Activation Of Proline-rich Tyrosine Kinase 2 Research Articles

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation.

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca2+ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca 2+ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca 2+ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca 2+ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca 2+ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca 2+ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH 4 Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca 2+ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.

Curcumin modulation of the activation of PYK2 in peripheral blood mononuclear cells from patients with lupus nephritis.

IntroductionProline-rich tyrosine kinase 2 (PYK2) provides important signals during the activation of lymphocytes, which is essential in autoimmune diseases. Systemic lupus erythematosus (SLE) is a representative autoimmune disease, and lupus nephritis (LN) is one of its most severe complications. Although glucocorticoid-binding immuno-suppression is the first-line therapy for patients with LN, the common and severe side effects of such treatment call for new strategies to improve long-term prognosis and life quality for these patients. Curcumin has been used to treat autoimmune disease with good curative effect, but little is known about the effect of curcumin on LN patients. Our aim was to investigate the mechanism of curcumin for management of LN, specifically regarding the PYK2 pathways.Material and methodsFreshly isolated peripheral blood mononuclear cells (PBMCs) from 20 LN patients and 20 healthy individuals were cultured and stimulated with either PMA, PMA+TyrA9 (PYK2 specific inhibitor), or PMA+Curcumin, and with PBS as control. After 48 hours of incubation, cells were harvested and the expression of PYK2, p-PYK2, CD40L, CTLA-4, and PBMCs proliferation were measured. Then the expression and activation of PYK2 was evaluated using Western blot, the expression of costimulatory molecules CD40L and CTLA-4 protein was evaluated using flow cytometry, and PBMC proliferation was assessed using a [3H]-thymidine incorporation assay.ResultsCurcumin inhibited the expression and activation of PYK2 in PBMCs in patients with LN in vitro. The inhibition rate of curcumin was negatively correlated with the level of serum complement, but positively correlated with 24-h proteinuria. Curcumin also suppressed the expression of costimulatory molecules CD40L and CTLA-4, as well as PBMC proliferation. Interestingly, these effects were not reproduced on PBMC cultures of healthy subjects.ConclusionsThe inhibition of PYK2 signalling protein may be one of the mechanisms underlying the action of curcumin in LN treatment.

PYK2 mediates BzATP-induced extracellular matrix proteins synthesis

Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2’(3)-Ο-(4-Benzoylbenzoyl) adenosine-5’-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts.BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression.These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation.

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.

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.

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.

Proline-Rich Tyrosine Kinase 2 Regulates Hippocampal Long-Term Depression

Proline-rich tyrosine kinase 2 (PYK2), also known as cell adhesion kinase beta or protein tyrosine kinase 2b, is a calcium-dependent signaling protein involved in cell migration. Phosphorylation of residue Y402 is associated with activation of PYK2 and leads to the recruitment of downstream signaling molecules. PYK2 was previously implicated in long-term potentiation (LTP); however, the role of PYK2 in long-term depression (LTD) is unknown. Here, we report that PYK2 is activated by NMDA receptor stimulation (chemical LTD) in cultured neurons. Small hairpin RNA-mediated knockdown of PYK2 blocks LTD, but not LTP, in hippocampal slice cultures. We find that the Y402 residue and, to a lesser extent, PYK2 kinase activity contribute to PYK2's role in LTD. Knockdown experiments indicate that PYK2 is required to suppress NMDA-induced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of PYK2 depresses NMDA-induced ERK phosphorylation and inhibits LTP, but not LTD. Our data indicate that PYK2 is critical for the induction of LTD, possibly in part by antagonizing ERK signaling in hippocampal neurons.

Functional activation of proline-rich tyrosine kinase2 (PYK2) in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

BackgroundSystemic 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.MethodsFreshly isolated PBMCs from 48 SLE patients, 32 patients with rheumatoid arthritis(RA) and 24 healthy individuals were analyzed for the expression and activation of PYK2 by western-blotting and immunocytochemistry. The other isolated PBMCs from patients with this condition were cultured and stimulated with PMA or TyrA9, and then the expression of costimulatory molecules CD40L and CTLA4 was evaluated using flow cytometry, PBMCs proliferation was determined with [3H]-thymidine incorporation (CPM).ResultsCompared with RA patients and healthy donors, PBMCs from SLE patients expressed more of both the total PYK2 protein and its activated/phosphorylated form. The increase of activated PYK2 protein in SLE PBMCs was correlated with the complication of nephritis and inversly associated the level of serum complements. In active SLE patients, activation of PYK2 in PBMCs is accompanying the increased cell proliferation and the induced expression of costimulatory molecules CD40L and CTLA4.ConclusionOur findings indicate that phosphorylated PYK2 in SLE PBMCs may induce the expression of CD40L and CTLA4, and subsequently the cell proliferation. PYK2 signaling enhances the autoreactive lymphocyte activation and plays an important role in the pathogenesis of SLE.

