Proline-rich Tyrosine Kinase 2 Phosphorylation Research Articles

Long Non-Coding RNA LINC00052 Targets miR-548p/Notch2/Pyk2 to Modulate Tumor Budding and Metastasis of Human Breast Cancer.

Abnormal expression of long non-coding RNAs (lncRNAs) is involved in many pathological processes of cancers. However, the role of lncRNA LINC00052 in breast cancer progression is still unclear. Here, LINC00052 expression was detected by in situ hybridization and quantitative real-time PCR assays. Cell Counting Kit-8, wound healing, and transwell assays were used to investigate changes in the proliferation, migration, and invasion of breast cancer cells. MiR-548p was found associated with LINC00052 or Notch2 by RNA pull-down, dual-luciferase reporter, and qRT-PCR assays. The effect of LINC00052 on lung metastasis was explored through in vivo experiments. High LINC00052 expression was observed in breast cancer tissues and cells. LINC00052 silencing inhibited the proliferation, migration, and invasion of MCF7 cells, and LINC00052 overexpression produced the opposite results. MiR-548p, a target gene of LINC00052, partially rescued the effects of LINC00052 on proliferation, migration, and invasion of MCF7. Notch2 was the target of miR-548p and LINC00052 could promote Notch2 expression. Moreover, the phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a downstream factor of Notch2, was increased by LINC00052, and a Pyk2 mutant could inhibit the cell migration and invasion induced by LINC00052 overexpression in MDA-MB-468 cells, which was similar to the function of the miR-548p mimic. We further demonstrated that LINC00052 exacerbated the metastases of breast cancer cells in vivo. Our research demonstrated that LINC00052 is highly expressed in breast cancer and promotes breast cancer proliferation, migration, and invasion via the miR-548p/Notch2/Pyk2 axis. LINC00052 could serve as a potential therapeutic target for breast cancer.

TAMI-50. PATHWAYS ACTIVATION IN GL261 MICE GLIOMA CELLS BY HIV-1 GLYCOPROTEIN GP120

Abstract Patients infected with human immunodeficiency virus type 1 (HIV-1) are more prone to developing cancers, including glioblastomas (GBMs). The median survival for GBM patients with HIV is significantly shorter than for HIV-negative GBM patients, even though they receive the same treatments. This difference indicates that HIV infection is associated with more aggressive tumor behavior and with treatment resistance. Earlier we demonstrated that gp120, a main glycoprotein in the HIV shell, stimulates glycolysis and protein synthesis in glioma cells. The purpose of this study was to evaluate the underlying gp120 dependent signaling mechanism in glioma cell. Mouse glioma cells GL-261 were continuously cultured for 7 days in medium with and without soluble gp120 Bal III (100ng/ml) and collected for Western blot and Cell cycle assays. Western blot analysis presented an increase in phosphorylation of Proline-rich tyrosine kinase (Pyk2(Y402)), p38(YT100/Y182) and p70s6(T421/S424), the key proteins of the Pyk2 pathway, along with the increased levels of Akt(S473) and Glycogen Synthase Kinase 3b (GSK3b (S9)) phosphorylation. Flow cytometry analysis of Cell Cycle revealed an increase of G2/M phase in cells cultured in gp120 Bal III when compared to control cells. Furthermore, GL-261 cells with knock-out of Pyk2 via CRISPR Cas 9 gene editing showed no significant change in cell cycle regulation when cultured with gp120 Bal III.Overall, these results demonstrate that gp120 triggers activation of Pyk2/MAPK and Akt/GSK3b pathways and alter cell cycle regulation in GBM. This research was made possible by NIH grant number 1SC1GM122691.

