title: Sequential activation of individual PKC isozymes in integrin-mediated muscle cell spreading: a role for MARCKS in an integrin signaling pathway.
abstract: To understand how muscle cell spreading and survival are mediated by integrins, we studied the signaling events initiated by the attachment of muscle cells to fibronectin (FN). We have previously demonstrated that muscle cell spreading on FN is mediated by alpha5beta1 integrin, is associated with rapid phosphorylation of focal adhesion kinase and is dependent on activation of protein kinase C (PKC). Here we investigated the role of individual PKC isozymes in these cellular processes. We show that alpha, delta and epsilonPKC are expressed in muscle cells and are activated upon integrin engagement with different kinetics - epsilonPKC was activated early, whereas alpha and deltaPKC were activated later. Using isozyme-specific inhibitors, we found that the activation of epsilonPKC was necessary for cell attachment to FN. However, using isozyme-specific activators, we found that activation of each of three isozymes was sufficient to promote the spreading of alpha5-integrin-deficient cells on FN. To investigate further the mechanism by which integrin signaling and PKC activation mediate cell spreading, we studied the effects of these processes on MARCKS, a substrate of PKC and a protein known to regulate actin dynamics. We found that MARCKS was localized to focal adhesion sites soon after cell adhesion and that MARCKS translocated from the membrane to the cytosol during the process of cell spreading. This translocation correlated with different phases of PKC activation and with reorganization of the actin cytoskeleton. Using MARCKS-antisense cDNA, we show that alpha5-expressing cells in which MARCKS expression is inhibited fail to spread on FN, providing evidence for the crucial role of MARCKS in muscle cell spreading. Together, the data suggest a model in which early activation of epsilonPKC is necessary for cell attachment; the later activation of alpha or deltaPKC may be necessary for the progression from attachment to spreading. The mechanism of PKC-mediated cell spreading may be via the phosphorylation of signaling proteins, such as MARCKS, that are involved in the reorganization of the actin cytoskeleton.
authors.affiliation: Department of Neurology and Neurological Sciences, Stanford University School of Medicine, CA 94305, USA.
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Disatnik
authors.forename: Stéphane C
authors.initials: SC
authors.lastname: Boutet
authors.forename: Christine H
authors.initials: CH
authors.lastname: Lee
authors.forename: Daria
authors.initials: D
authors.lastname: Mochly-Rosen
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Rando
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins; D002448:Cell Adhesion; D002460:Cell Line; D002465:Cell Movement; D048429:Cell Size; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005959:Glucosidases; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology* / Q000201:enzymology*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D002135:Calcium-Binding Proteins; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619710:MARCKS protein, human; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D010750:Phosphoproteins; C512949:Prkcsh protein, mouse; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse; D011493:Protein Kinase C; D005959:Glucosidases; C070368:PRKCSH protein, human