Activation Signal Transduction by Proline-rich Tyrosine Kinase 2 (PYK2) in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus

To explore the role of proline-rich tyrosine kinase 2 (PYK2) in systemic lupus erythematosus (SLE), we studied the expression, activation, and function of PYK2 in peripheral blood mononuclear cells (PBMC) from 36 SLE patients. As controls, samples from 19 patients with rheumatoid arthritis and 15 healthy individuals were studied simultaneously. We found a significant increase of both the total PYK2 protein and its activated/phosphorylated form in PBMCs from patients with SLE, particularly those with the complication of nephritis (WHO class IV). There is a clear correlation between the activation of PYK2 and the level of serum complements. In active SLE patients, activation of PYK2 in PBMCs accompanies the increased cell proliferation and the induced expression of co-stimulatory molecules CD40L and CTLA4. These results indicate that phosphorylated PYK2 may induce the expression of CD40L and CTLA4, and subsequently the cell proliferation. PYK2 signaling enhances the autoreactive lymphocyte activation and plays an important role in the pathogenesis of SLE.

Expression and Activation of Proline-Rich Tyrosine Kinase 2 (PYK2) in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus

This study analysed the expression and activation of proline-rich tyrosine kinase 2 (PYK2) in peripheral blood mononuclear cells (PBMCs) from 36 systemic lupus erythematosus (SLE) patients and explored whether activation of PYK2 correlates with disease activity or organ damage in SLE. Samples from 19 patients with rheumatoid arthritis (RA) and 15 healthy individuals were included as controls. There was a significant increase in both total PYK2 protein and its activated/phosphorylated form in PBMCs from patients with SLE, particularly in those with the complication of World Health Organization class IV lupus nephritis. There was a clear correlation between the activation of PYK2 and the level of serum complement, but no relationship was found between the activation of PYK2 and SLE Disease Activity Index (SLEDAI). As previous studies have shown that PYK2 provides important signals during the activation of lymphocytes, PYK2 could be a major contributor to the complex autoimmune pathogenesis of SLE.

Up-regulation of proline-rich tyrosine kinase 2 in non-small cell lung cancer

Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor tyrosine kinase, plays different roles in intracellular signaling pathways, that regulates a number of biological processes, such as cell proliferation, differentiation, adhesion and migration, which have been shown to correlate with tumor development and aggression. However, the involvement of PYK2 in human non-small cell lung cancer (NSCLC) has not yet been determined. In the present study, 90 patients with NSCLC (represented by adenocarcinoma and squamous cell carcinoma) were included retrospectively. NSCLC tissues were detected for the expression of PYK2 by immunohistochemistry. Correlation between the expression of PYK2 with the clinicopathological characteristics was analyzed. There were 64% (58 out of 90) of NSCLC patients with higher level of PYK2. Higher expression of PYK2 was significantly correlated with lymph node metastasis (node positive versus node negative, p=0.007). Patients with higher expression of PYK2 had advanced stage of NSCLCs (I+II versus III+IV, p=0.012). Protein level of PYK2 was also examined in 30 of these tumorous samples and matched non-tumorous counterparts by western blotting. PYK2 was apparently up-regulated in NSCLC tissues (tumor versus non-tumor, p=0.000). In the cell studies, extensive expression and activation of PYK2 were both found in higher metastatic BE1 cells. The activity of ERK1/2 in BE1 cells appeared extremely high as well. In conclusion, our results demonstrated that PYK2 is up-regulated in NSCLCs, and the higher expression and activation of PYK2 may play a role in modulating the activity of ERK1/2, and lead to the progression of NSCLC.

Mechanism of dietary salt-mediated increase in intravascular production of TGF-β1

Clinical and preclinical studies have demonstrated an important effect of arterial pathobiology on the progressive loss of renal function that occurs in chronic kidney disease. Chronic kidney disease, in turn, promotes alterations in vascular function. A modulating role for dietary salt has been suggested, with the amount of salt intake regulating endothelial cell production of transforming growth factor-beta1 (TGF-beta1), a fibrogenic growth factor that promotes arteriosclerosis and glomerulosclerosis. The purpose of the present studies was to determine how the interaction between dietary salt intake and vasculature promoted the production of TGF-beta1 in rats. Two different vascular tissues, aortic rings and glomeruli, were chosen for study. Dietary salt induced, in a dose-dependent fashion, activation of proline-rich tyrosine kinase-2 (Pyk2) and further identified c-Src as an important binding partner of Pyk2 in these tissues. Use of pharmacological inhibitors and dominant negative strategies confirmed that dietary salt induced complex formation of Pyk2 and c-Src with downstream activation of p38 and p42/44 mitogen-activated protein kinases and generation of TGF-beta1. The experiments defined the molecular signaling events that promoted the production of TGF-beta1, a key growth factor involved in the vascular response to increased salt intake.