Pyk2 tyrosine kinase conformations probed by molecular imprinted nanoparticles

Proline-rich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK) encompass a subfamily of nonreceptor tyrosine kinases. Whereas FAK is activated by clustering at focal adhesion membranes, PYK2 can be activated Ca2+-flux. Nevertheless, both kinases share some activation mechanistic details. The release of a FERM domain-mediated autoinhibitory conformation leads to autophosphorylation of a tyrosine located in the FERM-kinase linker. The phosphorylated tyrosine forms a docking site for Src kinase which contributes to full activation. While the conformation of the PYK2 autophosphorylation site remains unknown, the structure of the FAK FERM−kinase revealed that the corresponding regulatory site is sequestered in an abbreviated β sheet. We have recently reported that peptide-binding molecular imprinted nanoparticles (MINPs) targeting the consensus PYK2 phosphorylation site (Y402) and surroundings are able to inhibit autophosphorylation. However, the degree of inhibition was highly influenced by the putative conformation of the target binding site. We sought to further explore the association of MINP target conformational accessibility and inhibitor potency. In vitro kinase assays of PYK2 FERM-kinase indicate the ability of MINPs to inhibit the autophosphorylation of Y402. MINPs were designed to bind three distinct regions of the linker sequence. Autophosphorylation inhibition was highly dependent on linker region. Interestingly, the MINP impinging directly on the Y402 site exhibited the weakest inhibition among the three MINPs tested. The differential impact between linker target sites suggests that the PYK2 Y402 autophosphorylation site may also be constrained via secondary structure in the autoinhibited conformation. Taken together, we validate that MINPs can serve as inhibitors of protein kinase phosphorylation. We continue to explore the intriguing possibility that MINPs can serve as conformational probes.

Proline‐Rich Tyrosine Kinase Phosphorylation's Effect On the Na+/H+ Exchanger Isoform 1

The Na+‐H+ exchanger isoform 1 (NHE1) is a 12‐pass transmembrane protein that functions by exchanging one extracellular sodium ion for one intracellular proton. The transmembrane domain of NHE1 is involved in ion exchange, and the cytoplasmic tail is the regulatory domain which includes a series of phosphorylation sites as well as lipid and proteins binding sites that alter transport activity. NHE1 is a central driver of the hallmarks of cancer through its involvement in the regulation of cellular proliferation, migration, and invasion. While the exact number of phosphorylation sites on the cytoplasmic domain of NHE1 is still unclear, many studies have implicated 22 phosphorylation sites and 12 different kinases. One of the kinases believed to directly or indirectly lead to the phosphorylation of NHE1 at S602 and S605 is proline rich tyrosine kinase 2 (PyK2). To evaluate the role of PyK2 in NHE1 regulation we prepared three cell lines derived from Chinese hamster lung fibroblast lacking NHE1 expression (PS120 cells). These cell lines are PSN (PS120 cells expressing human NHE1), PSN S602A/S605A (PSN cells with serine to alanine mutations of the reported PyK2 phosphorylation sites) and PSN S602D/S605D (PSN cells with serine to aspartic acid mutations of the reported PyK2 phosphorylation sites to mimic phosphorylation). Some studies have found that PyK2 works inversely on NHE1 and causes inhibition upon phosphorylation, while other studies have shown PyK2 to enhance NHE1 activity. Here we evaluate the impact of PyK2 inhibition and mutation of the PyK2 phosphorylation sites on proliferation and migration. Western blot analysis is used to evaluate changes in Pyk2 expression and phosphorylation over time under low and normal serum conditions. Cell proliferation and cell migration are both evaluated. Our results suggest that serum stimulation increases Pyk2 phosphorylation and enhances both cell proliferation and cell migration. Inhibition of PyK2 by PF43I396 or the mutation of the Pyk2 phosphorylation sites on NHE1 decreases proliferation and migration rates in serum stimulated cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrogen Sulfide Preserves Endothelial Nitric Oxide Synthase Function by Inhibiting Proline-Rich Kinase 2: Implications for Cardiomyocyte Survival and Cardioprotection.

