Inhibition Of Pyk2 Research Articles

Increased vascular contractile responses to phenylephrine in Doca‐salt mice is normalized by Pyk2 blockade

Background: The calcium‐dependent proline‐rich tyrosine kinase (Pyk2), a nonreceptor protein activated by tyrosine phosphorylation, links G‐protein‐coupled receptors to vascular responses. DOCA‐salt hypertension is characterized by enhanced vascular reactivity. We tested the hypothesis that changes in vascular responses in DOCA‐salt mice are due to increased activation of Pyk2.Methods and results: Aorta and small mesenteric arteries from DOCA‐salt and uninephrectomized (UNI) male C57bl/6 mice were incubated with vehicle or Tyrphostin A‐9 (0.1μM, Pyk2 inhibitor). Systolic blood pressure (mmHg) was higher in DOCA (126±3) vs UNI (100±4). Vascular responses to PE (1nM to 10mM) were greater in vessels from DOCA‐salt than UNI, but treatment with Tyrphostin A‐9 abolished the difference among the groups (Figure 1). Pyk2 levels, as well as Pyk2Tyr402 phosphorylation, paxillin and paxillinTyr118 phosphorylation were increased in DOCA‐salt aorta (Figure2). Incubation with Tyrphostin A‐9 restored phosphorylation of Pyk2 and paxillin.Conclusion: Increased Pyk2 expression and activity are involved in increased vascular contractile‐responses in Doca‐salt mice.Financial Support: NIH (HL‐74167, RCW); FAPESP and CAPES, Brazil.

Essential role of Pyk2 and Src kinase activation in neuropeptide-induced proliferation of small cell lung cancer cells

Neuropeptide hormones like bombesin/gastrin-releasing peptide, galanin or bradykinin, acting via auto and paracrine growth loops, represent the principal mitogens of small cell lung cancer (SCLC). These mitogenic neuropeptides activate G(q/11)-coupled receptors which stimulate phospholipase Cbeta activity, followed by rises of the intracellular calcium concentration ([Ca2+](i)) and activation of protein kinase C (PKC). We report here that proline-rich tyrosine kinase Pyk2 is highly expressed in SCLC cells and provides a functional link between neuropeptide-induced increases in [Ca2+](i) and tumor cell proliferation. Activation of Pyk2 and its association with Src kinases critically depends on the elevation of [Ca2+](i), but is independent of PKC. Src kinase activities are crucial for neuropeptide-mediated GTP-loading of Ras and activation of extracellular signal-regulated kinases in SCLC cells. Pyk2 and Src kinases essentially contribute to anchorage-independent proliferation of SCLC cells. Inhibition of either Pyk2 or Src kinases by lentiviral RNAi or pharmacological inhibition with PP2, respectively, attenuated basal and neuropeptide-elicited survival and proliferation of SCLC cells in liquid culture and in soft agar. Thus, neuropeptides stimulate anchorage-independent survival and proliferation of SCLC cells via pathways involving Pyk2 and Src kinases. Therefore, Ca2+-induced Pyk2/Src complex formation may be a rewarding molecular target for novel therapeutic strategies in SCLC cells.

ICAM-1-Mediated, Src- and Pyk2-Dependent Vascular Endothelial Cadherin Tyrosine Phosphorylation Is Required for Leukocyte Transendothelial Migration

Leukocyte transendothelial migration (TEM) has been modeled as a multistep process beginning with rolling adhesion, followed by firm adhesion, and ending with either transcellular or paracellular passage of the leukocyte across the endothelial monolayer. In the case of paracellular TEM, endothelial cell (EC) junctions are transiently disassembled to allow passage of leukocytes. Numerous lines of evidence demonstrate that tyrosine phosphorylation of adherens junction proteins, such as vascular endothelial cadherin (VE-cadherin) and beta-catenin, correlates with the disassembly of junctions. However, the role of tyrosine phosphorylation in the regulation of junctions during leukocyte TEM is not completely understood. Using human leukocytes and EC, we show that ICAM-1 engagement leads to activation of two tyrosine kinases, Src and Pyk2. Using phospho-specific Abs, we show that engagement of ICAM-1 induces phosphorylation of VE-cadherin on tyrosines 658 and 731, which correspond to the p120-catenin and beta-catenin binding sites, respectively. These phosphorylation events require the activity of both Src and Pyk2. We find that inhibition of endothelial Src with PP2 or SU6656 blocks neutrophil transmigration (71.1 +/- 3.8% and 48.6 +/- 3.8% reduction, respectively), whereas inhibition of endothelial Pyk2 also results in decreased neutrophil transmigration (25.5 +/- 6.0% reduction). Moreover, overexpression of the nonphosphorylatable Y658F or Y731F mutants of VE-cadherin impairs transmigration of neutrophils compared with overexpression of wild-type VE-cadherin (32.7 +/- 7.1% and 38.8 +/- 6.5% reduction, respectively). Our results demonstrate that engagement of ICAM-1 by leukocytes results in tyrosine phosphorylation of VE-cadherin, which is required for efficient neutrophil TEM.