Proline-Rich Tyrosine Kinase-2 (PYK2): A New Target for the Treatment of Hematopoietic Neoplasms with TEL-FGFR3 Fusion or FGFR3 Active Mutation.

Constitutive activation of Fibroblast Growth Factor 3 (FGFR3) tyrosine kinase have been identified in various human cancers and have been reported to play an important role in some hematopoietic neoplasms. We have previously reported that TEL-FGFR3 in a patient with peripheral T-cell Lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates PLCγ, PK3K, STAT3, STAT5, MAPK through its constitutive tyrosine kinase activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). In KMS-11, human multiple myeloma cell line which expresses constitutively active mutant FGFR3, activations of PI3K and STAT3 pathways have been reported. However, little is known about how FGFR3 tyrosine kinase (TK) activates these downstream molecules. Here, we show that PYK2, a member of focal adhesion kinases, plays a pivotal role for the activation of PI3K, STAT3 and STAT5 in FGFR3 oncogenic pathways, and is a candidate for therapeutic target. PP1/PP2, a kinase inhibitor of SRC and PYK2, inhibited the cell growth of TF-V5 and KMS-11 cells in a dose-dependent manner (IC50=15μM, 25μM respectively), not affecting the cell growth of IL-3 dependent Ba/F3 cells. Another specific SRC inhibitor did not affect the cell growth of TF-V5 and KMS-11 cells. TEL-FGFR3 transfection to Ba/F3 cells led to the overexpression of PYK2 but not FAK. Expression and phosphorylation of PYK2 were identified in KMS-11 cells. Immunoprecipitation analysis using FGFR3 TK inhibitor SU5402 showed that the activation of PYK2 which was recruited to FGFR3 was dependent on the kinase activity of FGFR3. The cell growth of TF-V5 was completely inhibited at the concentration of PP1/PP2(30μM), which inhibited auto-phosphorylation of PYK2. PP1/PP2 suppressed the activation of PI3K-ATK pathway and decreased expression of C-MYC, inducing G1-arrest of TF-V5. PP1/PP2 induced intrinsic apoptosis of TF-V5 and did not affect activation of BAX but decrease expression of BCL-2 and BCL-XL through inactivation of STAT3 and STAT5. PP1/PP2 also inhibited the activation of PI3K and STAT3 in KMS-11 cells, inducing G1-arrest and apoptosis. PP1/PP2 inhibited tyrosine kinase of PYK2 mesured by in vitro kinase assay (IC50=23μM, 13μM, respectively). Further PYK2 C-terminus Associated Protein (PAP) siRNA expression plasmid significantly decreased the proliferation of TF-V5 but not mock transfected Ba/F3 cells. Our data demonstrates that PYK2 is an attractive molecular target for FGFR3 associated hematopoietic neoplasm.

Depolarization Activates ERK and Proline-rich Tyrosine Kinase 2 (PYK2) Independently in Different Cellular Compartments in Hippocampal Slices

In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. Both are also activated under pathological conditions following ischemia, convulsions, or electroconvulsive shock. Although in non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known. We addressed this question using K(+)-depolarized rat hippocampal slices. Depolarization increased the phosphorylation of PYK2, SFKs, and ERK. These effects resulted from Ca(2+) influx through voltage-gated Ca(2+) channels and were diminished by GF109203X, a protein kinase C inhibitor. Inhibition of SFKs with PP2 decreased PYK2 tyrosine phosphorylation dramatically, but not its autophosphorylation on Tyr-402. Moreover, PYK2 autophosphorylation and total tyrosine phosphorylation were profoundly altered in fyn-/- mice, revealing an important functional relationship between Fyn and PYK2 in the hippocampus. In contrast, ERK activation was unaltered by PP2, Fyn knock-out, or LY294002, a phosphatidyl-inositol-3-kinase inhibitor. ERK activation was prevented by MEK inhibitors that had no effect on PYK2. Immunofluorescence of hippocampal slices showed that PYK2 and ERK were activated in distinct cellular compartments in somatodendritic regions and nerve terminals, respectively, with virtually no overlap. Activation of ERK was critical for the rephosphorylation of a synaptic vesicle protein, synapsin I, following depolarization, underlining its functional importance in nerve terminals. Thus, in hippocampal slices, in contrast to cell lines, depolarization-induced activation of non-receptor tyrosine kinases and ERK occurs independently in distinct cellular compartments in which they appear to have different functional roles.

