A calmodulin-binding site on cyclin E mediates Ca2+-sensitive G1/s transitions in vascular smooth muscle cells.

Calcium transients are known to control several transition points in the eukaryotic cell cycle. For example， we have previously shown that a coordinate elevation in the intracellular free calcium ion concentration is required for G1- to S-phase cell cycle progression in vascular smooth muscle cells (VSMC). However， the molecular basis for this Ca2+ sensitivity was not known. Using buffers with differing [Ca2+]， we found that the kinase activity of mouse and human cyclin E/CDK2， but not other G1/S-associated cell cycle complexes， was responsive to physiological changes in [Ca2+]. We next determined that this Ca2+-responsive kinase activity was dependent on a direct interaction between calmodulin (CaM)， one of the major Ca2+-signal transducers of eukaryotic cells， and cyclin E. Pharmacological inhibition of CaM abrogated the Ca2+ sensitivity of cyclin E/CDK2 and retarded mouse VSMC proliferation by causing G1 arrest. We next defined the presence of a highly conserved 22 amino acid N-terminal CaM-binding motif in mammalian cyclin E genes (dissociation constant， 1.5+/-0.1 micromol/L) and showed its essential role in mediating Ca2+-sensitive kinase activity of cyclin E/CDK2. Mutant human cyclin E protein， lacking this CaM-binding motif， was incapable of binding CaM or responding to [Ca2+]. Taken together， these findings reveal CaM-dependent cyclin E/CDK2 activity as a mediator of the known Ca2+ sensitivity of the G1/S transition of VSMC.