Biochemical Journal

Research article

Phosphorylation of BATF regulates DNA binding: a novel mechanism for AP-1 (activator protein-1) regulation

Christopher D. DEPPMANN, Tina M. THORNTON, Fransiscus E. UTAMA, Elizabeth J. TAPAROWSKY

Abstract

BATF is a member of the AP-1 (activator protein-1) family of bZIP (basic leucine zipper) transcription factors that form transcriptionally inhibitory, DNA binding heterodimers with Jun proteins. In the present study, we demonstrate that BATF is phosphorylated in vivo on multiple serine and threonine residues and at least one tyrosine residue. Reverse-polarity PAGE revealed that serine-43 and threonine-48 within the DNA binding domain of BATF are phosphorylated. To model phosphorylation of the BATF DNA binding domain, serine-43 was replaced by an aspartate residue. BATF(S43D) retains the ability to dimerize with Jun proteins in vitro and in vivo, and the BATF(S43D):Jun heterodimer localizes properly to the nucleus of cells. Interestingly, BATF(S43D) functions like wild-type BATF to reduce AP-1-mediated gene transcription, despite the observed inability of the BATF(S43D):Jun heterodimer to bind DNA. These data demonstrate that phosphorylation of serine-43 converts BATF from a DNA binding into a non-DNA binding inhibitor of AP-1 activity. Given that 40% of mammalian bZIP transcription factors contain a residue analogous to serine-43 of BATF in their DNA binding domains, the phosphorylation event described here represents a mechanism that is potentially applicable to the regulation of many bZIP proteins.

  • activator protein-1 (AP-1)
  • basic leucine zipper
  • DNA binding domain
  • phosphorylation
  • transcription factor

Footnotes

  • Abbreviations used: AP-1, activator protein 1; ATF, activating transcription factor; BiFC, bimolecular fluorescence complementation; bJun, c-Jun-(257–318); bFos, c-Fos-(118–210); bZIP, basic leucine zipper; CBB, Coomassie Brilliant Blue; C/EBP, CCAAT/enhancer binding protein; CIAP, calf intestinal alkaline phosphatase; CMV, cytomegalovirus; DAPI, 4′,6′-diamidino-2-phenylindole; DBD, DNA binding domain; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; HA, haemagglutinin; IEF, isoelectric focusing; IPG, immobilized pH gradient; MT-BATF, Myc-tagged BATF; TBS, Tris-buffered saline; YFP, yellow fluorescent protein; YC, YFP-(155–238); YN, YFP-(1–154).