC proliferation.

Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation.

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.

184 Increasing Insulin Sensitivity in the Endothelium Leads to Reduced Nitric Oxide Bioavailability

<h3>Introduction</h3> Insulin resistance is known to precede Type 2 diabetes (T2DM). Insulin mediated release of the endothelial cell (EC) derived anti-atherosclerotic molecule, nitric oxide (NO) is blunted in patients suffering from insulin resistance T2DM. We examined the effects of enhancing EC insulin sensitivity <i>in vivo</i>, by generating a novel transgenic mouse, over-expressing Type A human Insulin Receptor (HIRECO) restricted to EC. <h3>Methods</h3> Western blotting and RT-PCR were carried out on tissues and isolated endothelial cells from lungs to measure protein levels and mRNA expression, respectively. NADPH-dependent lucigenin-enhanced chemiluminescence was used to measure superoxide anion levels. Isolated thoracic aortic rings suspended in an organ bath were used to determine vasomotor functions. eNOS activity was examined by citrulline assay with ¹⁴C-labelled L-arginine. HIRECO were compared to wild type littermates. <h3>Results</h3> Over-expressing human insulin receptors in EC had no significant effect on morphological features, metabolic phenotype or blood pressure of HIRECO. HIRECO demonstrated significant EC dysfunction measured by a blunted endothelium-dependent vasorelaxation to acetylcholine and reduced basal NO release. EC-independent response to sodium nitroprusside remained unchanged. EC dysfunction observed in the organ bath was normalised by a NADPH oxidase-specific inhibitor peptide, gp91ds-tat as well as the superoxide dismutase mimetic, MnTmPyp. HIRECO demonstrated significant increase in superoxide anion release compared to WT littermates. This data was supported by a concomitant increase in NADPH oxidase isoform, NOX2 protein expression. Basal eNOS and Akt phosphorylation levels in isolated EC of HIRECO mice were enhanced compared to WT mice. Interestingly, insulin-stimulated eNOS phosphorylation and activation was decreased, whereas Akt phosphorylation remained unchanged. eNOS tyrosine phosphorylation mediated by proline-rich tyrosine kinase (PYK2) was significantly enhanced in EC from HIRECO mice. In order to investigate, if the perturbations of insulin signalling in the EC have a pathological outcome, HIRECO mice were crossed with ApoE-knockout mice. These mice demonstrated a significant enhancement of plaque formation in aorta, as well as the aortic root. <h3>Conclusions/implications</h3> These data show that increasing EC insulin sensitivity leads to reduced bioavailability of NO. These data demonstrate for the first time that increased insulin signalling in EC increases the generation of superoxide anion via activation of NOX₂ NADPH oxidase and reduced NO production in response to insulin due at least in part to increased EC PYK2 activity leading to a pro-atherosclerotic state.

Hypoxia-driven sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) downregulation depends on low-density lipoprotein receptor-related protein 1 (LRP1)-signalling in cardiomyocytes

The maintenance of sarcoplasmic reticulum Ca2+ ATPase (SERCA2) activity is crucial for cardiac function and SERCA2 is dramatically reduced in the heart exposed to hypoxic/ischemic conditions. Previous work from our group showed that hypoxia upregulates the phosphorylated form of the Ca2+-dependent nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (pPyk2) protein levels in a low-density lipoprotein receptor-related protein (LRP1)-dependent manner. Pyk2 in turn may modulate SERCA2 in cardiomyocytes although this remains controversial. We therefore aimed to investigate the role of LRP1 on hypoxia-induced SERCA2 depletion in cardiomyocytes and to establish LRP1 signalling mechanisms involved. Western blot analysis showed that hypoxia reduced SERCA2 concomitantly with a sustained increase in LRP1 and pPyk2 protein levels in HL-1 cardiomyocytes. By impairing hypoxia-induced Pyk2 phosphorylation and HIF-1α accumulation, LRP1 deficiency prevented SERCA2 depletion and reduction of the sarcoplasmic reticulum calcium content in cardiomyocytes. Moreover, the inhibition of Pyk2 phosphorylation (with the Src-family inhibitor PP2) or the specific silencing of Pyk2 (with siRNA-anti Pyk2) preserved low HIF-1α and high SERCA2 levels in HL-1 cardiomyocytes exposed to hypoxia. We determined that the LRP1/Pyk2 axis represses SERCA2 mRNA expression via HIF-1α since HIF-1α overexpression abolished the protective effect of LRP1 deficiency on SERCA2 depletion. Our findings show a crucial role of LRP1/Pyk2/HIF-1α in hypoxia-induced cardiomyocyte SERCA2 downregulation, a pathophysiological process closely associated with heart failure.