Hydrogen sulfide (H2S) exhibits beneficial effects in the cardiovascular system, many of which depend on nitric oxide (NO). Proline-rich tyrosine kinase 2 (PYK2), a redox-sensitive tyrosine kinase, directly phosphorylates and inhibits endothelial NO synthase (eNOS). We investigated the ability of H2S to relieve PYK2-mediated eNOS inhibition and evaluated the importance of the H2S/PYK2/eNOS axis on cardiomyocyte injury in vitro and in vivo. Exposure of H9c2 cardiomyocytes to H2O2 or pharmacologic inhibition of H2S production increased PYK2 (Y402) and eNOS (Y656) phosphorylation. These effects were blocked by treatment with Na2S or by overexpression of cystathionine γ-lyase (CSE). In addition, PYK2 overexpression reduced eNOS activity in a H2S-reversible manner. The viability of cardiomyocytes exposed to Η2Ο2 was reduced and declined further after the inhibition of H2S production. PYK2 downregulation, l-cysteine supplementation, or CSE overexpression alleviated the effects of H2O2 on H9c2 cardiomyocyte survival. Moreover, H2S promoted PYK2 sulfhydration and inhibited its activity. In vivo, H2S administration reduced reactive oxygen species levels, as well as PYK2 (Y402) and eNOS (Y656) phosphorylation. Pharmacologic blockade of PYK2 or inhibition of PYK2 activation by Na2S reduced myocardial infarct size in mice. Coadministration of a PYK2 inhibitor and Na2S did not result in additive effects on infarct size. We conclude that H2S relieves the inhibitory effect of PYK2 on eNOS, allowing the latter to produce greater amounts of NO, thereby affording cardioprotection. Our results unravel the existence of a novel H2S-NO interaction and identify PYK2 as a crucial target for the protective effects of H2S under conditions of oxidative stress.

CF airway smooth muscle transcriptome reveals a role for PYK2.

Abnormal airway smooth muscle function can contribute to cystic fibrosis (CF) airway disease. We previously found that airway smooth muscle from newborn CF pigs had increased basal tone, an increased bronchodilator response, and abnormal calcium handling. Since CF pigs lack airway infection and inflammation at birth, these findings suggest intrinsic airway smooth muscle dysfunction in CF. In this study, we tested the hypothesis that CFTR loss in airway smooth muscle would produce a distinct set of changes in the airway smooth muscle transcriptome that we could use to develop novel therapeutic targets. Total RNA sequencing of newborn wild-type and CF airway smooth muscle revealed changes in muscle contraction-related genes, ontologies, and pathways. Using connectivity mapping, we identified several small molecules that elicit transcriptional signatures opposite of CF airway smooth muscle, including NVP-TAE684, an inhibitor of proline-rich tyrosine kinase 2 (PYK2). In CF airway smooth muscle tissue, PYK2 phosphorylation was increased and PYK2 inhibition decreased smooth muscle contraction. In vivo NVP-TAE684 treatment of wild-type mice reduced methacholine-induced airway smooth muscle contraction. These findings suggest that studies in the newborn CF pig may provide an important approach to enhance our understanding of airway smooth muscle biology and for discovery of novel airway smooth muscle therapeutics for CF and other diseases of airway hyperreactivity.

Tyrosine phosphorylation of eNOS regulates myocardial survival after an ischaemic insult: role of PYK2.

Endothelial nitric oxide (NO) synthase (eNOS) is known to play a cardioprotective protective. However, the molecular mechanisms regulating eNOS activity during ischaemia/reperfusion (I/R) injury are incompletely understood. eNOS is a substrate for several kinases that positively or negatively affect its enzymatic activity. Herein, we sought to correlate eNOS phosphorylation status with cardiomyocyte survival and we investigated the contribution of the proline-rich tyrosine kinase 2 (PYK2)/eNOS axis to the regulation of myocardial infarct size in vivo. Exposure of H9c2 cardiomyocytes to H2O2 lead to PYK2 phosphorylation on its activator site (Y402) and eNOS phosphorylation on the inhibitor site Y656 and the activator site S1176. Both H2O2-induced eNOS phosphorylation events were abolished by PYK2 pharmacological inhibition or gene knockdown. Activity assays demonstrated that phosphorylation of the tyrosine inhibitory site exerts a dominant effect over S1176. In cardiomyocytes subjected to oxidative stress or oxygen-glucose deprivation, inhibition of PYK2 limited cell injury; this effect was prevented by inhibition of NO production. In vivo, ischaemia-reperfusion induced an early activation of PYK2, leading to eNOS phosphorylation on Y656, which, in turn, reduced NO output, as judged by the low tissue levels of its downstream effector cGMP. Moreover, pharmacological blockade of PYK2 alleviated eNOS inhibition and prevented cardiac damage following I/R injury in wild-type, but not in eNOS KO mice. The current studies demonstrate that PYK2 is a pivotal regulator of eNOS function in myocardial infarction and identify PYK2 as a novel therapeutic target for cardioprotection.

CCL18 from ascites promotes ovarian cancer cell migration through proline-rich tyrosine kinase 2 signaling.

