Dual regulation of phosphorylation and dephosphorylation of C/EBPbeta modulate its transcriptional activation and DNA binding in response to growth hormone.
The phosphorylation state of transcription factors is a critical determinant of their function. C/EBPbeta 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/EBPbeta 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/EBPbeta contains a highly conserved MAPK consensus site that corresponds to Thr(188) in murine (m) LAP and Thr(37) in mLIP. Immunoblotting with antiphosphopeptide antibodies specific for Thr(188/37) of C/EBPbeta (anti-P-C/EBPbeta) 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 Thr(235) 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/EBPbeta 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/EBPbeta, in response to a single physiological stimulus such as GH, coordinately modulate the ability of C/EBPbeta to activate transcription by modulating its DNA binding activity and its transactivation capacity.
Piwien-Pilipuk
Graciela
G
Department of Physiology, University of Michigan, Ann Arbor 48109, USA.
MacDougald
Ormond
O
Schwartz
Jessica
J
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D020935:MAP Kinase Signaling System; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013912:Threonine; D015533:Transcriptional Activation
D022782:CCAAT-Enhancer-Binding Protein-beta; D013912:Threonine; D013006:Growth Hormone; D004247:DNA