CYCLIN-DEPENDENT KINASE-2 (CDK2) FORMS AN INACTIVE COMPLEX WITH CYCLIN D1 SINCE CDK2 ASSOCIATED WITH CYCLIN D1 IS NOT PHOSPHORYLATED BY CDK7-CYCLIN-H

Cyclin-dependent kinases (Cdks) form complexes with cyclins, and as a consequence they generally express kinase activities. One of these Cdk s, Cdk2, is known to bind with cyclins A and E, and plays an important role in the progression of the cell cycle via phosphorylation of targ et proteins such as the product of the retinoblastoma tumor-suppressor gene (pRB). It has been suggested that Cdk2 bound with cyclin D1 and Cdk2-cyclin-D1 complex show neither H1 histone nor pRB kinase activity . However, it is not clear whether Cdk2-cyclin-D1 has unknown targets and why Cdk2 is not activated by binding with cyclin D1. We investigat ed these questions using Cdk, cyclin and Cdk-cyclin complexes produced in a baculovirus expression system. Cdk2 formed a complex with cyclin D1 in this system. After extensive purification, Cdk2 was still bound to cyclin D. The Cdk2-cyclin-D1 complex did not phosphorylate any tes ted substrates, such as H1 histone, pRB, SV40 large T antigen, p53, E2 F-1 or a preparation of nuclear proteins from HeLa cells; in contrast, Cdk2-cyclin-E and Cdk2-cyclin-A phosphorylated these proteins. Moreov er, the Cdk2-cyclin-D1 complex was not activated by incubation with Cd k4 or cyclin E. Thus, Cdk2 and cyclin D1 formed a stable complex that was not activated. In order to determine why Cdk2-cyclin-D1 lacks kina se activity, we investigated the phosphorylation of Cdk2. Under-shifte d Cdk2, the active form of Cdk2, was not detected in the Cdk2-cyclin-D 1 complex in the baculovirus system. In human WI-38 cells, cyclin D1 b egan to form a complex with Cdk2 as well as with Cdk4 from the mid-G1 phase of the cell cycle. The Cdk2 bound to cyclin D1 in human cells wa s also the inactive form that was slowly migrated. Moreover, we found that Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7-cyclin-H, while Cdk2 bound to cyclin E, as well as free Cdk2, was phosphorylated by Cdk7-cyclin-H. Additionally, Cdk2 phosphorylated by Cdk7-cyclin-H did not bind to cyclin D1. These results strongly suggest that Cdk2 fo rms a stable complex with cyclin D1 but is not activated because the C dk2 molecule in the complex is not phosphorylated by Cdk7-cyclin-H and the phosphorylated Cdk2, an active form, does not bind to cyclin D1.