Src family kinases mediate neutrophil adhesion to adherent platelets

AbstractPolymorphonuclear leukocyte (PMN)–platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of “outside-in” signaling by Src-family tyrosine kinases (SFKs) in the regulation of αMβ2-integrin–dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activation-dependent epitopes in β2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from αMβ2−/−, hck−/−fgr−/−, and hck−/−fgr−/−lyn−/− mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by inhibition as well as by genetic deletion of αMβ2 integrin and SFKs. A Pyk2 inhibitor reduced platelet-PMN adhesion, indicating that Pyk2 may be a downstream effector of SFKs. Analysis of PMN-platelet interactions under flow revealed that SFK signaling was required for αMβ2-mediated shear-resistant adhesion of PMNs to adherent platelets, but was dispensable for P-selectin–PSGL-1–mediated recruitment and rolling. Finally, SFK activity was required to support PMN accumulation along adherent platelets at the site of vascular injury, in vivo. These results definitely establish a role for SFKs in PMN recruitment by activated platelets and suggest novel targets to disrupt the pathophysiologic consequences of platelet-leukocyte interactions in vascular disease.

Src and Pyk2 mediate angiotensin II effects in cultured rat astrocytes

Angiotensin II (Ang II)-induced proliferation of rat astrocytes is mediated by multiple signaling pathways. In the present study, we investigated the role of non-receptor tyrosine kinases on Ang II-signaling and proliferation of astrocytes cultured from neonatal rat pups. Ang II stimulated astrocyte growth, ERK1/2 phosphorylation and the phosphorylation of Src and proline-rich tyrosine kinase-2 (Pyk2), in astrocytes obtained from brainstem and cerebellum. Pretreatment with 10 μM PP2, a selective Src inhibitor, inhibited Ang II stimulated ERK1/2 phosphorylation by 59% to 91% both in brainstem and cerebellum astrocytes. PP2 also inhibited Ang II induction of brainstem (76% inhibition) and cerebellar (64% inhibition) astrocyte growth. Similarly, pretreatment with 25 μM dantrolene, the Pyk2 inhibitor, attenuated ERK1/2 activity in brainstem (62% inhibition) and in cerebellum astrocytes (44% inhibition). Interestingly, inhibition of Pyk2 inhibited Ang II-induced Src activation suggesting that these two non-receptor tyrosine kinases may be acting in concert to mediate Ang II effects in astrocytes. In summary, we found that Ang II stimulates the non-receptor tyrosine kinases Src and Pyk2 which mediate Ang II-induced ERK1/2 activation leading to stimulation of astrocyte growth. In addition, these two tyrosine kinases may be interacting to regulate effects of the peptide in these cells.

SDF‐1 Controls Pituitary Cell Proliferation through the Activation of ERK1/2 and the Ca2+‐Dependent, Cytosolic Tyrosine Kinase Pyk2

