Modulation of vascular smooth muscle cell migration by calcium/ calmodulin-dependent protein kinase

Pfleiderer, Paul J., Katherine Kun Lu, Michael T. Crow, Rebecca S. Keller, and Harold A. Singer. Modulation of vascular smooth muscle cell migration by calcium/calmodulin-dependent protein kinase II2. Am J Physiol Cell Physiol 286: C1238–C1245, 2004. First published February 4, 2004; 10.1152/ajpcell.00536.2003.—Previous studies demonstrated a requirement for multifunctional Ca /calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to 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 CaMKII 2 were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr, a CaMKII-selective phosphorylation site. Expression of kinasenegative CaMKII 2 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 CaMKII 2 autophosphorylation on Thr after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKII 2 phosphorylated substrate in vitro without added Ca /calmodulin and in the intact cell without added Ca -dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKII 2 on Thr. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKII 2, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKII 2 mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKII 2 isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKII 2 autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.