Covalent attachment of the SUMO-1 protein to the negative regulatory domain of the c-Myb transcription factor modifies its stability and transactivation capacity.
The transcription factor c-Myb is subject to several types of post-translational modifications, including phosphorylation, acetylation, and ubiquitination. These modifications regulate the transcription and transforming activity as well as the proteolytic stability of c-Myb. Here we report the covalent modification of c-Myb with the small ubiquitin-related protein SUMO-1. Mutational analysis identified two major sumolation sites (Lys(499) and Lys(523)) in the negative regulatory domain. Interestingly, the single mutation K523R completely abolished modification of c-Myb with SUMO-1, suggesting that sumolation of Lys(523) is required for modification of other lysines in c-Myb. In accordance with this observation, we found that the SUMO-1-conjugating enzyme Ubc9 interacted only with a region surrounding Lys(523) (also called the PEST/EVES motif). Experiments aimed at determining the proteolytic stability of sumolated and unmodified forms of c-Myb revealed that at least two covalently attached SUMO-1 molecules dramatically increased the stability of c-Myb. However, mutations of the SUMO-1 modification sites did not alter its stability, suggesting that a mechanism(s) other than competition of ubiquitin and SUMO-1 for the same lysine is involved in the stabilization of sumolated c-Myb protein. Finally, the K523R mutant of c-Myb, entirely deficient in sumolation, was shown to have an increased transactivation capacity on a Myb-responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of c-Myb. Thus, modification of c-Myb with SUMO-1 represents a novel mechanism through which the negative regulatory domain can exert its suppressing activity on c-Myb transactivation capacity.
Bies
Juraj
J
Department of Molecular Virology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovakia. exonbies@savba.sk
Markus
Ján
J
Wolff
Linda
L
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D051379:Mice; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020598:Proto-Oncogene Proteins c-myb / Q000737:chemistry* / Q000502:physiology; D025842:SUMO-1 Protein / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015534:Trans-Activators / Q000502:physiology*
D020598:Proto-Oncogene Proteins c-myb; D025842:SUMO-1 Protein; D015534:Trans-Activators; D012694:Serine