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2
1 Center for Cardiovascular Sciences, Albany Medical College, Albany, NY, USA
2 Division of Pulmonary and Critical Care Medicine, John Hopkins University, Baltimore, MD, USA
* To whom correspondence should be addressed. E-mail: singerh{at}mail.amc.edu.
Previous studies demonstrated a requirement for multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present molecular approaches were utilized to specifically assess the role of the predominant CaMKII isoform (
2 or C) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKII2 were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro using a peptide substrate and in intact cells by assessing phosphorylation of overexpressed phospholamban on Thr17, a CaMKII selective phosphorylation site. Expression of kinase-negative CaMKII2 inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKII2 autophosphorylation on Thr287 following activation of cells with ionomycin, and in fact these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKII2 phosphorylated substrate in vitro without added Ca2+/calmodulin and in the intact cell without added Ca2+-dependent stimuli, but inhibited autophosphorylation of endogenous CaMKII2 on Thr287. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKII2, an effect opposite of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKII2 mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKII2 isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKII2 autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.
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