BackgroundOvarian cancer (OC) ascites consist in a proinflammatory tumor environment that is characterized by the presence of various cytokines, chemokines and growth factors. The presence of these inflammatory-related factors in ascites is associated with a more aggressive tumor phenotype. CCL18 is a member of CCL chemokines and its expression has been associated with poor prognosis in some cancers. However, its role in OC progression has not been established. Therefore, the aim of the current study was to elucidate the role of ascites CCL18 in OC progression.MethodsELISA and tissue microarrays were used to assess CCL18 in ascites and phospho-Pyk2 expression in cancer tissues respectively. Cell migration was assessed using Boyden chambers. CCL18 and ascites signaling was examined in ovarian cancer cells utilizing siRNA and exogenous gene expression.ResultsHere, we show that CCL18 levels are markedly increased in advanced serous OC ascites relative to peritoneal effusions from women with benign conditions. Ascites and CCL18 dose-dependently enhanced the migration of OC cell lines CaOV3 and OVCAR3. CCL18 levels in ascites positively correlated with the ability of ascites to promote cell migration. CCL18 blocking antibodies significantly attenuated ascites-induced cell migration. Ascites and CCL18 stimulated the phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) in CaOV3 and OVCAR3 cells. Most importantly, the expression of phosphorylated Pyk2 in serous OC tumors was associated with shorter progression-free survival. Furthermore, enforced expression of Pyk2 promoted tumor cell migration while siRNA-mediated downregulation of Pyk2 attenuated cell migration. Downregulation of Pyk2 markedly inhibited ascites and CCL18-induced cell migration.ConclusionsTaken together, our findings establish an important role for CCL18, as a component of ascites, in the migration of tumor cells and identify Pyk2 as prognostic factor and a critical downstream signaling pathway for ascites-induced OC cell migration.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0542-2) contains supplementary material, which is available to authorized users.

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.

Abstract PR08: Microparticles derived from drug-resistant cells regulate miR-503 and PYK2 to promote migration and invasion in breast cancer

Abstract Introduction: Drug resistance and metastatic spread are two of the most malicious aspects of cancer progression. Microparticles (MPs) have previously been implicated in the spread of these phenotypes independently. We now demonstrate that these two characteristics are linked, with the MP-mediated acquisition of drug resistance correlating with the emergence of an enhanced metastatic capacity. In this way, MPs serve as a conduit between drug resistance and metastasis. Therefore, addressing the impact of MPs may be a means of managing both of these deleterious aspects simultaneously. This makes MPs a significant and viable target in the management of cancer. Methods and results: We used microarray analysis to identify regulatory microRNAs (miRNAs) contributing to the dissemination of the metastatic trait in our cancer model. miR-503 was downregulated in recipient cells following coculture with MPs isolated from drug resistant cells. This was inversely associated with metastatic capacity, as demonstrated in wound healing/scratch migration assays and Matrigel®-coated transwell invasion assays. We also found that the proline-rich tyrosine kinase 2 (PYK2) gene transcript and miRNAs associated with its regulation, were upregulated in recipient cells following co-culture with MPs derived from drug-resistant cells. Elevated PYK2 was accompanied with increased migration and invasion, with these phenotypes reversible in the presence of a pharmacological inhibitor of PYK2 phosphorylation, tyrphostin A9. However, the MP-mediated promotion of metastatic traits was not due to the direct transfer of effectors in the selectively packaged MP cargo. Rather, acquired PYK2 protein and transcripts were downstream effectors of components within the cargo itself. Therefore, the consideration of the role of MPs in trait acquisition extends beyond the direct transfer of components within the cargo to also include the transfer of intermediary regulators, resulting in alterations in the proteomic and transcriptional landscape of the recipient cells. This implicates the expanding role of MPs in the deleterious survival mechanisms of cancer pathogenesis. This research is funded by the NHMRC (APP1007613). Note: These results were published in Frontiers in Oncology as a full-length article before the online publication of this abstract. Citation information: Microparticles mediate the intercellular regulation of microRNA-503 and proline-rich tyrosine kinase 2 to alter the migration and invasion capacity of breast cancer cells. Gong J, Luk F, Jaiswal R, Bebawy M. Front Onc. 2014 Aug 15;4:220. doi: 10.3389/fonc.2014.00220. eCollection 2014. Citation Format: Joyce Gong, Frederick Luk, Ritu Jaiswal, Mary Bebawy. Microparticles derived from drug-resistant cells regulate miR-503 and PYK2 to promote migration and invasion in breast cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR08.

