title: Modulation of myocardin function by the ubiquitin E3 ligase UBR5.
abstract: Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.
authors.affiliation: Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, USA.
authors.forename: Guoqing
authors.initials: G
authors.lastname: Hu
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Wang
authors.forename: Darren N
authors.initials: DN
authors.lastname: Saunders
authors.forename: Michelle
authors.initials: M
authors.lastname: Henderson
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Russell
authors.forename: B Paul
authors.initials: BP
authors.lastname: Herring
authors.forename: Jiliang
authors.initials: J
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099251:Myocardin
chemical_list: D009687:Nuclear Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D015534:Trans-Activators; D044767:Ubiquitin-Protein Ligases; D000099251:Myocardin; C489244:UBR5 protein, mouse