Protein kinase Cepsilon-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 II, 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 PKCepsilon, PKCdelta, and PKCalpha. Only PKCepsilon increased phosphorylated MARCKS (pMARCKS). In contrast, Adv-mediated overexpression of a dominant-negative (dn) mutant of PKCepsilon reduced basal and ET-stimulated pMARCKS. dnPKCepsilon 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 PKCepsilon, and participates in the regulation of FAK phosphorylation and cell spreading.
Heidkamp
Maria C
MC
The Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, USA.
Iyengar
Rekha
R
Szotek
Erika L
EL
Cribbs
Leanne L
LL
Samarel
Allen M
AM
D000256:Adenoviridae; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D020536:Enzyme Activators / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D006352:Heart Ventricles / Q000166:cytology / Q000201:enzymology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation / Q000187:drug effects; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D014161:Transduction, Genetic
D020536:Enzyme Activators; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051744:Protein Kinase C-epsilon