Previous studies demonstrated a requirement for multifunctional Ca 2+/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 (δ2 or δc) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKIIδ2 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 Thr17, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKIIδ2 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 CaMKIIδ2 autophosphorylation on Thr287 after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKIIδ 2 phosphorylated substrate in vitro without added Ca 2+/calmodulin and in the intact cell without added Ca 2+-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKIIδ2 on Thr287. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKIIδ2, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKIIδ2 mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKIIδ2 isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKIIδ2 autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.
- Calcium/calmodulin-dependent protein kinase II
- Cell migration
- Platelet-derived growth factor
ASJC Scopus subject areas
- Cell Biology