Figure 5: CDK4/6 phosphorylates DUB3 at Ser 41.

(a) MDA-MB-231 cells stably expressing control or DUB3 shRNAs were treated with either vehicle or PD0332991 for 24 h. Western blot was performed with the indicated antibodies. (b) MDA-MB-231 cell lysates were subjected to immunoprecipitation with control IgG or anti-DUB3 antibodies. The immunoprecipitates were then blotted with the indicated antibodies. (c) Purified recombinant GST, GST-CDK4, GST-CDK6 and His-DUB3 were incubated in vitro as indicated. The interaction between DUB3 and CDK4/6 was then examined. CBS, Coomassie blue staining. (d) CDK4/6 phosphorylates DUB3 in vitro. Bacterial expressed GST-DUB3 WT and GST-DUB3 S41A fusion proteins were incubated with active CDK4 or CDK6 in the presence of [γ-³²P]ATP. Proteins were resolved by SDS–polyacrylamide gel electrophoresis; phosphorylated proteins were visualized with autoradiography. (e) FLAG-DUB3 WT or FLAG-DUB3 S41A was transfected in cells stably expressing DUB3 shRNA. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and the phosphorylation of Ser41 in DUB3 was then examined. (f) Cells were transfected with indicated plasmids and were treated with vehicle, or CDK4/6 inhibitor (PD0332991). Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and the phosphorylation of Ser41 was examined. (g) Cells were transfected with indicated plasmids and cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and western blot was performed. (h) MDA-MB-231 cells stably expressing indicated shRNAs were generated and western blot was performed with the indicated antibodies. (i) MDA-MB-231 cells stably expressing DUB3 shRNA were transfected with the WT, S41D or S41A mutant FLAG-DUB3 and treated with either vehicle or PD0332991. Western blot was performed with the indicated antibodies.