Numerous mutations of the Von Hippel-Lindau (have been found associated with

Numerous mutations of the Von Hippel-Lindau (have been found associated with intellectual disability and various types of cancers, such renal cell cancer, breast cancer and prostate cancer [39C41]. 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 284028-89-3 IC50 potential mediators of pVHL. A series 284028-89-3 IC50 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 in the protein pull-down list offered potential regulators of pVHL stability (Figure ?(Figure1A,1A, lane 284028-89-3 IC50 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 284028-89-3 IC50 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 time, immunofluorescence data showed co-localization of USP9X and pVHL (Supplementary Figure S1A). USP9X has a USP domain which consists of a conserved catalytic core essential for its deubiquitinase function. 284028-89-3 IC50 binding assays with recombinant GST-tagged pVHL and 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). Figure 1 USP9X physically interacts with pVHL 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 ligase in HEK293T cells. USP9X knockdown up-regulated pVHL, while HUWE1 showed no evidence of pVHL regulation at the protein level (Supplementary Figure S2A). Two other E3 ligases, UBR4 and Smurf1 also induced pVHL upon knockdown. As knockdown of USP9X in HEK293T cells significantly increased pVHL levels (Figure ?(Figure2A,2A, lane 1 lanes 2-3), ITSN2 both mRNA levels and protein half-life of pVHL were evaluated in HEK293T cells to further investigate the mechanism by which USP9X regulates pVHL. There were no significant alterations of pVHL mRNA after USP9X knockdown (Figure ?(Figure2B).2B). However, the half-life of pVHL was dramatically increased after USP9X knockdown (Figure ?(Figure2C).2C). Similar results were also obtained with the human hepatocellular carcinoma cell line HepG2 (Figure ?(Figure2D,2D, Supplementary Figure S2B and S2C). These results suggest USP9X influences pVHL levels through regulation of protein stability, rather than through alteration of mRNA levels. Figure 2 USP9X negatively regulates pVHL Negative regulation of pVHL by USP9X was further validated in the human prostate cancer cell line PC3 and the mouse melanoma cell line B16 (Figure 2D-2F and Supplementary Figure S2D, lane 1 lanes 2-3). In order to demonstrate USP9X negatively regulates pVHL in clear cell renal cell carcinoma (ccRCC) which is a VHL disease associated neoplasm, USP9X was knocked down in 786-0 cells stably expressing HA-tagged pVHL. The level of exogenous HA-tagged pVHL also increased after USP9X knockdown (Figure ?(Figure2G,2G, lane 1 lanes 2-3). These findings show both exogenous and endogenous pVHL can be regulated.