Central Role of Calcium-Dependent Tyrosine Kinase PYK2 in Endothelial Nitric Oxide Synthase–Mediated Angiogenic Response and Vascular Function

Matsui, Akihiro; Okigaki, Mitsuhiko; Amano, Katsuya; Adachi, Yasushi; Jin, Denan; Takai, Shinji; Yamashita, Tomoya; Kawashima, Seinosuke; Kurihara, Tatsuya; Miyazaki, Mizuo; Tateishi, Kento; Matsunaga, Shinsaku; Katsume, Asako; Honshou, Shoken; Takahashi, Toshifumi; Matoba, Satoaki; Kusaba, Tetsuro; Tatsumi, Tomohide; Matsubara, Hiroaki

doi:10.1161/circulationaha.106.645416

Cited by 45 publications

(50 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Generation of Pyk2-knockout mice allowed a better understanding of the role of this kinase in many physiologic context, including macrophage migration and osteoclast activation. 27,38,39 In the present study, we report that Pyk2-deficient platelets show a defective activation of PI3K␤ after integrin ␣2␤1-mediated adhesion. A possible role for Pyk2 in the regulation of platelet PI3K activity has been hypothesized previously on the basis of some circumstantial evidence.…”

supporting

confidence: 51%

“…Generation and characterization of PI3K␤ KD , PI3K␥ KD , and Pyk2-knockout mice was described previously. [25][26][27] The use of mice for our experimental work was approved by the Ethics Committee of the University of Pavia. The rabbit polyclonal Abs against Rap1 (121), against Pyk2 (N-19), as well as the mAb anti-tubulin (DM1A) were from Santa Cruz Biotechnology.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Consonni

Cipolla

Guidetti

et al. 2012

Blood

Self Cite

View full text Add to dashboard Cite

Integrin ␣2␤1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3K␤, but not of PI3K␥ or PI3K␣. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3K␤ (PI3K␤ KD ), but occurred normally in PI3K␥ KD platelets. Integrin ␣2␤1 failed to stimulate PI3K␤ in platelets from phospholipase C␥2 (PLC␥2)-knockout mice, and we found that intracellular Ca 2؉ linked PLC␥2 to PI3K␤ activation. Integrin ␣2␤1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLC␥2 and intracellular Ca 2؉ . Whereas activation of Pyk2 occurred normally in PI3K␤ KD platelets, stimulation of PI3K␤ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3K␤ was required for ␣2␤1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin ␣IIb␤3 were reduced after inhibition of PI3K␤ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3K␤ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3K␤ downstream of integrin ␣2␤1, and document a novel role for Pyk2 and PI3K␤ in integrin ␣2␤1 promoted inside-out activation of integrin ␣IIb␤3 and thrombus formation. (Blood. 2012;119(3):847-856) IntroductionClass I PI3Ks are key signaling enzymes that phosphorylate the inositol ring of membrane phospholipids and generate different 3-phosphoinositides, important intracellular messengers that regulate several cellular processes through the downstream activation of the protein Ser/Thr kinase Akt. 1 Circulating blood platelets express all members of the class I PI3K family, which includes the PI3K␣, PI3K␤, PI3K␦, and PI3K␥ isoforms. PI3K activity is essential for platelet aggregation and thrombus formation, 1,2 and therefore these enzymes are potential novel targets for antithrombotic agents. For this reason, it is essential to recognize the precise contribution of every PI3K isoform in platelet activation induced by different extracellular agonists.Pharmacologic and genetic evidence indicates that PI3K␤ plays a predominant role in the regulation of platelet function. [3][4][5][6][7] Selective inactivation of PI3K␤ completely prevents platelet aggregation induced by the collagen receptor glycoprotein VI (GPVI) and reduces occlusive thrombus formation. 4,5 PI3K␤ is also implicated in the platelet response to agonists that stimulate G-protein coupled receptors (GPCRs) such as ADP or thromboxane A 2 (TxA 2 ). 3,4,8,9 Whereas PI3K␦ has been demonstrated to play a minor role in platelet activation, 10 PI3K␣ was recently proposed to be as important as PI3K␤ in GPVI signaling. 7 Similarly, several reports have documented that, in addition to PI3K␤, PI3K␥ is also implicated in GPCR-mediated platelet activation. 4,9,11,12 These observations are indicative of a still poorly appreciated interplay...

show abstract

supporting

confidence: 51%

Section: Methodsmentioning

confidence: 99%

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Consonni

Cipolla

Guidetti

et al. 2012

Blood

Self Cite

View full text Add to dashboard Cite

show abstract

“…Previous studies (15,18) have reported that Pyk2 is activated by vagal stimulation through the ACh receptor. We explored this phenomenon in the whole heart to probe its possible role in arrhythmogenesis.…”

