INHIBITION OF CYCLIN D1 PHOSPHORYLATION ON THREONINE-286 PREVENTS ITSRAPID DEGRADATION VIA THE UBIQUINTIN-PROTEASOME PATHWAY

The expression of D-type G1 cyclins and their assembly with their cata lytic partners, the cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), into active holoenzyme complexes are regulated by growth factor-induce d signals. In turn, the ability of cyclin D-dependent kinases to trigg er phosphorylation of the retinoblastoma (Rb) protein in the mid- to l ate G(1) phase of the cell cycle makes the inactivation of Rb's growth suppressive function a mitogen-dependent step. The ability of D-type cyclins to act as growth factor sensors depends not only on their rapi d induction by mitogens but also on their inherent instability, which ensures their precipitous degradation in cells deprived of growth fact ors. However, the mechanisms governing the turnover of D-type cyclins have not yet been elucidated. We now show that cyclin D1 turnover is g overned by ubiquitination and proteasomal degradation, which are posit ively regulated by cyclin D1 phosphorylation on threonine-286. Althoug h ''free'' or CDK4-bound cyclin D1 molecules are intrinsically unstabl e (t1/2 < 30 min), a cyclin D1 mutant (T286A) containing an alanine fo r threonine-286 substitution fails to undergo efficient polyubiquitina tion in an in vitro system or in vivo, and it is markedly stabilized ( t1/2 similar to 3.5 hr) when inducibly expressed in either quiescent o r proliferating mouse fibroblasts. Phosphorylation of cyclin D1 on thr eonine-286 also occurs in insect Sf9 cells, and although the process i s enhanced significantly by the binding of cyclin D1 to CDK4, it does not depend on CDK4 catalytic activity. This implies that another kinas e can phosphorylate cyclin D1 to accelerate its destruction and points to yet another means by which cyclin D-dependent kinase activity may be exogenously regulated.