Abstract 69: Possible Involvement Of Proline-rich Tyrosine Kinase 2 (pyk2) In The Pathogenesis Of Kawasaki Disease

Introduction: Although etiology of Kawasaki disease (KD) remains elusive, a line of recent experimental studies implies that some kinds of infectious stimuli are implicate in the vasculitis through uncontrolled innate immune systems such as pattern recognition receptor (PPR)-mediated inflammatory signaling. It has already known that Candida albicans water-soluble fraction (CAWS) inducing KD-like vasculitis in mice function through PRP. Furthermore, it is reported that proline-rich tyrosine kinase (Pyk2), which is molecule involved in the PRPs-dependent signaling pathways, plays an important role in activation of NF-κB. Therefore, we investigated a possible relevance of Pyk2 in the pathogenesis of KD. Methods: Pyk2-knock out (Pyk2-KO) and wild-type C57BL/6 mice (WT) were administered CAWS to induce KD-like vasculitis. Extension of the experimental vasculitis was immunohistochemically determined with anti-MPO antibody. CAWS-stimulated NF-κB activation was evaluated by quantifying nuclear translocation of NF-κB p65 subunit in peritoneal macrophages isolated from PYK2-KO and wild-type mice in vitro. Cytokines and chemokines across each mice were compared by cytokine array. Results: Pyk2-KO mice didn’t show any apparent defective phenotype. While marked inflammation was observed in the aortic root of CAWS-treated WT mice, such vasculitis was barely detected in CAWS-treated Pyk2-KO mice. CAWS-induced NF-κB activation was also less observed in macrophages from Pyk2-KO mice. There were differences in some cytokines and chemokines production between mice. Conclusion: We speculate that Pyk2 is involved in the pathogenesis of KD. Pyk2 might be a potential therapeutic target for KD.

S-Nitrosylation of proline-rich tyrosine kinase 2 involves its activation induced by oxygen–glucose deprivation

Previous studies have demonstrated that activation of proline-rich tyrosine kinase 2 (PYK2) in cerebral ischemia is involved in the modulation of N-methyl-d-aspartate-type (NMDA) glutamate receptor activity and Ca2+ dynamics, resulting in ischemic neuron death ultimately. A number of reports indicate that PYK2 is a redox sensitive kinase that must be activated by an estrogen-induced reactive oxygen species (ROS). However, the mechanism of PYK2 activation remains incompletely illustrated. Accumulating attention is focused on nitric oxide (NO, a free radical) which plays a critical role in cellular signal transduction through stimulus-coupled S-nitrosylation of cysteine residues. Here we reported that PYK2 over-expressed in human embryonic kidney (HEK293) cells was S-nitrosylated (forming SNO–PYK2) by reacting with GSNO, an exogenous NO donor, at one critical cysteine residue (Cys534) with a biotin switch assay. Moreover, our results showed that S-nitrosylation and phosphorylation of PYK2 over-expressed in SH-SY5Y cells was significantly increased after oxygen–glucose deprivation (OGD). We further investigated whether the activation (phosphorylation) of PYK2 was associated with S-nitrosylation following SH-SY5Y cells OGD. Our results showed that the cysteine534 residue (site of S-nitrosylation) mutant PYK2 over-expressed in SH-SY5Y cells diminished S-nitrosylation of PYK2 and inhibited its phosphorylation induced by OGD. In addition, overexpression of the mutant PYK2 protein could prevent nuclear accumulation and abrogate neuronal cell death compared to wild type PYK2 in SH-SY5Y cells induced by OGD. These data suggest that the activation of PYK2 following OGD may be modulated by S-nitrosylation, which provides a new avenue for stroke therapy by targeting the post-translational modification machinery.

Regulation of NHE3 by lysophosphatidic acid is mediated by phosphorylation of NHE3 by RSK2.

Na(+)/H(+) exchange by Na(+)/H(+) exchanger 3 (NHE3) is a major route of sodium absorption in the intestine and kidney. We have shown previously that lysophosphatidic acid (LPA), a small phospholipid produced ubiquitously by all types of cells, stimulates NHE3 via LPA5 receptor. Stimulation of NHE3 activity by LPA involves LPA5 transactivating EGF receptor (EGFR) in the apical membrane. EGFR activates proline-rich tyrosine kinase 2 (Pyk2) and ERK, both of which are necessary for NHE3 regulation. However, Pyk2 and ERK are regulated by EGFR via independent pathways and appear to converge on an unidentified intermediate that ultimately targets NHE3. The p90 ribosomal S6 kinase (RSK) family of Ser/Thr protein kinases is a known effector of EGFR and ERK. Hence, we hypothesized that RSK may be the convergent effector of Pyk2 and ERK although it is not known whether Pyk2 regulates RSK. In this study, we show that Pyk2 is necessary for the maintenance of phosphoinositide-dependent kinase 1 (PDK1) autophosphorylation, and knockdown of Pyk2 or PDK1 mitigated LPA-induced phosphorylation of RSK and stimulation of NHE3 activity. Additionally, we show that RSK2, but not RSK1, is responsible for NHE3 regulation. RSK2 interacts with NHE3 at the apical membrane domain, where it phosphorylates NHE3. Alteration of S663 of NHE3 ablated LPA-induced phosphorylation of NHE3 and stimulation of the transport activity. Our study identifies RSK2 as a new kinase that regulates NHE3 activity by direct phosphorylation.