Protein Kinase A Represses Skeletal Myogenesis by Targeting Myocyte Enhancer Factor 2D

Abstract

Activation of protein kinase A (PKA) by elevation of the intracellular cyclic AMP (cAMP) level inhibits skeletal myogenesis. Previously, an indirect modulation of the myogenic regulatory factors (MRFs) was implicated as the mechanism. Because myocyte enhancer factor 2 (MEF2) proteins are key regulators of myogenesis and obligatory partners for the MRFs, here we assessed whether these proteins could be involved in PKA-mediated myogenic repression. Initially, in silico analysis revealed several consensus PKA phosphoacceptor sites on MEF2, and subsequent analysis by in vitro kinase assays indicated that PKA directly and efficiently phosphorylates MEF2D. Using mass spectrometric determination of phosphorylated residues, we document that MEF2D serine 121 and serine 190 are targeted by PKA. Transcriptional reporter gene assays to assess MEF2D function revealed that PKA potently represses the transactivation properties of MEF2D. Furthermore, engineered mutation of MEF2D PKA phosphoacceptor sites (serines 121 and 190 to alanine) rendered a PKA-resistant MEF2D protein, which efficiently rescues myogenesis from PKA-mediated repression. Concomitantly, increased intracellular cAMP-mediated PKA activation also resulted in an enhanced nuclear accumulation of histone deacetylase 4 (HDAC4) and a subsequent increase in the MEF2D-HDAC4 repressor complex. Collectively, these data identify MEF2D as a primary target of PKA signaling in myoblasts that leads to inhibition of the skeletal muscle differentiation program.

PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy

“…In NRVMs infected with adenovirus expressing GFP alone and in GFP-tagged HDAC5-WT, cAMP significantly inhibited PE-stimulated MEF2 transcriptional activity (Fig. 4A), a result that is consistent with previous reports showing that cAMP inhibits MEF2 activity in skeletal myocytes and neuronal cells (29,30). Interestingly, coinfection by the adenoviral MEF2 luciferase construct and adenoviral GFP-HADC5-S280A abolished the cAMP-induced decrease in MEF2 transcriptional activation (Fig.…”

Dephosphorylation at a Conserved SP Motif Governs cAMP Sensitivity and Nuclear Localization of Class IIa Histone Deacetylases*

“…1B). This is reminiscent of what was reported previously (45). Unlike HDAC4, HDAC5 exhibited a basal nuclear localization pattern (Fig.…”

“…As shown for HDAC5, cAMP also promotes nuclear localization of HDAC4 in C2C12 myoblasts and MLB14 chondrocytes (45,46). Two possible mechanisms have been suggested: (i) cAMP activates PKA, phosphorylates MEF2D, and then stimulates MEF2D binding and nuclear localization of HDAC4; and (ii) cAMP indirectly activates PP2A, which in turn dephosphorylates HDAC4 and inhibits its 14-3-3 binding and cytoplasmic localization (45,46).…”

“…Two possible mechanisms have been suggested: (i) cAMP activates PKA, phosphorylates MEF2D, and then stimulates MEF2D binding and nuclear localization of HDAC4; and (ii) cAMP indirectly activates PP2A, which in turn dephosphorylates HDAC4 and inhibits its 14-3-3 binding and cytoplasmic localization (45,46). We and others have recently demonstrated that LKB1 activates SIK2 and SIK3 to promote phosphorylation at 14-3-3 binding sites of class IIa HDACs and stimulate their cytoplasmic localization (47,48,89).…”

cAMP signalling inhibits p53 acetylation and apoptosis via HDAC and SIRT deacetylases

“…The cAMP signalling pathway has been linked to both SIRT activation and subcellular localization of HDACs (20)(21)(22)(23), and we therefore hypothesized that cAMP antagonizes the IR-mediated acetylation of p53 through HDACs and/or SIRT1. To test this possibility, we examined the effects of the deacetylase inhibitors trichostatin A (TSA) and …”