The stability and transactivation potential of the mammalian MafA transcription factor are regulated by serine 65 phosphorylation.
The level of the MafA transcription factor is regulated by a variety of effectors of beta cell function, including glucose, fatty acids, and insulin. Here, we show that phosphorylation at Ser(65) of mammalian MafA influences both protein stability and transactivation potential. Replacement of Ser(65) 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 Ser(65) 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 Ser(14) 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 Ser(65) phosphorylation. Our observations not only illustrate the sensitivity of the cellular transcriptional and degradation machinery to phosphomimetic mutants at Ser(65), but also demonstrate the singular importance of phosphorylation at this amino acid in regulating MafA activity.
Guo
Shuangli
S
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
Burnette
Ryan
R
Zhao
Li
L
Vanderford
Nathan L
NL
Poitout
Vincent
V
Hagman
Derek K
DK
Henderson
Eva
E
Ozcan
Sabire
S
Wadzinski
Brian E
BE
Stein
Roland
R
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D051473:Maf Transcription Factors, Large / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010768:Phosphoserine / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D015533:Transcriptional Activation / Q000235:genetics*; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination
D051473:Maf Transcription Factors, Large; C485160:Mafa protein, mouse; D025801:Ubiquitin; D010768:Phosphoserine; D038362:Glycogen Synthase Kinase 3