Bayer Allison L, Heidkamp Maria C, Howes Amy L, Heller Brown Joan, Byron Kenneth L, Samarel Allen M
The Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, Maywood, IL 60153, USA.
J Mol Cell Cardiol. 2003 Sep;35(9):1121-33. doi: 10.1016/s0022-2828(03)00228-1.
Proline-rich tyrosine kinase 2 (PYK2) is a nonreceptor protein tyrosine kinase that links G-protein-coupled receptors to activation of MAPK cascades and cellular growth. In smooth muscle and other cell types, PYK2 activation is dependent on either Ca(2+) or protein kinase C (PKC), and we have previously shown that endothelin-1 (ET) activates PYK2 in adult and neonatal rat ventricular myocytes (NRVM). However, ET both alters intracellular Ca(2+) (Ca(2+)), and activates the novel, Ca(2+)-independent PKCs. Therefore, immunoprecipitation and western blotting experiments were used to examine the PKC and Ca(2+) dependence of PYK2 activation in NRVM. PYK2 was activated by ET (100 nM; 2-30 min) and phenylephrine (50 microM; 2-30 min), which are both hypertrophic agonists that activate Gq-coupled receptors. Moreover, adenoviral (Adv)-mediated overexpression of constitutively active (ca) Galphaq increased PYK2-Y(402) phosphorylation as early as 8 h post-infection, as compared to NRVM infected with a control Adv encoding beta-galactosidase. caGalphaq overexpression also induced PKC epsilon and PKCdelta (but not PKCalpha) translocation, followed by downregulation of both novel PKC isoenzymes. Phorbol myristate acetate (PMA; 200 nM), a direct activator of Ca(2+)-dependent and Ca(2+)-independent PKCs, activated PYK2 within 10 min, and PYK2 phosphorylation remained elevated after 30 min of stimulation. Adv-mediated overexpression of caPKC epsilon increased PYK2 phosphorylation, whereas Adv-mediated overexpression of a kinase-inactive mutant of PKC epsilon markedly inhibited ET-induced, but not basal PYK2 phosphorylation. In contrast, both basal and ET-induced PYK2 phosphorylation were blocked by treatment with the Src-family protein kinase inhibitor PP2. Although reducing Ca(2+) with either nifedipine (10 microM) or BAPTA-AM (50 microM) decreased basal PYK2 phosphorylation, it did not prevent ET-induced PYK2 activation. Furthermore, increasing Ca(2+) with ionomycin (10 microM), K(+) depolarization, or BayK8644 (1 microM) was not sufficient to further activate PYK2. These data demonstrate that ET-induced PYK2 activation is Gq, PKC epsilon, and Src dependent, describing a distinct signaling pathway leading to agonist-induced PYK2 activation in cardiomyocytes.
富含脯氨酸的酪氨酸激酶2（PYK2）是一种非受体蛋白酪氨酸激酶，它将G蛋白偶联受体与MAPK级联反应的激活及细胞生长联系起来。在平滑肌和其他细胞类型中，PYK2的激活依赖于Ca(2+)或蛋白激酶C（PKC），并且我们之前已经表明内皮素-1（ET）可激活成年和新生大鼠心室肌细胞（NRVM）中的PYK2。然而，ET既会改变细胞内Ca(2+)（Ca(2+)），又会激活新型的、不依赖Ca(2+)的PKC。因此，采用免疫沉淀和蛋白质印迹实验来检测NRVM中PYK2激活对PKC和Ca(2+)的依赖性。ET（100 nM；2 - 30分钟）和去氧肾上腺素（50 microM；2 - 30分钟）可激活PYK2，这两种物质都是激活Gq偶联受体的肥大激动剂。此外，与感染编码β-半乳糖苷酶的对照腺病毒（Adv）的NRVM相比，腺病毒介导的组成型活性（ca）Galphaq过表达早在感染后8小时就增加了PYK2 - Y(402)的磷酸化。caGalphaq过表达还诱导了PKCε和PKCδ（但不是PKCα）的易位，随后这两种新型PKC同工酶均下调。佛波酯（PMA；200 nM），一种Ca(2+)依赖性和Ca(2+)非依赖性PKC的直接激活剂，在10分钟内激活了PYK2，并且在刺激30分钟后PYK2磷酸化仍保持升高。腺病毒介导的caPKCε过表达增加了PYK2磷酸化，而腺病毒介导的PKCε激酶失活突变体过表达则显著抑制了ET诱导的而非基础水平的PYK2磷酸化。相反，Src家族蛋白激酶抑制剂PP2处理可阻断基础水平和ET诱导的PYK2磷酸化。虽然用硝苯地平（10 microM）或BAPTA - AM（50 microM）降低Ca(2+)会降低基础水平的PYK2磷酸化，但它并不能阻止ET诱导的PYK2激活。此外，用离子霉素（10 microM）、K(+)去极化或BayK8644（1 microM）增加Ca(2+)不足以进一步激活PYK2。这些数据表明ET诱导的PYK2激活是Gq、PKCε和Src依赖性的，描述了一条导致心肌细胞中激动剂诱导的PYK2激活的独特信号通路。
