Muscle A-kinase–anchoring protein-β–bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D

Abstract

Edited by Roger J. ColbranMyocyte enhancer factor 2 (MEF2) transcription factors are key regulators of the development and adult phenotype of diverse tissues, including skeletal and cardiac muscles. Controlled by multiple post-translational modifications, MEF2D is an effector for the Ca 2؉ /calmodulin-dependent protein phosphatase calcineurin (CaN, PP2B, and PPP3). CaN-catalyzed dephosphorylation promotes the desumoylation and acetylation of MEF2D, increasing its transcriptional activity. Both MEF2D and CaN bind the scaffold protein muscle A-kinaseanchoring protein ␤ (mAKAP␤), which is localized to the nuclear envelope, such that C2C12 skeletal myoblast differentiation and neonatal rat ventricular myocyte hypertrophy are inhibited by mAKAP␤ signalosome targeting. Using immunoprecipitation and DNA-binding assays, we now show that the formation of mAKAP␤ signalosomes is required for MEF2D dephosphorylation, desumoylation, and acetylation in C2C12 cells. Reduced MEF2D phosphorylation was coupled to a switch from type IIa histone deacetylase to p300 histone acetylase binding that correlated with increased MEF2D-dependent gene expression and ventricular myocyte hypertrophy. Together, these results highlight the importance of mAKAP␤ signalosomes for regulating MEF2D activity in striated muscle, affirming mAKAP␤ as a nodal regulator in the myocyte intracellular signaling network. 3 The abbreviations used are: by guest on July 9, 2020 http://www.jbc.org/ Downloaded from Figure 5. Regulation of MEF2D-HDAC5 binding. A, C2C12 cells co-transfected with expression plasmids for GFP-and FLAG-tagged MEF2D and GFP-tagged HDAC5 were cultured in GM or DM for 3 h before immunoprecipitation using FLAG antibodies and Western blotting using MEF2D and HDAC5 antibodies. p (one-way ANOVA) ϭ 0.003. B, HEK293 cells were transfected with GFP-and FLAG-tagged MEF2D mutants and GFP-HDAC5 before immunoprecipitation using FLAG antibodies and Western blotting as in A. p (one-way ANOVA) Ͻ 0.0001. C, same as A except using C2C12 cells transfected with mAKAP or control siRNA. p (two-way ANOVA for both factors and interaction) Ͻ 0.04. D, same as A except using C2C12 cells co-expressing mCherry or CBD-mCherry. p (two-way ANOVA for growth media and interaction) Ͻ 0.04. n ϭ 3 independent experiments for all panels. Downloaded fromFigure 7. Calcineurin-dependent regulation of MEF2D-p300 DNA complexes. A, C2C12 cells expressing GFP-and FLAG-tagged MEF2D were cultured in GM or DM in the absence or presence of cyclosporin A (500 nM) for 3 h before pulldown assay using a biotinylated oligonucleotide based upon a Myh7 MyoD cis-active element (24) and Western blotting using MEF2D antibodies. Myh7m is a mutant oligonucleotide control. p (one-way ANOVA) ϭ 0.004. B, same as in A except using HEK293 cells co-expressing mutant MEF2D proteins. p (two-way ANOVA for MEF2D mutants and interaction) Ͻ 0.04. C, same as A except C2C12 cells transfected with mAKAP or control siRNA. D, same as A except using C2C12 cells co-expressing mCherry or CBD-mCherry. p (two-way ANOVA f...

Metoprolol Mitigates Ischemic Heart Remodeling and Fibrosis by Increasing the Expression of akap5 in Ischemic Heart

“…PP2B is widely expressed in the heart and elsewhere, and is reported to complex with various AKAPs . (20) Consistent with this, AKAP5 and PP2B were co-localized by immunoprecipitation in cardiac samples, and this relationship appeared to change under ischemia and metoprolol treatment (Fig. 5A).…”

MEF2 transcription factors differentially contribute to retinal ganglion cell loss after optic nerve injury

“…We next asked whether MEF2A gain-of-function would, conversely, worsen RGC survival after optic nerve crush. In addition, MEF2A Ser-408 phosphorylation is a well-established mechanism for control of MEF2A activity in cells, promoting a switch between MEF2A-dependent gene activation and repression [ 13 , 27 , 28 ]. Thus, flag-tagged MEF2A wildtype and mutant proteins were expressed by AAV2 intravitreal injection of wild type mice.…”

“…Together these data support a role for MEF2A-regulated gene expression that opposes RGC survival after axon injury. We previously found that expression of the perinuclear scaffold protein muscle A-kinase anchoring protein α (mAKAPα) is required for neurotrophic factor- and cyclic-adenosine mononucleotide (cAMP)-dependent RGC neuroprotection after optic nerve crush injury, but not during RGC development [ 20 ], while MEF2 regulation in myocytes is regulated by mAKAP signalosomes [ 27 , 39 ]. The previous finding that MEF2A expression mediates survival signaling downstream of the stress-induced DLK/LZK pathway similarly places MEF2A at a central locus in the regulation of stress-induced survival signaling [ 17 ].…”

The Osteogenesis Imperfecta Type V Mutant BRIL/IFITM5 Promotes Transcriptional Activation of MEF2, NFATc, and NR4A in Osteoblasts

“…This was supported by the results showing the synergistic effects of BRIL and CnA, and the repressive effects of RCAN1, HDAC4, and the immunosuppressive drug FK506. Previous studies have provided evidence that Ca 2+ -calcineurin is a common player leading to activation of MEF2 and NFATc [48][49][50][51][52]. The roles of MEF2C and NFATc1/c2 in skeletal biology, studied in several bone cell types (osteoblast, chondrocytes, osteocytes, and osteoclasts), could in part explain the variable cellular and tissue anomalies, and phenotypes, observed in OI type V. MEF2C is a transcription factor involved in osteoblast differentiation and gene regulation, thought to be upstream of bone-specific transcription factors RUNX2 and Osterix (OSX/SP7) [53].…”