Stromal cell-derived factor-1 (SDF-1) is a chemokine of the CXC subfamily that exerts its effects via CXCR4, a G-protein-coupled receptor. CXCR4 is often expressed by tumor cells, and its activation causes tumor cell proliferation. Using GH4C1 cells, here we show that SDF-1 induced cell proliferation in a dose-dependent manner. Thus, we evaluated the intracellular signaling involved in this effect. SDF-1 increased cytosolic [Ca2+] and activated Pyk2, ERK1/2, and BKCa channels. To correlate these intracellular effectors with the proliferative activity of SDF-1, we inhibited their activity using BAPTA-AM (Ca2+ chelator), PD98059 (MEK inhibitor), salicylate (Pyk2 inhibitor), and TEA (K+ channel blocker). All these compounds reverted SDF-1-induced proliferation, suggesting the involvement of multiple intracellular pathways. To identify a possible crosstalk and a molecular ordering among these pathways, we tested these antagonists on SDF-1-dependent activation of ERK1/2, Pyk2, and BKCa channels. We report that the inhibition of [Ca2+]i increase or the blockade of BKCa channel activity did not affect ERK1/2 activation by SDF-1; Pyk2 activation was purely Ca2+-dependent, not involving ERK1/2 or BKCa channels; and BKCa channel activity was antagonized by Pyk2 but not by ERK1/2 inhibitors. These data suggest that SDF-1-dependent increase of [Ca2+]i activates Pyk2, which, in turn, regulates BKCa channel activity. Conversely, ERK1/2 activation is an independent phenomenon. In conclusion, we demonstrate that SDF-1 induces proliferation of GH4C1 cells, suggesting that the activation of CXCR4 may represent a novel regulatory mechanism for pituitary cell proliferation which may contribute to pituitary adenoma development.

The Tyrosine Kinase, Pyk2/RAFTK, Mediates LPS-Induced IL-8 and MCP-1 Expression in Human Endothelial Cells.

Lipopolysaccharide (LPS), an essential component of the surface of Gram-negative bacteria, has been shown to induce various inflammatory cytokines, including IL-8 and MCP-1, in endothelial cells. However, the mechanism by which LPS mediates the production of these inflammatory cytokines is not well known. In the present study, we investigated the role of proline-rich tyrosine kinase 2 (Pyk2), also known as RAFTK, in LPS-induced IL-8 and MCP-1 expression in endothelial cells. We observed the concentration-dependent and time-dependent tyrosine phosphorylation of Pyk2 following LPS treatment in human umbilical vein endothelial cells (HUVEC). We next analyzed the site of Pyk2 phosphorylation using antibodies specific to the individual phosphorylated tyrosine residues in Pyk2: pY402 (Src binding site), pY881 (Grb2 binding site) and pY580 (autophosphorylation site). LPS stimulation significantly enhanced phosphorylation of the pY402 and pY580 residues, but not of the pY881 site. The role of Pyk2 in LPS-induced IL-8 and MCP-1 expression was further investigated using a recombinant adeno-associated virus (AAV) expressing kinase-defective mutant Pyk2. Endothelial cells infected with the catalytically inactive Pyk2 mutant significantly attenuated LPS-induced IL-8 and MCP-1 production in HUVEC, compared to cells infected with the control AAV vector. Treatment of endothelial cells with a specific inhibitor of Pyk2, Tyrphostin A9, also blocked the expression of IL-8 and MCP-1 in a dose-dependent manner. Both p42/p44 and p38 MAP kinase pathways have been implicated in the production of inflammatory cytokines in endothelial cells. We found that blocking of Pyk2 using Tyrphostin A9 inhibited the phosphorylation of p38 MAP kinase but merely altered the kinetics of p42/p44 MAPK phosphorylation, indicating that p38 MAP kinase is an important downstream molecule of Pyk2 in LPS-induced IL-8 production. The involvement of the p38 MAP kinase pathway was confirmed by the dose-dependent blocking of IL-8 production upon treatment with a specific inhibitor of p38 MAP kinase. In summary, our data illustrate a key role for Pyk2 in LPS-mediated signaling, resulting in IL-8 and MCP-1 secretion in human endothelial cells. These findings may have important implications for the treatment of sepsis and other inflammatory disorders.

