Protein kinase Cε-dependent MARCKS phosphorylation in neonatal and adult rat ventricular myocytes

The myristoylated, alanine-rich protein kinase C substrate (MARCKS) is a cytoskeletal protein implicated in the regulation of cell spreading, stress fiber formation, and focal adhesion assembly in nonmuscle cells. However, its precise role in cardiomyocyte growth, and its PKC-dependent regulation have not been fully explored. In this report, we show that MARCKS is expressed and phosphorylated under basal conditions in cultured neonatal and adult rat ventricular myocytes (NRVM and ARVM, respectively). The PKC activators phenylephrine, angiotensin IT, and endothelin-1 (ET) further increased MARCKS phosphorylation, with ET inducing the greatest response. To determine which PKC isoenzyme was responsible for agonist-induced MARCKS phosphorylation, NRVM and ARVM were infected with replication-defective adenoviruses (Adv) encoding wildtype (wt) and constitutively active (ca) mutants of PKC epsilon, PKC delta, and PKC alpha. Only PKC epsilon increased phosphorylated MARCKS (pMARCKS). In contrast, Adv-mediated overexpression of a dominant-negative (dn) mutant of PKC epsilon reduced basal and ET-stimulated pMARCKS. dnPKC epsilon; overexpression also prevented ET-induced, apparent co-localization of pMARCKS with f-actin staining structures. Adv-mediated overexpression of GFP-tagged, wtMARCKS (wtMARCKS-GFP) increased phosphorylation of focal adhesion kinase (FAK) and also increased NRVM surface area. In contrast, overexpression of a GFP-tagged, non-phosphorylatable (np) MARCKS mutant (npMARCKS-GFP) decreased basal and ET-induced endogenous MARCKS and FAK phosphorylation, and blocked the ET-induced increase in NRVM surface area. We conclude that MARCKS is expressed in cardiomyocytes, is phosphorylated by PKC epsilon, and participates in the regulation of EAK phosphorylation and cell spreading. (c) 2006 Elsevier Inc. All rights reserved.