Section: Discussionmentioning

confidence: 95%

“…Pyk2 is expressed in cardiomyocytes, cardiac fibroblasts, vascular smooth muscle cells, and vascular endothelial cells (3,8,18,22,27). Pyk2 signaling has been implicated in the induction of pressure overload-induced left ventricular (LV) hypertrophy and the transition to heart failure (4).…”

mentioning

confidence: 99%

Role of Pyk2 in cardiac arrhythmogenesis

Lang

Glukhov

Efimova

et al. 2011

American Journal of Physiology-Heart and Circulatory Physiology

View full text Add to dashboard Cite

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein kinase regulated by intracellular Ca2+, CaMK, and PKC and can be activated by different stress signals involved in heart failure. However, Pyk2 has not been investigated in the human heart, and the functional role of Pyk2 signaling at the whole heart level has not been elucidated. We hypothesize that Ca2+-dependent activation of Pyk2 is involved in cardiac electrophysiology. We examined the expression of Pyk2 in nonfailing versus ischemic and nonischemic failing human hearts ( n = 6 hearts/group). To investigate Pyk2 function, we optically mapped perfused hearts from wild-type (WT; n = 7) and knockout (Pyk2−/−; n = 8) mice during autonomic stimulation. Experiments were done in control mice and after 1 wk of transverse aortic constriction. We used the Illumina beadarray approach for transcriptional profiling of WT and Pyk2−/− mouse ventricles. Western blot analysis revealed a doubling of Pyk2 activation in nonischemic failing versus nonfailing human hearts. In mouse hearts, we observed a much higher probability of ventricular tachyarrhythmia during ACh perfusion in Pyk2−/− versus WT mice. Parasympathetic stimulation resulted in a dose-dependent decrease of atrial action potential duration (APD) in both WT and Pyk2−/− mice, whereas in ventricles it induced APD shortening in Pyk2−/− mice but not in WT mice. Deficiency of Pyk2 abolished ACh-induced prolongation of atrioventricular delay in Pyk2−/− mouse hearts but did not affect heart rate. Lower mRNA and protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase 2 and higher mRNA levels of Na+/Ca2+ exchanger 1 were detected in Pyk2−/− hearts compared with WT hearts. The transverse aortic constriction protocol did not change the phenotype. In conclusion, our results indicate a protective role of Pyk2 with respect to ventricular tachyarrhythmia during parasympathetic stimulation by regulation of gene expression related to Ca2+ handling. We hypothesize that activation of Pyk2 in the human heart during heart failure may contribute to protection against arrhythmia.

show abstract

“…19 Moreover, endothelial cell migration and tube formation promoted by VEGF was found to be strongly impaired in the absence of Pyk2. 20 A recent study also showed that Pyk2 is implicated in reactive oxygen species generation and reactive oxygen speciesmediated proinflammatory events involved in atherosclerosis. 21 These previous observations clearly point to a relevant role for Pyk2 in the cardiovascular system.…”

Section: Introductionmentioning

confidence: 98%

Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

Canobbio¹,

Cipolla

Consonni³

et al. 2013

Blood

Self Cite

View full text Add to dashboard Cite

Key Points• The tyrosine kinase Pyk2 regulates a p38MAPK-cPLA2 pathway required for thrombin-induced thromboxane A2 production and platelet aggregation.• Pyk2 deletion in mice confers protection against pulmonary thromboembolism and arterial thrombosis.In the present study, we used a knockout murine model to analyze the contribution of the Ca 2؉ -dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin ␣ IIb ␤ 3 and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A 2 production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA 2 IntroductionPyk2, also known as RAFTK or CAD␤, is a nonreceptor tyrosine kinase that is highly homologous to the focal adhesion kinase FAK and is predominantly expressed in the CNS and hematopoietic cells. 1-3 Like FAK, Pyk2 does not possess SH2 or SH3 domains, but has a centrally located catalytic domain flanked by an N-terminal FERM domain and a C-terminal focal adhesion targeting FAT domain. Pyk2 can be tyrosine phosphorylated and activated by a variety of different cellular stimuli, including cytokines, growth factors, agonists of G-protein-coupled receptors (GPCRs), integrin ligands, and stress stimuli. [3][4][5][6] Typically, Pyk2 activation is mediated by both Src kinase-and cytosolic Ca 2ϩ -dependent pathways. Src kinases phosphorylate Pyk2 at Tyr579, Tyr580, and Tyr881, increasing the catalytic activity of the kinase and promoting autophosphorylation on Tyr402 in the FERM domain. 5,6 Even in the absence of Src-mediated phosphorylation, Pyk2 can be activated on increase of the cytosolic Ca 2ϩ concentration. [4][5][6][7] It has been shown that Ca 2ϩ and calmodulin bind to the N-terminal FERM domain of Pyk2, triggering Pyk2 dimerization, activation, and autophosphorylation at Tyr402. 8 Phosphorylated Tyr402 is a binding site for the SH2 domain of Src. 5,6 Therefore, Pyk2 is a kinase that can link Ca 2ϩ -based signaling pathways to protein tyrosine phosphorylation.Some observations reported over the past 2 decades have implicated Pyk2 in platelet activation. Pyk2 is highly expressed in megakaryocytes and platelets and is rapidly phosphorylated on stimulation with se...

show abstract

Central Role of Calcium-Dependent Tyrosine Kinase PYK2 in Endothelial Nitric Oxide Synthase–Mediated Angiogenic Response and Vascular Function

Abstract: Background-The involvement of Ca 2ϩ -dependent tyrosine kinase PYK2 in the Akt/endothelial NO synthase pathway remains to be determined. Methods and Results-Blood flow recovery and neovessel formation after hind-limb ischemia were impaired in PYK2

Cited by 45 publications

References 39 publications

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Role of Pyk2 in cardiac arrhythmogenesis

Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

Contact Info

Product

Resources

About