E. is likely that the majority of USP9X related cellular activities in cancer are as yet undiscovered, and to address the complexities of these mechanisms will be challenging. In this study, we identify USP9X as a pVHL interacting protein which regulates pVHL turnover through a newly identified pVHL E3 ligase designated Smurf1. This study shows that inhibition of USP9X function by either shRNA or a chemical inhibitor significantly enhances pVHL levels and suppresses tumor cell proliferation. Our findings reveal USP9X functions in cell proliferation through regulation of the pVHL-HIF pathway, and raise the possibility of therapeutic targeting of USP9X for rescue of unstable pVHL mutants from degradation for treatment of VHL-related tumors. RESULTS USP9X physically interacts with pVHL To identify genes involved in the regulation of pVHL levels, Flag pull-down assay was performed to search for potential mediators of pVHL. A series of known pVHL associated proteins such as Elongin B, Elongin C, and TRiC/CCT family proteins were identified by mass spectrometry (MS) analysis, confirming the reliability of this assay. A wide variety of E3 ligases and deubiquitinases Tcfec in the protein pull-down list offered potential regulators of pVHL stability (Figure ?(Figure1A,1A, lane 2), including HUWE1 E3 ligase and USP9X deubiquitinase which have been reported to interact [44]. We initially hypothesized that pVHL, USP9X, and HUWE1 interact with each other. To validate this supposition, we first verified interaction of pVHL and USP9X. HA-tagged pVHL was overexpressed and immunoprecipitated in 786-0 cells, which are a pVHL-defective renal cell carcinoma cell line. As shown in Figure ?Figure1B,1B, HA-tagged pVHL binds to endogenous USP9X under MG132 treatment (lane 1 lane 2). In addition, immunoblotting using an anti-VHL antibody identified pVHL in the immunoprecipitant of endogenous USP9X in HEK293T cells (Figure ?(Figure1C,1C, lane 2 lane 3). At the same Tilbroquinol time, immunofluorescence data showed co-localization of USP9X and pVHL (Supplementary Figure S1A). USP9X has a USP domain which consists of Tilbroquinol a conserved catalytic core essential for its deubiquitinase function. binding assays with recombinant GST-tagged pVHL and Tilbroquinol the His-tagged USP9X USP domain suggested there is direct binding of pVHL and USP9X through the USP domain (Figure ?(Figure1D,1D, lane 1 lane 2). To verify interaction of pVHL and HUWE1, exogenous co-immunoprecipitation assays were carried out after transiently transfecting human kidney HEK293T cells with Flag-tagged HUWE1 and HA-tagged pVHL. Two co-immunoprecipitation results showed pVHL associates with HUWE1 after treatment with MG132 (Supplementary Figure S1B and S1C). Open in a separate window Figure 1 USP9X physically interacts with pVHLA. Flag pull-down analysis. Empty or Flag-pVHL vector was transfected into HEK293T cells for 24 hours. Harvested cells were subjected to Flag pull-down assay. Samples were run on a gradient gel followed by silver staining. Indicated bands were excised for MS analysis. B. binding of pVHL with USP9X. 786-0 cells infected with HA empty control or Tilbroquinol HA-VHL were treated with MG132 (10 M) for 4 hours. Cells were harvested and then immunoprecipitated with anti-HA antibody followed by Tilbroquinol immunoblotting with anti-USP9X and anti-HA antibodies. C. binding of endogenous pVHL with USP9X. HEK293T cells treated with MG132 (10 M for 4 hours) were immunoprecipitated with an anti-USP9X antibody and immunoblotted with indicated antibodies. The indicated pVHL is the shorter isoform of endogenous pVHL C pVHL19. D. binding of pVHL with the USP9X USP domain. GST control or GST-tagged pVHL was incubated with His-tagged USP9XUSP (USP9X USP domain). USP9X negatively regulates pVHL In order to determine whether these two proteins regulate pVHL levels, we knocked down or which is a known pVHL E3.