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 G 1 - to S-phase cell cycle progression in vascular smooth muscle cells (VSMC). However, the molecular basis for this Ca 2+ sensitivity was not known. Using buffers with differing [Ca 2+ ], we found that the kinase activity of mouse and human cyclin E/CDK2, but not other G 1 /S-associated cell cycle complexes, was responsive to physiological changes in [Ca 2+ ]. We next determined that this Ca 2+ -responsive kinase activity was dependent on a direct interaction between calmodulin (CaM), one of the major Ca 2+ -signal transducers of eukaryotic cells, and cyclin E. Pharmacological inhibition of CaM abrogated the Ca 2+ sensitivity of cyclin E/CDK2 and retarded mouse VSMC proliferation by causing G 1 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 μmol/L) and showed its essential role in mediating Ca 2+ -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 [Ca 2+ ]. Taken together, these findings reveal CaM-dependent cyclin E/CDK2 activity as a mediator of the known Ca 2+ sensitivity of the G 1 /S transition of VSMC.