Store-operated Ca(2+) entry regulates glioma cell migration and invasion via modulation of Pyk2 phosphorylation.

BackgroundThe ubiquitous second messenger Ca2+ has been demonstrated to play an important role in cancer progression. Store-operated Ca2+ entry (SOCE) is the main Ca2+ entry pathway regulating intracellular Ca2+ concentration in a variety of cancer types. The present study aimed to explore the specific mechanisms of SOCE in the processes of glioma migration and invasion.MethodsThe expression of Orai1, a key component of SOCE, was examined in glioma samples and glioma cell lines by immunohistochemistry and western blot analysis. Both pharmacological intervention and RNA interference were employed to investigate the role of SOCE in glioma cell migration and invasion in vitro. The intracellular Ca2+ was certified through Fluo-4/AM based Ca2+ measurement. The effect of SOCE on cell viability, migration, and invasion was explored by methyl thiazolyl tetrazolium (MTT) assay, wound healing assay, transwell invasion assay. Western blot analysis and immunofluorescence assay were used to observe the changes of downstream related protein and cell morpholog.ResultsOrai1 expression was elevated in glioma tissues and several glioma cell lines compared with non-neoplastic brain tissues. Either inhibition of SOCE by a pharmacological inhibitor or Orai1 downregulation suppressed glioma cell migration and invasion. However, re-expression of Orai1 could rescue glioma cell motility. Furthermore, phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) participated in the mechanisms by which SOCE regulated focal adhesion turnover and epithelial-to-mesenchymal (−like) transition in glioma cells, both of which are considered to be critical for tumor progression.ConclusionsThe SOCE-Pyk2 pathway is essential for glioma migration and invasion. The study indicates the potential value of Orai1 as a molecular target for anti-invasion therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-014-0098-1) contains supplementary material, which is available to authorized users.

Microparticles Mediate the Intercellular Regulation of microRNA-503 and Proline-Rich Tyrosine Kinase 2 to Alter the Migration and Invasion Capacity of Breast Cancer Cells.

The successful treatment of cancer is hampered by drug resistance and metastasis. While these two obstacles were once considered separately, recent evidence associates resistance with an enhanced metastatic capacity. However, the underlying mechanisms remain undefined. We previously described the intercellular transfer of drug resistance via submicron vesicles called microparticles (MPs). We now propose that MPs derived from drug-resistant cells are also involved in the intercellular transfer of components to enhance the migration and invasion capacity of cells. Thus, MPs may be a conduit between resistance and metastasis. We used microarray analysis to identify regulatory microRNAs (miRNAs), which contribute to the dissemination of metastatic traits. miR-503 was downregulated in recipient cells following co-culture with MPs isolated from drug-resistant cells. miR-503 was inversely associated with metastasis, as demonstrated using wound healing/scratch migration assays and Matrigel®-coated transwell invasion assays. Proline-rich tyrosine kinase 2 (PYK2) was upregulated in recipient cells and associated with increased migration and invasion, with these phenotypes being reversed using a pharmacological inhibitor of PYK2 phosphorylation, tyrphostin A9. However, the MP-mediated promotion of metastatic traits was not due to the presence of these effectors in the MP cargo but rather due to down stream effector molecules in these pathways. This is the first demonstration that the role of MPs in trait acquisition extends beyond the direct transfer of vesicle components and also includes transfer of intermediary regulators that induce down stream mediators following transfer to recipient cells. This implicates an expanding role of MPs in cancer pathogenesis.

PYK2 selectively mediates signals for growth versus differentiation in response to stretch of spontaneously active vascular smooth muscle.

Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium‐dependent signals. We studied the role of the calcium‐ and integrin‐activated proline‐rich tyrosine kinase 2 (PYK2) in stretch‐induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr‐402 was increased both by a 10‐min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF‐4594755 (0.5–1 μmol/L), while still sensitive to stretch. In 3‐day organ culture, PF‐4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high‐K+ (60 mmol/L) or to α1‐adrenergic stimulation by cirazoline. Basal and stretch‐induced PYK2 phosphorylation in culture were inhibited by PF‐4594755, closely mimicking inhibition of non‐voltage‐dependent calcium influx by 2‐APB (30 μmol/L). In contrast, the L‐type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch‐induced but not basal PYK2 phosphorylation. Stretch‐induced Akt and ERK1/2 phosphorylation was eliminated by PF‐4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch‐sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 μmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage‐ and non‐voltage‐dependent calcium influx. A small‐molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle.

