Direct Interaction between Myocyte Enhancer Factor 2 (MEF2) and Protein Phosphatase 1α Represses MEF2-Dependent Gene Expression

Abstract

The myocyte enhancer factor 2 (MEF2) transcription factors play important roles in neuronal, cardiac, and skeletal muscle tissues. MEF2 serves as a nuclear sensor, integrating signals from several signaling cascades through protein-protein interactions with kinases, chromatin remodeling factors, and other transcriptional regulators. Here, we report a novel interaction between the catalytic subunit of protein phosphatase 1␣ (PP1␣) and MEF2. Interaction occurs within the nucleus, and binding of PP1␣ to MEF2 potently represses MEF2-dependent transcription. The interaction utilizes uncharacterized domains in both PP1␣ and MEF2, and PP1␣ phosphatase activity is not obligatory for MEF2 repression. Moreover, a MEF2-PP1␣ regulatory complex leads to nuclear retention and recruitment of histone deacetylase 4 to MEF2 transcription complexes. PP1␣-mediated repression of MEF2 overrides the positive influence of calcineurin signaling, suggesting PP1␣ exerts a dominant level of control over MEF2 function. Indeed, PP1␣-mediated repression of MEF2 function interferes with the prosurvival effect of MEF2 in primary hippocampal neurons. The PP1␣-MEF2 interaction constitutes a potent locus of control for MEF2-dependent gene expression, having potentially important implications for neuronal cell survival, cardiac remodeling in disease, and terminal differentiation of vascular, cardiac, and skeletal muscle.The myocyte enhancer factor 2 (MEF2) transcription factors play important roles in T-cell selection, neuronal survival, and terminal differentiation of cardiac and skeletal muscle (3, 47). The MEF2 family proteins are encoded by four genes, MEF2A to -D, which demonstrate tissue-specific and temporally dependent developmental expression patterns. Expression and activity of MEF2 factors in both cardiac and skeletal muscle lineages are vital for activation and maintenance of genes representing the structural components of sarcomeric muscle. Gene-targeted ablation of MEF2A or MEF2C results in aberrant heart formation and premature death (32, 46), whereas loss of the single MEF2 gene in Drosophila melanogaster (Dmef2) results in complete loss of all muscle tissues (31). In neurons, MEF2 transcriptional activity plays a critical role for prevention of apoptosis due to neurocytotoxicity signals (1,5,20,37).The amino-terminal (NT) region of MEF2 transcription factors is composed of a highly conserved MADS (MCM1, agamous, deficiens, and serum response factor) domain, responsible for DNA binding to the consensus DNA binding element (T/C)TA(A/T) 4 TA(G/A), and the MEF2 domain, which is required for homo-and heterodimerization of MEF2 factors. The carboxyl terminus (CT) of all MEF2 factors is subject to alternative splicing, represents the target for several signal transduction pathways, and is required for transcriptional activation properties (3).MEF2 transcriptional activity is stimulated by the mitogenactivated protein kinase (MAPK) p38 signaling module (25, 50), Ca 2ϩ /calmodulin kinases (CaMKs) (52), extracellular signal-regulated kinase ...

A Novel RhoA/ROCK-CPI-17-MEF2C Signaling Pathway Regulates Vascular Smooth Muscle Cell Gene Expression

“…In VSMCs, PP1␣ is targeted to the myosin light chains by physical interaction with MYPT1; however, this RVXF domain is also conserved among MADS-box proteins, such as MEF2A-D and may serve to target PP1␣ to nuclear MEF2 proteins (13,14). Interestingly, SRF also contains a conserved RVXF domain within its MADS-box, yet our data suggest that PP1␣ cannot overcome myocardin or TGF-␤ induction of SRF-target genes (Fig.…”

“…Plasmids-MEF2, PP1␣, p38 MAPK (p38 MAPK), MKK6EE, and luciferase constructs were described previously (6,13). The RhoA L63 and C3 transferase expression vectors were kindly provided by A.…”

“…Immunoblot Analysis-Protein extractions were achieved using an Nonidet P-40 lysis buffer described previously (13). Protein concentrations were determined by Bradford assay, and 15 g were resolved using SDS-PAGE and transferred to an Immobilon-P membrane (Millipore, Inc.).…”

“…PP1␣ Regulates MEF2-dependent Gene Expression in VSMCsWe have recently identified PP1␣ as a dominant repressor of MEF2 transcriptional activity (13). In smooth muscle, PP1␣ is the catalytic subunit of the myosin light chain phosphatase complex (MLCP) (21).…”

“…Yet, the signaling pathways that regulate MEF2-dependent myocardin expression in VSMCs remain unknown; however, recent studies suggest that RhoA signaling may be involved (11,12). We recently identified protein phosphatase 1␣ (PP1␣) as a potent trans-dominant repressor of MEF2 activity (13). Interestingly, in VSMCs PP1␣ serves as the catalytic subunit of the myosin light chain phosphatase complex (MLCP) and is regulated by RhoA signaling to control calcium sensitivity during contraction (14).…”