Knockdown of SENP1 drastically induced cell cycle arrest and suppressed cell proliferation in several tested human malignancy cell lines. (4, 10), as well as deubiquitinating enzymes (DUBs), including the ubiquitin-specific protease (USP) family members USP28, USP36, USP37, USP13, and USP22 (10C14). Thus, c-Myc levels and activity are tightly controlled by a dynamic balance between ubiquitination and deubiquitination. Recently, it has been shown that c-Myc is also regulated by SUMOylation (15C18), a posttranslational modification of proteins by small ubiquitin-like modifiers (SUMOs), including SUMO1, SUMO2, and SUMO3. While c-Myc lacks a sequence consensus SUMOylation lysine (Lys, K), it can be SUMOylated on at least 10 SUMO acceptor Ks, including K323 and K326 (15, 16, 18). However, mutating K323 and K326 or all of the 10 Ks did not abolish c-Myc SUMOylation and did not significantly alter its levels and activity (15, 16, 18). Thus, how SUMOylation regulates c-Myc stability and activity Naspm remains elusive. Further, SUMO modification is usually highly dynamic Naspm and the effects of c-Myc deSUMOylation remain unknown. In an attempt to understand the deSUMOylation regulation of c-Myc, we identified SENP1 in Naspm a screen for deSUMOylating enzymes interacting with c-Myc. We confirmed that SENP1 directly binds to and deSUMOylates c-Myc in vitro and in cells. Interestingly, overexpression of wild-type (WT) SENP1, but not its catalytically inactive C603S mutant (SENP1C603S, Cys-603 mutated to Ser), stabilizes c-Myc and enhances c-MycCdriven transcription. Knockdown of SENP1 reduces c-Myc levels and drastically induces cell cycle arrest and suppresses cell proliferation. We showed that c-Myc is usually comodified by ubiquitin and SUMO, and c-Myc deSUMOylation by SENP1 reduces its polyubiquitination, indicating that SUMOylation promotes c-Myc degradation through the proteasome system. Consistent with this functional role, we find that SENP1 is frequently overexpressed in human breast malignancy cell lines and primary breast malignancy tissues, correlating with the up-regulation of c-Myc. Together, these results reveal that SENP1 is usually a crucial c-Myc deSUMOylating enzyme that positively regulates c-Myc stability and activity. Results SENP1 Interacts with c-Myc in Cells and in Vitro. To examine whether c-Myc is usually regulated by deSUMOylation, we screened all known deSUMOylating enzymes for their ability to bind to c-Myc using coimmunoprecipitation (co-IP)Cimmunoblot (IB) assays and found that c-Myc Naspm interacted with SENP1, SENP3, and USPL1 (and and and and and and and and 0.05, compared with c-Myc only. ( 0.05, ** 0.01, compared with the absence of doxycycline. SENP1 Stabilizes c-Myc Protein and Increases Its Levels. We then sought to test whether SENP1 regulates c-Myc levels. As shown in Fig. 2and and mRNA levels. Consistently, induced expression of WT SENP1, but not SENP1C603S, in U2OS cells also induced the levels of endogenous c-Myc in multiple clones (and genes, but not the and genes (Fig. 2and genes (and and and and and and and and and and and and and 0.05, ** 0.01, compared with the scrambled control. EPOR (and Naspm and common colony numbers were shown in mRNA is usually overexpressed in half of the tested breast malignancy cell lines relative to nontransformed MCF10A cells (Fig. 6and and and and correlation between SENP1 and c-Myc expression was determine by 2 test (and and and em F /em ) c-Myc, indicating that a previously reported effect of SENP7 knockdown on c-Myc SUMOylation (15) may be indirect or cell-type dependent. Adding to the complexity of the SUMO regulation of c-Myc, a recent report showed that RNF4, a SUMO-targeted ubiquitin ligase (STUbL) that targets SUMO2-altered PML for ubiquitination and proteasome degradation, increases c-Myc stability by promoting K11- and K33-linked polyubiquitination, which does not depend on c-Myc SUMOylation, but requires preexisting c-Myc S62 phosphorylation (31). These results strongly argue that SUMOylation dynamically regulates c-Myc activity and levels in a cell context-dependent manner, similar to c-Myc ubiquitination. Functionally, we show that SENP1 is essential for cancer.