Store-operated Ca 2+ entry regulates glioma cell migration and invasion via modulation of Pyk2 phosphorylation

The ubiquitous second messenger Ca2+ has been demonstrated to play an important role in cancer progression. Store-operated Ca2+ entry (SOCE) is the main Ca2+ entry pathway regulating intracellular Ca2+ concentration in a variety of cancer types. The present study aimed to explore the specific mechanisms of SOCE in the processes of glioma migration and invasion. The expression of Orai1, a key component of SOCE, was examined in glioma samples and glioma cell lines by immunohistochemistry and western blot analysis. Both pharmacological intervention and RNA interference were employed to investigate the role of SOCE in glioma cell migration and invasion in vitro. The intracellular Ca2+ was certified through Fluo-4/AM based Ca2+ measurement. The effect of SOCE on cell viability, migration, and invasion was explored by methyl thiazolyl tetrazolium (MTT) assay, wound healing assay, transwell invasion assay. Western blot analysis and immunofluorescence assay were used to observe the changes of downstream related protein and cell morpholog. Orai1 expression was elevated in glioma tissues and several glioma cell lines compared with non-neoplastic brain tissues. Either inhibition of SOCE by a pharmacological inhibitor or Orai1 downregulation suppressed glioma cell migration and invasion. However, re-expression of Orai1 could rescue glioma cell motility. Furthermore, phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) participated in the mechanisms by which SOCE regulated focal adhesion turnover and epithelial-to-mesenchymal (−like) transition in glioma cells, both of which are considered to be critical for tumor progression. The SOCE-Pyk2 pathway is essential for glioma migration and invasion. The study indicates the potential value of Orai1 as a molecular target for anti-invasion therapy.

Hypoxia Induces Metalloproteinase-9 Activation and Human Vascular Smooth Muscle Cell Migration Through Low-Density Lipoprotein Receptor–Related Protein 1–Mediated Pyk2 Phosphorylation

Hypoxia disturbs vascular function by promoting extracellular matrix remodeling. Extracellular matrix integrity and composition are modulated by metalloproteinases (MMPs). Our aim was to investigate the role of low-density lipoprotein receptor-related protein 1 (LRP1) in regulating MMP-9/MMP-2 activation and vascular smooth muscle cells (VSMCs) migration in response to hypoxia, and to elucidate the LRP1-signaling pathways involved in this process. Western blot analysis showed that hypoxia induced a sustained phosphorylation of proline-rich tyrosine kinase 2 concomitantly with LRP1 overexpression in human VSMCs (hVSMCs). Deletion of LRP1 using small-interfering RNA technology or treatment of hVSMCs with the Src family kinase inhibitor PP2 impaired hypoxia-induced phosphorylation of proline-rich tyrosine kinase 2 levels. Coimmunoprecipitation experiments showed that the higher amounts of phosphorylation of proline-rich tyrosine kinase 2/LRP1β immunoprecipitates in hypoxic hVSMCs were abolished in PP2-treated hVSMCs. Both LRP1 silencing and PP2 treatment were highly effective in the prevention of hypoxia-induced MMP-9 activation and hVSMC migration. Cellular subfractionation experiments revealed that PP2 effects may be caused by impairment of hypoxia-induced nuclear factor-κβ translocation to the nucleus. ELISA measurements showed that LRP1 silencing but not PP2 treatment increased interleukin-1β, interleukin-6, and monocyte chemoattractant protein-1 secretion by hypoxic hVSMCs. Our findings determine a crucial role of LRP1-mediated Pyk2 phosphorylation on hypoxia-induced MMP-9 activation and hVSMC migration and therefore in hypoxia-induced vascular remodeling. Both LRP1 silencing and PP2 treatments also influence hypoxia-induced proinflammatory effects in hVSMCs. Therefore, further studies are required to establish therapeutical strategies that efficiently modulate vascular remodeling and inflammation associated with hypoxia-vascular diseases.

