SPECIFIC REGULATION OF E2F FAMILY MEMBERS BY CYCLIN-DEPENDENT KINASES

The transcription factor E2F-1 interacts stably with cyclin A via a sm all domain near its amino terminus and is negatively regulated by the cyclin A-dependent kinases, Thus, the activities of E2F, a family of t ranscription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators: the retinoblastoma prote in family and the cyclin-dependent kinase family. To investigate furth er the regulation of E2F by cyclin-dependent kinases, we have extended our studies to include additional cyclins and E2F family members, Usi ng purified components in an in vitro system, we show that the E2F-1-D P-1 heterodimer, the functionally active form of the E2F activity, is not a substrate for the active cyclin D-dependent kinases but is effic iently phosphorylated by the cyclin B-dependent kinases, which do not form stable complexes with the E2F-1-DP-1 heterodimer. Phosphorylation of the E2F-1-DP-1 heterodimer by cyclin B-dependent kinases, however, did not result in down-regulation of its DNA-binding activity, as is readily seen after phosphorylation by cyclin A-dependent kinases, sugg esting that phosphorylation per se is not sufficient to regulate E2F D NA-binding activity, Furthermore, heterodimers containing E2F-4, a fam ily member lacking the cyclin A binding domain found in E2F-1, are not efficiently phosphorylated or functionally down-regulated by cyclin A -dependent kinases, However, addition of the E2F-1 cyclin A binding do main to E2F-1 conferred cyclin A-dependent kinase-mediated down-regula tion of the E2F-4-DP-1 heterodimer. Thus, both enzymatic phosphorylati on and stable physical interaction are necessary for the specific regu lation of E2F family members by cyclin-dependent kinases.