The role of cyclin D3-dependent kinase in the phosphorylation of p130 in mouse BALB/c 3T3 fibroblasts

We have observed that cyclin D3-dependent kinase activity is increased in the late G(1), phase in BALB/c 3T3 fibroblasts. The profile of cyclin D3-associated activity closely parallels that of cyclin D1, which is also induced after mitogenic stimulation of quiescent cells. These activities correlate with the appearance of hyperphosphorylated p130, an Rb family member important in regulating E2F-4 and E2F-5 activity in fibroblastic cells. We demonstrated, however, that only the cyclin D3 activity efficiently phosphorylated p130 in an in vitro kinase assay. This apparent specificity was further demonstrated by experiments which demonstrated that cyclin D3 was physically associated with p130 at the times when D3-dependent kinase activity and p130 hyperphosphorylation were observed. Examination of E2F by electrophoretic mobility shift assay revealed that E2F-4 DNA binding activity existed in a p130.E2F complex at times before D3-dependent kinase activity was apparent and in a free E2F 4 complex after D3 activity developed. Thus, our data suggest that cyclin D3 preferentially phosphorylates p130 and is thereby specifically targeted to overcoming growth-suppressive control mediated through p130 pathways.