Fibronectin Fragment Activation of Proline-rich Tyrosine Kinase PYK2 Mediates Integrin Signals Regulating Collagenase-3 Expression by Human Chondrocytes through a Protein Kinase C-dependent Pathway

Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the alpha5beta1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the alpha5beta1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCdelta inhibitor rottlerin. Furthermore, PKCdelta translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after cotransfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves FN-f stimulation of the alpha5beta1 integrin and activation of the nonreceptor tyrosine kinase PYK2 by PKC, most likely PKCdelta

Chronic ethanol feeding impairs endothelin-1-stimulated glucose uptake via decreased G alpha 11 expression in rat adipocytes.

Chronic ethanol feeding decreases insulin-stimulated glucose uptake in rat adipocytes. Here, we show that chronic ethanol also decreases endothelin-stimulated glucose uptake. Endothelin-1 increased uptake of 2-deoxyglucose 2.4-fold in adipocytes isolated from pair-fed rats. However, in adipocytes isolated from rats that had consumed a diet containing 35% ethanol for 4 wk, endothelin-1 did not increase glucose uptake. Although endothelin-1 increased GLUT4 quantity at the plasma membrane in adipocytes from pair-fed rats, there was no increase in GLUT4 after chronic ethanol feeding. Loss of endothelin-1-stimulated glucose uptake after ethanol feeding was associated with a specific decrease in the quantity of Galpha11 in plasma membranes, with no change in Galphaq quantity. Activation of proline-rich tyrosine kinase 2 (PYK2), a downstream target of Galphaq/11 that is required for endothelin-1-stimulated GLUT4 translocation in 3T3-L1 adipocytes, was also suppressed after chronic ethanol feeding. In contrast, activation of p38 MAPK by endothelin-1 was not affected by chronic ethanol exposure. These data demonstrate that chronic ethanol feeding suppresses endothelin-1-stimulated glucose uptake and suggest that decreased expression of Galpha11 coupled to impaired endothelin-1-dependent activation of PYK2 contributes to this response.

Cyclic Strain Activates Redox-sensitive Proline-rich Tyrosine Kinase 2 (PYK2) in Endothelial Cells

Proline-rich tyrosine kinase 2 (PYK2), structurally related to focal adhesion kinase, has been shown to play a role in signaling cascades. Endothelial cells (ECs) under hemodynamic forces increase reactive oxygen species (ROS) that modulate signaling pathways and gene expression. In the present study, we found that bovine ECs subjected to cyclic strain rapidly induced phosphorylation of PYK2 and Src kinase. This strain-induced PYK2 and Src phosphorylation was inhibited by pretreating ECs with an antioxidant N-acetylcysteine. Similarly, ECs exposed to H(2)O(2) increased both PYK2 and Src phosphorylation. An increased association of Src to PYK2 was observed in ECs after cyclic strain or H(2)O(2) exposure. ECs treated with an inhibitor to Src (PPI) greatly reduced Src and PYK2 phosphorylation, indicating that Src mediated PYK2 activation. Whereas the protein kinase C (PKC) inhibitor (calphostin C) pretreatment was shown to inhibit strain-induced NADPH oxidase activity, ECs treated with either calphostin C or the inhibitor to NADPH oxidase (DPI) reduced strain-induced ROS levels and then greatly inhibited the Src and PYK2 activation. In contrast to the activation of PYK2 and Src with calcium ionophore (ionomycin), ECs treated with a Ca(2+) chelator inhibited both phosphorylation, indicating that PYK2 and Src activation requires Ca(2+). ECs transfected with antisense to PKCalpha, but not antisense to PKCepsilon(,) reduced cyclic strain-induced PYK2 activation. These data suggest that cyclic strain-induced PYK2 activity is mediated via Ca(2+)-dependent PKCalpha that increases NADPH oxidase activity to produce ROS crucial for Src and PYK2 activation. ECs under cyclic strain thus activate redox-sensitive PYK2 via Src and PKC, and this PYK2 activation may play a key role in the signaling responses in ECs under hemodynamic influence.

