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 (Lys499 and Lys523) in the negative regulatory domain. Interestingly, the single mutation K523R completely abolished modification of c-Myb with SUMO-1, suggesting that sumolation of Lys523 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 Lys523 (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.