Dual regulation of phosphorylation and dephosphorylation of C/EBP β modulate its transcriptional activation and DNA binding in response to growth hormone

Abstract

The phosphorylation state of transcription factors is a critical determinant of their function. C/EBPβ occurs in cells as the transcriptional activator liver-enriched activating protein (LAP) and in the truncated form liver-enriched inhibitory protein (LIP) that inhibits transcription. Analysis of C/EBPβ phosphorylation by isoelectric focusing (IEF) shows that LAP is present in multiple forms, each with a different degree of phosphorylation in 3T3-F442A fibroblasts. Growth hormone (GH) treatment induces a new band near the negative pole, consistent with GH-promoted dephosphorylation of LAP. In addition, bands near the positive pole are rapidly and transiently induced, suggesting that GH also stimulates phosphorylation at some site(s) on LAP. C/EBPβ contains a highly conserved MAPK consensus site that corresponds to Thr188 in murine (m) LAP and Thr37 in mLIP. Immunoblotting with antiphosphopeptide antibodies specific for Thr188/37 of C/EBPβ (anti-P-C/EBPβ) shows that GH rapidly and transiently promotes phosphorylation of mLAP and mLIP on the MAPK site. MEK inhibitors prevent this GH-promoted phosphorylation of LAP and LIP, suggesting that such phosphorylation depends on GH-activated MAPK signaling. Mutation of Thr235 to Ala in the homologous MAPK site of human (h) LAP (hLAPT235A) inhibits transcription mediated by the c-fos promoter in response to GH, indicating that phosphorylation at the MAPK site is required for LAP to be transcriptionally active in the context of GH-stimulated activation of the c-fos promoter. Complexes bound to the c-fos C/EBP site transiently contain C/EBPβ phosphorylated at the MAPK site. As phosphorylation subsides, the binding of less transcriptionally active forms of LAP increases, consistent with the transient nature of c-fos stimulation by GH and other growth factors. Thus, both phosphorylation and dephosphorylation of C/EBPβ, in response to a single physiological stimulus such as GH, coordinately modulate the ability of C/EBPβ to activate transcription by modulating its DNA binding activity and its transactivation capacity.