A Role for Ca 2+ /Calmodulin-Dependent Protein Kinase II in the Mitogen-Activated Protein Kinase Signaling Cascade of Cultured Rat Aortic Vascular Smooth Muscle Cells

Abstract

Abstract Exposure of cultured rat aortic vascular smooth muscle (VSM) cells to the Ca 2+ ionophore ionomycin produced an increase in extracellular signal–regulated kinase 1/2 (ERK1/2) activity that was maximal between 2 and 5 minutes but then declined to basal values within 20 minutes of stimulation. Elevation of [Ca 2+ ] i in VSM cells leads to an even more rapid activation of Ca 2+ /calmodulin-dependent protein kinase II (CaM kinase II); thus, it was postulated that the Ca 2+ -dependent component of ERK1/2 activation was mediated by CaM kinase II. Transient ERK1/2 activation by ionomycin was almost completely abolished by pretreating cells with 30 μmol/L KN-93, a CaM kinase II inhibitor. Treatment of cells with KN-93 did not antagonize the ability of ionomycin to mobilize intracellular Ca 2+ but prevented CaM kinase II and ERK1/2 activation with almost identical potencies. Consistent with a role for Ca 2+ and calmodulin in intracellular Ca 2+ –induced activation of ERK, cells pretreated with calmodulin inhibitors (W-7 or calmidazolium) exhibited an attenuated ERK response to ionomycin. ERK1/2 activation in response to phorbol esters and platelet-derived growth factor were not significantly affected by KN-93, whereas the response to angiotensin II and thrombin were attenuated by 60% and 40%, respectively. Transient expression of wild-type δ 2 CaM kinase II in COS-7 cells resulted in increased ERK2 activity, whereas coexpression of wild-type and a kinase-negative mutant resulted in a diminution of this response. These data suggest that regulation of cellular responses by Ca 2+ -dependent pathways in VSM cells may be mediated in part by CaM kinase II–dependent activation of ERK1/2.