IndraLab

"CDK4/6-mediated serine-41 phosphorylation of DUB3 has been shown to be essential for the catalytic activity of DUB3 ( xref )."

"We next tested how CDK4/6 and S41 phosphorylation of DUB3 affects SNAIL1 ubiquitination."

"As shown in xref , mutation at S41 abolished CDK4/6-mediated phosphorylation of DUB3 in vitro ."

"Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and the phosphorylation of Ser41 in DUB3 was then examined."

"We found that Ser41 is a major phosphorylation site on DUB3, which matches with a CDK consensus motif (Supplementary Fig. 6b). To test whether S41 is a CDK4/6 phosphorylation site, GST-fused WT DUB3 and S41A mutants were incubated with active CDK4 or CDK6 and an in vitro kinase assay was performed. As shown in Fig. 5d, mutation at S41 abolished CDK4/6-mediated phosphorylation of DUB3 in vitro. A phospho-Ser41-specific antibody was generated to further study the phosphorylation of Ser41 in cells."

"Phosphorylation of Ser41 regulates DUB3 activity."

"We next tested how CDK4/6 and S41 phosphorylation of DUB3 affects SNAIL1 ubiquitination. We transfected cells with FLAG-DUB3 and HA-SNAIL1, treated cells with vehicle or CDK4/6 inhibitor PD0332991, and SNAIL1 ubiquitination was determined. As shown in Fig. 6a, SNAIL1 ubiquitination was stronger in cells treated with PD0332991 compared to vehicle, suggesting that CDK4/6 activity is important for the catalytic activity of DUB3. We further tested whether phosphorylation of S41 on DUB3 could affect the ubiquitination of SNAIL1 in cells. As shown in Fig. 6b,c, overexpression of both WT and S41D mutant DUB3 in cells efficiently decreased the ubiquitination of SNAIL1; however, S41A mutant failed to do so. Collectively, these results suggest that phosphorylation of S41 is important for the deubiquitinase activity of DUB3 towards SNAIL1."

"We further tested whether phosphorylation of S41 on DUB3 could affect the ubiquitination of SNAIL1 in cells."

"We next tested how CDK4/6 and S41 phosphorylation of DUB3 affects SNAIL1 ubiquitination."

"We further tested whether phosphorylation of S41 on DUB3 could affect the protein level of SNAIL1 in cells."

"We further tested whether phosphorylation of S41 on DUB3 could affect the ubiquitination of SNAIL1 in cells."

"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."

"We demonstrate that phosphorylation of DUB3 on Ser41 is critical for activation of the enzymatic function."

"We found that CDK4/6 inhibition markedly reduces the phosphorylation of DUB3 at Ser41 (Fig. 5f)."

"We demonstrate that phosphorylation of DUB3 on Ser41 is critical for activation of the enzymatic function. S41A mutant decreases DUB3 catalytic activity towards the fluorogenic substrate, Ub-AMC. Ser41 is located in an unstructured region of the protein, which is outside of the ubiquitin hydrolase domain. There are several reports implicating the regulatory phosphorylation of DUBs5758. These post-translational modifications may activate or modulate regulatory subunits, which can control DUB activity and possibly substrate specificity. In addition, the phosphorylation modification of DUBs may trigger structural changes and affect ubiquitin recognition. Further detailed structural studies on DUB3 should be carried out to better understand the role of phosphorylation on its catalytic activity. Although we show that CDK4/6-mediated Ser41 phosphorylation is essential for DUB3 activation, phosphorylation of the other potential sites should be further investigated."

"We demonstrate that phosphorylation of DUB3 on Ser41 is critical for activation of the enzymatic function."

"CDK4/6-mediated serine-41 phosphorylation of DUB3 has been shown to be essential for the catalytic activity of DUB3 ( Liu et al., 2017 )."

"We found that Ser41 is a major phosphorylation site on DUB3, which matches with a CDK consensus motif (Supplementary Fig. 6b)."

"We found that CDK4/6 inhibition markedly reduces the phosphorylation of DUB3 at Ser41 ( xref )."

"A phospho-Ser41-specific antibody was generated to further study the phosphorylation of Ser41 in cells. HEK293T cells were transfected with DUB3 WT or the S41A mutant. As shown in Fig. 5e, WT DUB3 was phosphorylated in cells. However, S41A mutation completely abrogated the phosphorylation of DUB3 at this site, indicating the specificity of this antibody."

"This result suggests that S41 phosphorylation is essential for the deubiquitinase activity of DUB3. We next tested how CDK4/6 and S41 phosphorylation of DUB3 affects SNAIL1 ubiquitination."

"We further tested whether phosphorylation of S41 on DUB3 could affect the protein level of SNAIL1 in cells."