Stability and Transactivation Potential of the Mammalian MafA Transcription Factor Are Regulated by Serineí 65 Phosphorylation

The level of the MafA transcription factor is regulated by a variety of effectors of β cell function, including glucose, fatty acids, and insulin. Here, we show that phosphorylation at Ser⁶⁵ of mammalian MafA influences both protein stability and transactivation potential. Replacement of Ser⁶⁵ with Glu to mimic phosphorylation produced a protein that was as unstable as the wild type, whereas Asp or Ala mutation blocked degradation. Analysis of MafA chimeric and deletion constructs suggests that protein phosphorylation at Ser⁶⁵ alone represents the initial degradation signal, with ubiquitinylation occurring within the C terminus (amino acids 234-359). Although only wild type MafA and S65E were polyubiquitinylated, both S65D and S65E potently stimulated transactivation compared with S65A. Phosphorylation at Ser¹⁴ also enhanced activation, although it had no impact on protein turnover. The mobility of MafA S65A was profoundly affected upon SDS-PAGE, with the S65E and S65D mutants influenced less due to their ability to serve as substrates for glycogen synthase kinase 3, which acts at neighboring N-terminal residues after Ser⁶⁵ phosphorylation. Our observations not only illustrate the sensitivity of the cellular transcriptional and degradation machinery to phosphomimetic mutants at Ser⁶⁵, but also demonstrate the singular importance of phosphorylation at this amino acid in regulating MafA activity.