title: Complex SUMO-1 regulation of cardiac transcription factor Nkx2-5.
abstract: Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggested that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a "shifting" site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity.
authors.affiliation: Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.
authors.forename: Mauro W
authors.initials: MW
authors.lastname: Costa
authors.forename: Stella
authors.initials: S
authors.lastname: Lee
authors.forename: Milena B
authors.initials: MB
authors.lastname: Furtado
authors.forename: Li
authors.initials: L
authors.lastname: Xin
authors.forename: Duncan B
authors.initials: DB
authors.lastname: Sparrow
authors.forename: Camila G
authors.initials: CG
authors.lastname: Martinez
authors.forename: Sally L
authors.initials: SL
authors.lastname: Dunwoodie
authors.forename: Eleonora
authors.initials: E
authors.lastname: Kurtenbach
authors.forename: Tim
authors.initials: T
authors.lastname: Mohun
authors.forename: Nadia
authors.initials: N
authors.lastname: Rosenthal
authors.forename: Richard P
authors.initials: RP
authors.lastname: Harvey
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D024202:Electrophoretic Mobility Shift Assay; D005455:Fluorescent Antibody Technique; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D009206:Myocardium / Q000378:metabolism*; D050828:Protein Inhibitors of Activated STAT / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism
chemical_list: D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; C106529:PIAS1 protein, human; C501001:PIAS2 protein, human; D050828:Protein Inhibitors of Activated STAT; D025842:SUMO-1 Protein; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; C470301:UBA2 protein, human; D044764:Ubiquitin-Activating Enzymes