CALMODULIN INHIBITOR W13 INDUCES SUSTAINED ACTIVATION OF ERK2 AND EXPRESSION OF P21(CIP1)

One of the major signaling pathways by which extracellular signals ind uce cell proliferation and differentiation involves the activation of extracellular signal-regulated kinases (ERKs), Because calmodulin is e ssential for quiescent cells to enter cell cycle, the role of calmodul in on ERK2 activation was studied in cultured fibroblasts. Serum, phor bol esters, or active Ras induced ERK2 activation in NIH 3T3 fibroblas ts. This activation was not inhibited by calmodulin blockade. Surprisi ngly, inhibition of calmodulin prior to fetal bovine serum addition pr olonged activation of ERK2, Furthermore, inactivation of calmodulin in serum-starved cells induced ERK2 phosphorylation that was dependent o n MAP kinase kinase (MEK). Inactivation of calmodulin in serum-starved cells also induced activation of Ras, Raf, and MEK. On the contrary, tyrosine phosphorylation of tyrosine kinase receptors was not observed . These results indicate that calmodulin inhibits ERK2 activation path way at the level of Ras. Calmodulin inhibition induced overexpression of p21(cip1) which was dependent on MEK activity. We propose that inhi bition of Ras by calmodulin prevents the activation of ERK2 at low ser um concentration. Thus, entering into the cell cycle after serum addit ion would imply the overcoming of the inhibitory effect of calmodulin and consequently ERK2 activation. Furthermore, down-regulation of Ras by calmodulin may be also important to determine the duration of ERK2 activation and to prevent a high p21(cip1) expression that would lead to an inhibition of cell proliferation.