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

Sabire Özcan,Roland Stein,Shuangli Guo,Ryan Burnette,Vincent Poitout,Nathan L. Vanderford,Eva Henderson,Li Zhao,Brian E. Wadzinski,Derek K. Hagman

doi:10.1074/jbc.m806314200

Abstract

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.

Highlights

MafA appears to act as a barometer of adult ␤ cell function
MafA levels are unusually sensitive in relation to other islet regulators to metabolic effectors of islet ␤ cell function, such as glucose (5, 14 –16), fatty acids (15, 17), and insulin (18)
The phosphoamino acid composition of MafA was assessed in HeLa cells infected with an adenovirus-expressing mouse MafA in medium containing [32P]orthophosphate

Summary

EXPERIMENTAL PROCEDURES

DNA Constructs—The S14A, S14E, S65A, S65E, S65D, and T267A mutants were prepared in cytomegalovirus-driven Myc-MafA expression plasmid using the QuikChangeTM sitedirected mutagenesis kit (Stratagene, La Jolla, CA). Wild type Gal4-MafA (amino acids 1–359) and the 1–75 and 1–233 mutants were constructed by subcloning PCR-generated mouse MafA sequences into the simian virus 40 promoter/ enhancer-driven Gal[4] expression plasmid pSG424 (22) to create in-frame Gal[4] DNA-binding domain fusion proteins. Nuclear extracts were prepared as described (5) and immunoblotted using an anti-MafA antibody (1:1000; Bethyl Laboratories, Montgomery, TX). To measure the turnover rate of MafA, ␤TC-3 cells were cultured in 100-mm dishes and first transfected with 4 ␮g of the MafA expression construct. The beads were washed three times with radioimmune precipitation assay buffer, and the eluted proteins were separated by SDS-PAGE and analyzed by immunoblotting using an anti-HA antibody (Sigma). The eluted protein was hydrolyzed with 6 N HCl as described previously (26). 32P-Amino acids were separated by two-dimensional electrophoresis (27), and cold phosphoserine (Sigma), phosphothreonine (Sigma), and phosphotyrosine (Sigma) served as position markers

RESULTS

25 S65D MafA

DISCUSSION

75 Gal4 TAD