Glycogen synthase kinase-3β is critical for Interferon-α-induced serotonin uptake in human Jurkat T cells

Dysregulation of glycogen synthase kinase (GSK)-3β contributes to the pathophysiology of mood disorders. However, how its regulation is responsible for the functioning of serotonin (5-HT) requires further investigation. Although enhancement of T-cell function may present an alternative strategy to treat depression, the precise mechanisms have yet to be established. Our previous studies have found that interferon-alpha (IFN-α) up-regulates serotonin transporter (5-HTT) expression and induces 5-HT uptake in T cells. The present study is to examine GSK-3β regulation on IFN-α-induced 5-HTT functions. GSK-3β short hairpin RNAs (shRNAs) or GSK-3β inhibitors decreased IFN-α-induced 5-HT uptake and 5-HTT expression. Src activation and calcium/calcium-activated calmodulin kinase II (CaMKII) were involved in IFN-α-induced phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) (Tyr402) and GSK-3β (Tyr216), which regulated 5-HT uptake. GSK-3β knockdown blocked the IFN-α-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) and signal transducer and transactivator (STAT) 1. In addition to inhibiting ERK, a selective 5-HTT inhibitor fluoxetine blocked IFN-α-induced activations of Src, CaMKII-regulated Pyk2/GSK-3β cascade, as well as STAT1 activation and translocation. These results indicated that calcium/CaMKII- and Src-regulated Pyk2 participated in IFN-α-induced GSK-3β activation and GSK-3β-regulated 5-HT uptake. GSK-3β signaling facilitated IFN-α-activated STAT1 by regulating ERK1/2, which controlled 5-HT uptake. Fluoxetine interfered with the Pyk2/GSK-3β cascade, thereby inhibiting IFN-α-induced 5-HT uptake.

Focal Adhesion Kinase–Related Nonkinase Inhibits Vascular Smooth Muscle Cell Invasion by Focal Adhesion Targeting, Tyrosine 168 Phosphorylation, and Competition for p130 Cas Binding

Focal adhesion kinase-related nonkinase (FRNK), the C-terminal domain of focal adhesion kinase (FAK), is a tyrosine-phosphorylated, vascular smooth muscle cell (VSMC)-specific inhibitor of cell migration. FRNK inhibits both FAK and proline-rich tyrosine kinase 2 (PYK2) in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling. Adenovirally mediated gene transfer of green fluorescent protein-tagged, wild-type (wt) FRNK into balloon-injured rat carotid arteries confirmed that FRNK overexpression inhibited both FAK and PYK2 phosphorylation and downstream signaling in vivo. To identify which kinase was involved in regulating VSMC invasion, adenovirally mediated expression of specific short hairpin RNAs was used to knock down FAK versus PYK2 in cultured VSMCs, but only FAK short hairpin RNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Tyr168Phe-, Tyr232Phe-, and Tyr168,232Phe-) green fluorescent protein-FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Tyr168 phosphorylation was required for FRNK to inhibit invasion. Preventing Tyr168 phosphorylation also increased FRNK-paxillin interaction, as determined by coimmunoprecipitation, total internal reflection fluorescence microscopy, and fluorescence recovery after photobleaching. Furthermore, wtFRNK competed with FAK for binding to p130(Cas) (a critically important regulator of cell migration) and prevented its phosphorylation. However, Tyr168Phe-FRNK was unable to bind p130(Cas). We propose a 3-stage mechanism for FRNK inhibition: focal adhesion targeting, Tyr168 phosphorylation, and competition with FAK for p130 binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.

Neuropilin-1 Signaling through p130Cas Tyrosine Phosphorylation Is Essential for Growth Factor-Dependent Migration of Glioma and Endothelial Cells

