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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.

Original publication

DOI

10.1371/journal.pone.0024812

Type

Journal article

Journal

PLoS One

Publication Date

2011

Volume

6

Keywords

Animals, Blotting, Western, COS Cells, Cell Line, Chlorocebus aethiops, Electrophoretic Mobility Shift Assay, Fluorescent Antibody Technique, Homeobox Protein Nkx-2.5, Homeodomain Proteins, Humans, Immunohistochemistry, Immunoprecipitation, Mice, Myocardium, Protein Inhibitors of Activated STAT, SUMO-1 Protein, Small Ubiquitin-Related Modifier Proteins, Transcription Factors, Ubiquitin-Activating Enzymes