CRNK Overexpression Alters Hypertrophic Gene Expression In Cultured Neonatal Rat Ventricular Myocytes

We previously showed that overexpression of PYK2, a Ca2+-dependent nonreceptor protein tyrosine kinase, activated the stress-activated protein kinases JNK1/2 and p38MAPK, and down-regulated SERCA2 gene transcription in neonatal rat ventricular myocytes (NRVM) (Heidkamp et al., AJP:Cell 289:C471-C482, 2005). In the present study, we examined the effects of adenovirally (Adv)-mediated overexpression of a C-terminal, dominant-negative inhibitor of PYK2, known as CRNK (Cell adhesion kinase-β Related NonKinase) on cellular growth and hypertrophic gene expression in similarly cultured NRVM. NRVM were maintained in serum-free culture medium (UI), or infected with either Adv-CRNK or control Adv expressing green fluorescent protein (Adv-GFP; 5 moi, 24–48h). Cell extracts were analyzed for JNK and p38MAPK phosphorylation by Western blotting, for total protein and DNA content by spectrophotometry and fluorometry, and for SERCA2 and ANF mRNA levels by real-time RT-PCR. CRNK overexpression had no effect on basal JNK or p38MAPK, but blocked the H2O2-induced increase in JNK1/2 (but not p38MAPK) phosphorylation. CRNK overexpression also did not affect basal total protein/DNA ratio, and did not prevent the increase in total protein/DNA ratio produced by treatment with phorbol myristate acetate (200nM, 24h). However, CRNK overexpression significantly (P<0.05) increased SERCA2 mRNA levels 2.1±0.5-fold as compared to UI or GFP-expressing NRVM. In contrast, ANF mRNA levels were significantly decreased to 36±14% of UI by CRNK overexpression. These data indicate that PYK2 is necessary to elicit specific aspects of the hypertrophic phenotype in cultured NRVM. Supported by NIH RO1 HL34328 and the Dr. Ralph and Marian Falk Medical Research Trust.

Integrin α4β1 Promotes Focal Adhesion Kinase-Independent Cell Motility via α4 Cytoplasmic Domain-Specific Activation of c-Src

The fibronectin binding integrins alpha5beta1 and alpha4beta1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For alpha5beta1, beta1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for alpha5beta1-stimulated cell motility and that exogenous expression of human alpha4 in FAK-null fibroblasts forms a functional alpha4beta1 receptor that promotes robust cell motility equal to the alpha5beta1 stimulation of wild-type and FAK-reconstituted fibroblasts. alpha4beta1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the alpha4 cytoplasmic domain, independent of direct paxillin binding to alpha4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. alpha4 cytoplasmic domain-initiated signaling led to a approximately 4-fold activation of c-Src which did not require paxillin binding to alpha4. Notably, alpha4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase alpha overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. alpha4beta1-stimulated cell motility of triple-null Src(-/-), c-Yes(-/-), and Fyn(-/-) fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for alpha5beta1-stimulated FAK activation, our results support the existence of a novel alpha4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.

Chemokine Stromal Cell-Derived Factor 1α Induces Proliferation and Growth Hormone Release in GH4C1 Rat Pituitary Adenoma Cell Line through Multiple Intracellular Signals

We used GH4C1 cells as a model to study the effects of the chemokine stromal cell-derived factor 1 (SDF1) in pituitary functions. In these cells, SDF1alpha induced proliferation and growth hormone secretion, suggesting a possible regulatory role for this chemokine at pituitary level. We evaluated the intracellular signaling involved in these effects: SDF1alpha increased cytosolic [Ca(2+)] and activated Pyk2, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) channels. To correlate these intracellular effectors with the proliferative and secretory effects, we inhibited their activity using BAPTA-AM (Ca(2+) chelator), 2'-amino-3'-methoxyflavone (PD98059; a mitogen-activated protein kinase kinase inhibitor), salicylate (Pyk2 inhibitor), and tetraethyl ammonium (K(+) channel blocker). All of these compounds reverted SDF1alpha-induced proliferation, suggesting the involvement of multiple intracellular pathways. Conversely, only BAPTA-AM reverted growth hormone secretion. To identify a possible cross-talk and a molecular ordering among these pathways, we tested these antagonists on SDF1alpha-dependent activation of ERK1/2, Pyk2, and BK(Ca) channels. From these experiments, we observed that the inhibition of [Ca(2+)](i) increase or BK(Ca) channel activity did not affect ERK1/2 activation by SDF1alpha; Pyk2 activation was purely Ca(2+)-dependent, not involving ERK1/2 or BK(Ca) channels; and BK(Ca) channel activity was antagonized by Pyk2 but not by ERK1/2 inhibitors. These data suggest that an SDF1alpha-dependent increase of [Ca(2+)](i) activates Pyk2, which in turn regulates BK(Ca) channel activity. Conversely, ERK1/2 activation is an independent phenomenon. In conclusion, we demonstrate that SDF1alpha causes both proliferation and growth hormone release from pituitary adenoma cells, suggesting that the activation of CXCR4 may represent a novel regulatory mechanism for growth hormone secretion and pituitary cell proliferation, which may contribute to pituitary adenoma development.

