Mechanism for repression of cyclin A-CDK activity in early G1 phase of mammalian cell cycle

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Destruction of cyclin A during mitotic (M) phase depends on a destruction box (D box) which is conserved among A- and B-type cyclins. We have constructed a D-box mutant of human cyclin A and investigated its fate and function during the cell cycle in mouse fibroblasts. When expressed at a nearly physiological level, this D-box mutant was stabilized during the mitosis and subsequent entry into G1 phase but did not interfere the progression of M-to-G1 transition. Thus the destruction of cyclin A is not essential for the M-to-G1 transition. Even though the D-box mutant of cyclin A was stable, we found its associated cyclin-dependent kinase (CDK) activity was downregulated at the conclusion of mitosis. The stabilized cyclin A-associated CDK activity was also repressed during the M-to-G1 transition in fibroblasts derived from mice nullizygous for CDK inhibitors p21 and/or p27, indicating that neither p21 nor p27 is essential for the repression. In addition, phosphoamino acid analyses strongly suggested that CDKs associated with the D-box mutant cyclin A are not inactivated by phosphorylation at tyrosines during the M-to-G1 transition. We found that an Rb-family tumor suppressor p107 binds to the D-box mutant cyclin A at the early G1 phase when both p21 and p27 are missing, and that the stabilized cyclin A-associated CDK activity is deprepressed in p21-/-p27-/-p107-/- fibroblasts. These results suggest that p21 and p27 function as primary inhibitors and p107 serves as a secondary inhibitor in the downregulation of the stabilized cyclin A-associated CDK during the M-to-G1 transition. This alternative downregulation pathway might be a "fail-safe" mechanism which maintains normal progression through the cell cycle independent of the cyclin destruction pathway.