Published online before print September 1, 2005, doi: 10.1161/01.RES.0000184684.88750.FE
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© 2005 American Heart Association, Inc.
Molecular Medicine |
Glycogen Synthase Kinase 3ß Inhibits Myocardin-Dependent Transcription and Hypertrophy Induction Through Site-Specific Phosphorylation
From Molecular Cardiology, Department of Internal Medicine III, University of Frankfurt, Germany.
Correspondence to Stefanie Dimmeler, Department of Molecular Cardiology, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. E-mail Dimmeler{at}em.uni-frankfurt.de
Cardiomyocyte hypertrophy is transcriptionally controlled and inhibited by glycogen synthase kinase 3ß (GSK3ß). Myocardin is a muscle-specific transcription factor with yet unknown relation to hypertrophy. Therefore, we investigated whether myocardin is sufficient to induce cardiomyocyte hypertrophy and whether myocardin is regulated by GSK3ß through site-specific phosphorylation. Adenoviral myocardin overexpression induced cardiomyocyte hypertrophy in neonatal rat cardiomyocytes, with increased cell size, total protein amount, and transcription of atrial natriuretic factor (ANF). In vitro and in vivo (HEK 293 cells) kinase assays with synthetic peptides and full-length myocardin demonstrated that myocardin was a "primed" GSK3ß substrate, with serines 455 to 467 and 624 to 636 being the major GSK3ß phosphorylation sites. Myocardin-induced ANF transcription and increase in total protein amount were enhanced by GSK3ß blockade (10 mmol/L LiCl), indicating that GSK3ß inhibits myocardin. A GSK3ß phosphorylation-resistant myocardin mutant (8xA) activated ANF transcription twice as potently as wildtype myocardin under basal conditions with GSK3ß being active. Conversely, a GSK3ß phospho-mimetic myocardin mutant (8xD) was transcriptionally repressed after GSK3ß blockade, indicating that GSK3ß phosphorylation at the sites identified inhibits myocardin transcriptional activity. GAL4-myocardin fusion constructs demonstrated that GSK3ß phosphorylation reduced the intrinsic myocardin transcriptional activity. A cell-permeable (Antennapedia protein transduction tag) peptide containing the mapped myocardin GSK3ß motifs 624 to 636 induced hypertrophy of cultured cardiomyocytes, suggesting that the peptide acted as substrate-based GSK3ß inhibitor in cardiomyocytes. Therefore, we conclude that the GSK3ß–myocardin interaction constitutes a novel molecular control of cardiomyocyte hypertrophy. Phosphorylation by GSK3ß comprises a novel post-transcriptional regulatory mechanism of myocardin.
Key Words: cardiomyocytes • hypertrophy • myocardin • transcription factors
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