Role of USF1 phosphorylation on cardiac alpha-myosin heavy chain promoter activity.
Contractile activity of the cardiac myocyte is required for maintaining cell mass and phenotype, including expression of the cardiac-specific alpha-myosin heavy chain (alpha-MHC) gene. An E-box hemodynamic response element (HME) located at position -47 within the alpha-MHC promoter is both necessary and sufficient to confer contractile responsiveness to the gene and has been shown to bind upstream stimulatory factor-1 (USF1). When studied in spontaneously contracting cardiac myocytes, there is enhanced binding of USF1 to the HME compared with quiescent cells, which correlates with a threefold increase in alpha-MHC promoter activity. A molecular mechanism by which contractile function modulates alpha-MHC transcriptional activity may involve signaling via phosphorylation of USF1. The present studies showed that purified rat USF1 was phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) but not casein kinase II. Phosphorylated USF1 by either PKC or PKA had increased DNA binding activity to the HME. PKC-mediated phosphorylation also leads to the formation of USF1 multimers as assessed by gel shift assay. Analysis of in vivo phosphorylated nuclear proteins from cultured ventricular myocytes showed that USF1 was phosphorylated, and resolution by two-dimensional gel electrophoresis identified at least two distinct phosphorylated USF1 molecules. These results suggest that endogenous kinases can covalently modify USF1 and provide a potential molecular mechanism by which the contractile stimulus mediates changes in myocyte gene transcription.
Xiao
Qianxun
Q
Division of Endocrinology, Department of Medicine, North Shore-Long Island Jewish Research Institute, New York University School of Medicine, Manhasset, New York 11030, USA.
Kenessey
Agnes
A
Ojamaa
Kaie
K
D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics*; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D020218:Response Elements / Q000502:physiology; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D051720:Upstream Stimulatory Factors; D024723:Ventricular Myosins / Q000235:genetics*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D024723:Ventricular Myosins; D018995:Myosin Heavy Chains; D013755:Tetradecanoylphorbol Acetate