Figure 4: Cyclin F regulates B-Myb activity via its hydrophobic patch motif.

(a) HEK293 cells were transfected with empty vector (EV) or mouse FLAG B-Myb expression plasmids, and subjected to FLAG-immunoprecipitation followed by immunoblotting with anti-cyclin F and anti-FLAG antibodies. (b) HEK293 cells were transfected with EV, FLAG Cyclin F wt or cyclin F hydrophobic patch mutant (MR/AA) expression plasmids, and subjected to immunoprecipitation with FLAG beads. Lysates and immunoprecipitates were analysed by immunoblotting. The arrow indicates the co-precipitated endogenous B-Myb, and numbers below the panels represent quantification of the B-Myb band. The asterisk denotes an unspecific band. (c) U2OS cells or cells expressing siRNA-resistant Frt-Cyclin F wild-type (wt) or MR/AA mutant were transfected with indicated siRNAs. Cells were then synchronized with a double thymidine block and released for 8 h followed by IR as indicated (6 Gy). After 3 h, cells were harvested and analysed by immunoblotting with phospho-B-Myb-T487, B-Myb, cyclin F and actin antibodies. (d) U2OS cells, Frt-cyclin F wild-type or MR/AA mutant siRNA-resistant cells were transfected with siRNA. After 48 h, cells were treated with IR (6 Gy) and harvested 3 h later. RNA was extracted, cDNA generated and relative numbers of transcripts were analysed by quantitative PCR. Values were normalized to the IR-treated control (UNC transfected) sample. Error bars indicate s.e.m. (n=5). (e) U2OS or Frt-Cyclin F MR/AA mutant siRNA-resistant cells were depleted for 48 h as indicated. Twenty-four hours after transfection, doxycycline was added to induce the siRNA-resistant version of cyclin F. After another 24 h, cells were irradiated with 6 Gy of IR, left for 2 h, treated with Nocodazole for 7 h and harvested for flow cytometry. Error bars indicate s.e.m. (n=3). (f) Cyclin F regulates B-Myb transcriptional activity to guard against unscheduled mitotic entry in the presence of unrepaired DNA lesions. In the absence of damage, cyclin F-mediated control of B-Myb has little impact on cell proliferation (left side of the figure). However, when cells are challenged with IR, cyclin F suppresses the activating CDK–cyclin A-mediated phosphorylation of B-Myb and thereby the transcriptional programme that promotes progression through the G2 phase into mitosis (right side of the figure).