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF) and plays an important role in mediating cell motility. However, the NRP1 signaling pathways important for cell motility are poorly understood. Here we report that p130(Cas) tyrosine phosphorylation is stimulated by hepatocyte growth factor and platelet-derived growth factor in U87MG glioma cells and VEGF in endothelial cells and is dependent on NRP1 via its intracellular domain. In endothelial cells, NRP1 silencing reduced, but did not prevent, VEGF receptor 2 (VEGFR2) phosphorylation, while expression of a mutant form of NRP1 lacking the intracellular domain (NRP1ΔC) did not affect receptor phosphorylation in U87MG cells or human umbilical vein endothelial cells (HUVECs). In HUVECs, NRP1 was also required for VEGF-induced phosphorylation of proline-rich tyrosine kinase 2, which was necessary for p130(Cas) phosphorylation. Importantly, knockdown of NRP1 or p130(Cas) or expression of either NRP1ΔC or a non-tyrosine-phosphorylatable substrate domain mutant protein (p130(Cas15F)) was sufficient to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular domain in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and identify a critical role for a novel NRP1-p130(Cas) pathway in the regulation of chemotaxis.

Hypertension Promotes Phosphorylation of Focal Adhesion Kinase and Proline-Rich Tyrosine Kinase 2 in Rats: Implication for the Pathogenesis of Hypertensive Vascular Disease

Atherosclerosis is initiated by adhesion and infiltration of inflammatory leukocytes into the intima, where non-receptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), play important roles as intracellular messengers of mechanical and biochemical signals. In the present study, we examined whether FAK and PYK2 are up-regulated by elevated blood pressure or circulating humoral factors in hypertension. We used a rat model of abdominal aortic banding that allows separate evaluation of elevated blood pressure (upper body) and circulating humoral factors (lower body). We obtained the proximal and distal aortas of the banding site, 6 hours, 3 days, and 1 and 4 weeks after the banding procedure, for evaluation of phosphorylation of FAK and PYK2 by Western blotting. Arterial pressure was significantly elevated only in the upper body throughout the experimental period. The expression of FAK and the FAK phosphorylation were significantly increased at 1 and 4 weeks only in the proximal aorta. This was also the case for the expression of total PYK2 and the PYK2 phosphorylation. In contrast, there was no significant change in FAK or PYK2 phosphorylation in the distal aorta, whereas plasma levels of angiotensin II were systemically elevated. In sham-operated rats, no change in FAK or PYK2 phoshorylation was noted in the proximal and distal aortas. These results indicate that phosphorylation of FAK and PYK2 is upregulated by elevated blood pressure but not by humoral factors in the rat aorta, demonstrating novel aspects of atherogenesis in hypertension.

SOCS3 inhibiting migration of A549 cells correlates with PYK2 signaling in vitro

BackgroundSuppressor of cytokine signaling 3 (SOCS3) is considered to inhibit cytokine responses and play a negative role in migration of various cells. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor kinase and has been found crucial to cell motility. However, little is known about whether SOCS3 could regulate PYK2 pro-migratory function in lung cancer.MethodsThe methylation status of SOCS3 was investigated in HBE and A549 cell lines by methylation-specific PCR. A549 cells were either treated with a demethylation agent 5-aza-2'-deoxycytidine or transfected with three SOCS3 mutants with various functional domains deleted. Besides, cells were pretreated with a proteasome inhibitor β-lactacystin where indicated. The effects of SOCS3 up-regulation on PYK2 expression, PYK2 and ERK1/2 phosphorylations were assessed by western blot using indicated antibodies. RT-PCR was used to estimate PYK2 mRNA levels. Transwell experiments were performed to evaluate cell migration.ResultsSOCS3 expression was found impaired in A549 cells and higher PYK2 activity was correlated with enhanced cell migration. We identified that SOCS3 was aberrantly methylated in the exon 2, and 5-aza-2'-deoxycytidine restored SOCS3 expression. Reactivation of SOCS3 attenuated PYK2 expression and phosphorylation, cell migration was inhibited as well. Transfection studies indicated that exogenous SOCS3 interacted with PYK2, and both the Src homology 2 (SH2) and the kinase inhibitory region (KIR) domains of SOCS3 contributed to PYK2 binding. Furthermore, SOCS3 was found to inhibit PYK2-associated ERK1/2 activity in A549 cells. SOCS3 possibly promoted degradation of PYK2 in a SOCS-box-dependent manner and interfered with PYK2-related signaling events, such as cell migration.ConclusionThese data indicate that SOCS3 negatively regulates cell motility and decreased SOCS3 induced by methylation may confer a migration advantage to A549 cells. These results also suggest a negative role of SOCS3 in PYK2 signaling, and a previously unidentified regulatory mechanism for PYK2 function.