LFA-1 integrin and the microtubular cytoskeleton are involved in the Ca2+-mediated regulation of the activity of the tyrosine kinase PYK2 in T cells

Lymphocyte function-associated antigen (LFA-1) is a member of the beta2 family of integrins that is selectively expressed on leukocytes. Herein, we show that Ca(2)(+) mobilizing agents A23187, thapsigargin, and ionomycin induce an increase in adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) and activation and redistribution of the proline-rich tyrosine kinase-2 (PYK2) to the microtubule-organizing center (MTOC) in T-lymphoblasts. These effects are similar to those observed upon direct induction of activation of LFA-1 with the stimulatory mAb KIM-127. Most importantly, Ca(2)(+) mobilization did not induce activation of PYK2 when the LFA-1/ICAM-1 interaction was prevented with function-blocking mAb, implying that the Ca(2)(+)-induced activation of PYK2 requires integrin engagement. Furthermore, pretreatment of the cells with the Ca(2)(+) chelator EGTA, which depletes the intracellular Ca(2)(+), inhibited the effects of mAb KIM-127 on cell morphology and PYK2 activation. This inhibition with EGTA was not reversed by cross-linking integrin LFA-1 with specific antibodies, indicating that Ca(2)(+) exerts its effects through a target downstream of this integrin. In this regard, immunofluorescence and Western blot analysis showed that Ca(2)(+) chelators affect the organization of the microtubular cytoskeleton and the localization of PYK2 to the MTOC area, suggesting that these agents could inhibit the activation of PYK2 by interfering with the microtubular network of T cells. Taken together, our results demonstrate for the first time an important role for the integrin LFA-1 and the microtubular cytoskeleton in the Ca(2)(+)-mediated activation of PYK2 in T-lymphocytes.

Altered PYK2 phosphorylation by ANG II in hypertensive vascular smooth muscle

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit increased cell growth compared with normotensive Wistar-Kyoto rats (WKY). ANG II stimulates growth via G(q)-protein-coupled signaling that involves changes in cytosolic intracellular Ca(2+) concentration ([Ca(2+)](i)) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. This study examines the role of the proline-rich tyrosine kinase 2 (PYK2) in hypertensive VSMC. Basal PYK2 phosphorylation in SHR VSMC was increased compared with WKY (0.44 +/- 0.02 vs. 0.20 +/- 0.02-fold). ANG II-induced activation of PYK2 in SHR VSMC was of greater magnitude (2.2 +/- 0.2-fold in SHR; 1.4 +/- 0.1-fold in WKY) and occurred more rapidly (peak activation at 2 min in SHR vs. 5 min in WKY). This effect was blocked by pretreatment with the [Ca(2+)](i) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or the PKC inhibitor chelerythrine. Basal and ANG II-stimulated c-Fos expression was increased in SHR versus WKY VSMC. PYK2 downregulation with antisense oligonucleotides blocked ANG II-induced c-Fos expression. Increased PYK2 activation may be altered signaling cascades that regulate cell growth in hypertensive VSMC.

Expression and characterization of splice variants of PYK2, a focal adhesion kinase-related protein

Focal adhesion kinase and the recently identified proline-rich tyrosine kinase 2 (PYK2), also known as cell adhesion kinase &bgr ;, related adhesion focal tyrosine kinase or calcium-dependent protein tyrosine kinase, define a new family of non-receptor protein tyrosine kinases. Activation of PYK2 has been implicated in multiple signaling events, including modulation of ion channels, T- and B-cell receptor signaling and cell death. Mechanisms underlying the functional diversity of PYK2 are unclear. Here, we provide evidence for two novel alternatively expressed isoforms of PYK2. One isoform, designated PYK2s (PYK2 splice form), appears to be a splice variant of PYK2 lacking 42 amino acids within the C-terminal domain. A second isoform, referred to as PRNK (PYK2-related non-kinase), appears to be specified by mRNAs that encode only part of the C-terminal domain of PYK2. Northern blot analysis indicates that the unspliced PYK2 is expressed at high levels in the brain and poorly expressed in the spleen, whereas PYK2s and PRNK are expressed in the spleen. In situ hybridization studies of rat brain demonstrate that the unspliced PYK2 is selectively expressed at high levels in hippocampus, cerebral cortex and olfactory bulb, whereas PYK2s and PRNK are expressed at low levels in all regions of rat brain examined. Immunofluorescence analysis of ectopically expressed PRNK protein shows that PRNK, in contrast to full-length PYK2, is localized to focal adhesions by sequences within the focal adhesion targeting domain. In addition, PYK2, but not PRNK, interacts with p130(cas )and Graf. These results imply that PRNK may selectively regulate PYK2 function in certain cells by binding to some but not all PYK2 binding partners, and the functional diversity mediated by PYK2 may be due in part to complex alternative splicing.