Signal Transduction of Physiological Concentrations of Vasopressin in A7r5 Vascular Smooth Muscle Cells: A ROLE FOR PYK2 AND TYROSINE PHOSPHORYLATION OF K+CHANNELS IN THE STIMULATION OF Ca2+ SPIKING

The signal transduction pathway linking physiological concentrations of [Arg(8)]vasopressin (AVP) to an increase in frequency of Ca(2+) spiking was examined in confluent cultures of A7r5 vascular smooth muscle cells. Immunoprecipitation/Western blot studies revealed a robust increase in tyrosine phosphorylation of the non-receptor tyrosine kinase, PYK2, in A7r5 cells treated with 4beta-phorbol 12-myristate 13-acetate or ionomycin. 100 pm AVP also induced PYK2 tyrosine phosphorylation, and this effect was inhibited by protein kinase C inhibitors Ro-31-8220 (1-10 microm) or chelerythrine chloride (1-20 microm). In fura-2-loaded A7r5 cells, the stimulation of Ca(2+) spiking by 100 pm AVP or 1 nm 4beta-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 microm, a Src family kinase inhibitor). Salicylate (20 mm, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 microm), but not its inactive analog, tyrphostin A63, also blocked AVP-stimulated Ca(2+) spiking. PYK2 phosphorylation was inhibited by both PP2 and salicylate, whereas tyrphostin A47 failed to inhibit PYK2 tyrosine phosphorylation. ERK1/2 kinases did not appear to be involved because 1) 100 pm AVP did not appreciably increase ERK1/2 phosphorylation and U-0126 (2.5 microm) did not inhibit AVP-stimulated Ca(2+) spiking; and 2) epidermal growth factor (10 nm) robustly stimulated ERK1/2 phosphorylation but did not induce Ca(2+) spiking. Delayed rectifier K(+) channels may mediate the PYK2 activity because Kv1.2 channel protein co-immunoprecipitated with PYK2 and tyrosine phosphorylation of Kv1.2 was stimulated by AVP and inhibited by Ro-31-8220, PP2, and salicylate but not tyrphostin A47. Our findings are consistent with a role for PYK2 and phosphorylation of K(+) channels in the stimulation of Ca(2+) spiking by physiological concentrations of AVP.

Suppression of Pyk2 kinase and cellular activities by FIP200.

Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic tyrosine kinase implicated to play a role in several intracellular signaling pathways. We report the identification of a novel Pyk2-interacting protein designated FIP200 (FAK family kinase-interacting protein of 200 kD) by using a yeast two-hybrid screen. In vitro binding assays and coimmunoprecipitation confirmed association of FIP200 with Pyk2, and similar assays also showed FIP200 binding to FAK. However, immunofluorescent staining indicated that FIP200 was predominantly localized in the cytoplasm. FIP200 bound to the kinase domain of Pyk2 and inhibited its kinase activity in in vitro kinase assays. FIP200 also inhibited the kinase activity of the Pyk2 isolated from SYF cells (deficient in Src, Yes, and Fyn expression) and the Pyk2 mutant lacking binding site for Src, suggesting that it regulated Pyk2 kinase directly rather than affecting the associated Src family kinases. Consistent with its inhibitory effect in vitro, FIP200 inhibited activation of Pyk2 and Pyk2-induced apoptosis in intact cells, which correlated with its binding to Pyk2. Finally, activation of Pyk2 by several biological stimuli correlated with the dissociation of endogenous FIP200-Pyk2 complex, which provided further support for inhibition of Pyk2 by FIP200 in intact cells. Together, these results suggest that FIP200 functions as an inhibitor of Pyk2 via binding to its kinase domain.