Abstract 1389: PKA Constitutes a Novel Activator of Cardiac Protective Pak1 Signaling
Pak1, the best characterized member of the p21 activated kinase (Pak) family, is an important downstream effector of the small GTPases, cdc42 and Rac1, and plays a crucial role in various vital cellular processes, including cell growth and cell survival. However, the exact mechanism governing Pak1 activation remains elusive. Here we provide several lines of evidence to define PKA (cAMP-dependent protein kinase A) as a primary regulator of Pak1 activation in the heart. First, beta-adrenergic receptor (B-AR) stimulation by isoproterenol (ISO) leads to a profound increase in Pak1 activation, as evidenced by its phosphorylation, in a time- and dose-dependent manner. ISO-induced Pak1 activation is abolished by inhibition of PKA with PKI (a peptide inhibitor of PKA), suggesting B-AR induced Pak1 activation is dependent on phosphorylation of Pak1 by PKA. Second, stimulation of adenylate cyclase by forskolin also markedly increases Pak1 phosphorylation by 7-fold in a PKI-sensitive manner. Importantly, using in vitro kinase assays, we have demonstrated that PKA overtly increases Pak1 phosphorylation, and that a point mutation of Pak1 to replace Thr 423 with a glutamic acid fully blocks PKA-mediated phosphorylation of Pak1, indicating Thr 423 is the specific phosphorylation site for PKA. Indeed, co-immunoprecipitation analysis shows that PKA associates with WT Pak1 but not the Pak1 mutant. Functional studies have revealed that enhanced Pak1 activation protects cardiomyocytes against hypoxia- or ischemia-induced cell death. Cardiac protection by Pak1 is mediated, at least in part, by up-regulating a panel of genes involved in cell growth and survival, including AKT, NF-kB, and ERK1/2, as manifested by gene expression profiles. These results provide compelling evidence that PKA, an evolutionarily conserved and ubiquitously expressed Ser/Thr protein kinase, constitutes a primary regulator of cardiac protective Pak1 signaling, shedding new light on our understanding of the fundamental function and underlying signaling mechanism of Pak1.