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

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.

SUBMITTER: Deppmann CD

PROVIDER: S-EPMC1223616 | biostudies-other | 2003 Sep

REPOSITORIES: biostudies-other

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Project description:Binding free energies are calculated for the phosphorylated and unphosphorylated complexes between the kinase inducible domain (KID) of the DNA transcriptional activator cAMP response element binding (CREB) protein and the KIX domain of its coactivator, CREB-binding protein (CBP). To our knowledge, this is the first application of a method based on a potential of mean force (PMF) with restraining potentials to compute the binding free energy of protein-protein complexes. The KID:KIX complexes are chosen here because of their biological relevance to the DNA transcription process and their relatively small size (81 residues for the KIX domain of CBP, and 28 residues for KID). The results for pKID:KIX and KID:KIX are -9.55 and -4.96 kcal/mol, respectively, in good agreement with experimental estimates (-8.8 and -5.8 kcal/mol, respectively). A comparison between specific contributions to protein-protein binding for the phosphorylated and unphosphorylated complexes reveals a dual role for the phosphorylation of KID at Ser-133 in effecting a more favorable free energy of the bound system: 1), stabilization of the unbound conformation of phosphorylated KID due to favorable intramolecular interactions of the phosphate group of Ser-133 with the charged groups of an arginine-rich region spanning both ?-helices, which lowers the configurational entropy; and 2), more favorable intermolecular electrostatic interactions between pSer-133 and Arg-131 of KID, and Lys-662, Tyr-658, and Glu-666 of KIX. Charge reduction through ligand phosphorylation emerges as a possible mechanism for controlling the unbound state conformation of KID and, ultimately, gene expression. This work also demonstrates that the PMF-based method with restraining potentials provides an added benefit in that important elements of the binding pathway are evidenced. Furthermore, the practicality of the PMF-based method for larger systems is validated by agreement with experiment. In addition, we provide a somewhat differently structured exposition of the PMF-based method with restraining potentials and outline its generalization to systems in which both protein and ligand may adopt unbound conformations that are different from those of the bound state.

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

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