Modulation of vascular smooth muscle cell migration by calcium/ calmodulin-dependent protein kinase II-delta 2.
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 (delta(2) or delta(C)) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKII delta(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(17), a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKII delta(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 delta(2) autophosphorylation on Thr(287) after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKII delta(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 CaMKII delta(2) on Thr(287). Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKII delta(2), an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKII delta(2) mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKII delta(2) isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKII delta(2) autophosphorylation may play an important role in PDGF-stimulated VSM cell migration.
Pfleiderer
Paul J
PJ
Center for Cardiovascular Sciences, Albany Medical College, NY 12208, USA.
Lu
Katherine Kun
KK
Crow
Michael T
MT
Keller
Rebecca S
RS
Singer
Harold A
HA
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013912:Threonine / Q000378:metabolism
D002147:Calmodulin; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D013912:Threonine; C517660:CAMK2D protein, human; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517677:Camk2d protein, rat; D002118:Calcium