Figure 6: Phosphorylation of Ser41 regulates DUB3 activity.

(a) MDA-MB-231 cells were cotransfected with indicated plasmids and treated with vehicle or PD0332991. Cell lysates were boiled and immunoprecipitated with HA beads and immunoblotted as indicated. (b) Cells were transfected with indicated plasmids and the polyubiquitylated SNAIL1 protein was examined. (c) Cells were cotransfected with indicated plasmids and Ni-NTA beads were used to pull down His-tagged ubiquitin, and the polyubiquitylated SNAIL1 protein was examined. (d) Cells were transfected with indicated plasmids and western blot was performed as indicated. (e,f) The migratory ability of cells as in d was analysed by wound-healing assay. Scale bars, 100 μm. Results are quantified in f. The results represent the means±s.d. of three independent experiments. ^##P<0.01. (g) Cells stably expressing DUB3 shRNA were transfected with the WT, S41D or S41A mutant FLAG-DUB3 and one million cells from c were injected into the lateral tail vein of immunodeficient mice (n=8). After 12 weeks, mice were killed and the development of lung metastases was determined by counting the number of metastatic lung nodules. Data are represented as mean±s.d. Statistical analyses were performed with the Student’s t-test. ^##P<0.01. (h) The working model to illustrate that CDK4/6 phosphorylation-dependent activation of DUB3 regulates epithelial–mesenchymal transition and metastasis through SNAIL1.