title: c-Myc is targeted to the proteasome for degradation in a SUMOylation-dependent manner, regulated by PIAS1, SENP7 and RNF4.
abstract: c-Myc is the most frequently overexpressed oncogene in tumors, including breast cancer, colon cancer and lung cancer. Post-translational modifications comprising phosphorylation, acetylation and ubiquitylation regulate the activity of c-Myc. Recently, it was shown that c-Myc-driven tumors are strongly dependent on the SUMO pathway. Currently, the relevant SUMO target proteins in this pathway are unknown. Here we show that c-Myc is a target protein for SUMOylation, and that SUMOylated c-Myc is subsequently ubiquitylated and degraded by the proteasome. SUMO chains appeared to be dispensable for this process, polymerization-deficient SUMO mutants supported proteolysis of SUMOylated c-Myc. These results indicate that multiple SUMO monomers conjugated to c-Myc could be sufficient to direct SUMOylated c-Myc to the ubiquitin-proteasome pathway. Knocking down the SUMO-targeted ubiquitin ligase RNF4 enhanced the levels of SUMOylated c-Myc, indicating that RNF4 could recognize a multi-SUMOylated protein as a substrate in addition to poly-SUMOylated proteins. Knocking down the SUMO E3 ligase PIAS1 resulted in reduced c-Myc SUMOylation and increased c-Myc transcriptional activity, indicating that PIAS1 mediates c-Myc SUMOylation. Increased SUMOylation of c-Myc was noted upon knockdown of the SUMO protease SENP7, indicating that it also could regulate a multi-SUMOylated protein in addition to poly-SUMOylated proteins. C-Myc lacks KxE-type SUMOylation consensus motifs. We used mass spectrometry to identify 10 SUMO acceptor lysines: K52, K148, K157, K317, K323, K326, K389, K392, K398 and K430. Intriguingly, mutating all 10 SUMO acceptor lysines did not reduce c-Myc SUMOylation, suggesting that SUMO acceptor lysines in c-Myc act promiscuously. Our results provide novel insight into the complexity of c-Myc post-translational regulation.
authors.affiliation: a Department of Molecular Cell Biology; Leiden University Medical Center ; Leiden , The Netherlands.
authors.forename: Román
authors.initials: R
authors.lastname: González-Prieto
authors.forename: Sabine Ag
authors.initials: SA
authors.lastname: Cuijpers
authors.forename: Ramesh
authors.initials: R
authors.lastname: Kumar
authors.forename: Ivo A
authors.initials: IA
authors.lastname: Hendriks
authors.forename: Alfred Co
authors.initials: AC
authors.lastname: Vertegaal
mesh_terms: D020816:Amino Acid Motifs; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D050828:Protein Inhibitors of Activated STAT / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000235:genetics; D059748:Proteolysis; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D058207:Sumoylation; D014157:Transcription Factors / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: C489427:MYC protein, human; D009687:Nuclear Proteins; C106529:PIAS1 protein, human; D050828:Protein Inhibitors of Activated STAT; D016271:Proto-Oncogene Proteins c-myc; C114070:RNF4 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; D010450:Endopeptidases; C533959:SENP7 protein, human; D046988:Proteasome Endopeptidase Complex; D008239:Lysine
keywords: PIAS1; RNF4; SENP7; SUMO; c-Myc; proteasome