Viral K-cyclin derived from Kaposi's sarcoma-associated herpesvirus is homologous with mammalian D-type cyclins. Here, we demonstrated the regulatory mechanisms for K-cyclin function and degradation in human embryonic kidney HEK293 and primary effusion lymphoma JSC-1 cell lines. Proteasome inhibitor MG132 treatment induced an accumulation of ubiquitinated K-cyclin in these cells, and co-expression of CDK6 prevented K-cyclin ubiquitination. Also K-cyclin mutants incompetent for CDK6-binding were destabilized by proteasome pathway. Furthermore, silencing of p16INK4a promoted K-cyclin-CDK6 complex formation and hence induced K-cyclin-associated kinase activity in HEK293 cells. These observations indicate that CDK6-bound K-cyclin is functionally stable but monomeric K-cyclin is targeted to ubiquitin-dependent degradation pathway in these cells. Our data suggest that the balance between CDK6 and p16INK4a regulates the availability of functional K-cyclin in human cells. (C) 2010 Elsevier Inc. All rights reserved.