Regulation of cyclin-dependent kinase 2 activity by ceramide

Cyclin-dependent kinases have been implicated in the inactivation of retino blastoma (Rb) protein and cell cycle progression. Recent studies have demon strated that the lipid molecule ceramide is able to induce Rb hypophosphory lation leading to growth arrest and cellular senescence. In this study, we examined the underlying mechanisms of Rb hypophosphorylation and cell cycle progression utilizing the antiproliferative molecule ceramide. C-6-Ceramid e induced a G0/G1 arrest of the cell cycle in WI38 human diploid fibroblast s, Employing immunoprecipitation kinase assays, we found that ceramide spec ifically inhibited cyclin-dependent kinase CDK2, with a mild effect on CDC2 and significantly less effect on CDK4. The effect of ceramide was specific such that C-6-dihy-droceramide was not effective. Ceramide did not directl y inhibit CDK2 in vitro but caused activation of p21, a major class of CDK- inhibitory proteins, and led to a greater association of p21 to CDK2. Using purified protein phosphatases, we showed that ceramide activated both prot ein phosphatase 1 and protein phosphatase 2A activities specific for CDK2 i n vitro. Further, calyculin A and okadaic acid, both potent protein phospha tase inhibitors, together almost completely reversed the effects of ceramid e on CDK2 inhibition. Taken together, these results demonstrate a dual mech anism by which ceramide inhibits the cell cycle. Ceramide causes an increas e in p21 association with CDK2 and through activation of protein phosphatas es selectively regulates CDK2. These events may lead to activation of Rb pr otein and subsequent cell cycle arrest. (C) 2000 Academic Press.