Myocyte Enhancer Factor 2A and 2D Undergo Phosphorylation and Caspase-Mediated Degradation during Apoptosis of Rat Cerebellar Granule Neurons

Abstract

Myocyte enhancer factor 2 (MEF2) proteins are important regulators of gene expression during the development of skeletal, cardiac, and smooth muscle. MEF2 proteins are also present in brain and recently have been implicated in neuronal survival and differentiation. In this study we examined the cellular mechanisms regulating the activity of MEF2s during apoptosis of cultured cerebellar granule neurons, an established in vitro model for studying depolarization-dependent neuronal survival. All four MEF2 isoforms (A, B, C, and D) were detected by immunoblot analysis in cerebellar granule neurons. Endogenous MEF2A and MEF2D, but not MEF2B or MEF2C, were phosphorylated with the induction of apoptosis. The putative sites that were phosphorylated during apoptosis are functionally distinct from those previously reported to enhance MEF2 transcription. The increased phosphorylation of MEF2A and MEF2D was followed by decreased DNA binding, reduced transcriptional activity, and caspase-dependent cleavage to fragments containing N-terminal DNA binding domains and C-terminal transactivation domains. Expression of the highly homologous N terminus of MEF2A (1-131 amino acids) antagonized the transcriptional activity and prosurvival effects of a constitutively active mutant of MEF2D (MEF2D-VP16). We conclude that MEF2A and MEF2D are prosurvival factors with high transcriptional activity in postmitotic cerebellar granule neurons. When these neurons are induced to undergo apoptosis by lowering extracellular potassium, MEF2A and MEF2D are phosphorylated, followed by decreased DNA binding and cleavage by a caspase-sensitive pathway to N-terminal fragments lacking the transactivation domains. The degradation of MEF2D and MEF2A and the generation of MEF2 fragments that have the potential to act as dominant-inactive transcription factors lead to apoptotic cell death.

Oxidation of Survival Factor MEF2D in Neuronal Death and Parkinson's Disease

“…Together, these findings indicate that CMA preferentially degrades oxidized and nonfunctional MEF2D. Since proteolytic cleavage of MEF2D has been shown to generate a dominant negative fragment (17,38,39), an accumulation of non-functional MEF2D may lead to its aberrant processing and the generation of fragments that are detrimental to neuronal viability. Based on all these observations, we propose a model in which moderate oxidative stress activates CMA and removes oxidized non-functional MEF2D.…”

MEF2C is required for the normal allocation of cells between the ventricular and sinoatrial precursors of the primary heart field

“…TUNEL assays were performed to investigate the possibility of increased apoptosis in the mutant. We had considered apoptosis a likely possibility since the MEF2s are anti-apoptotic in neurons (Mao et al, 1999;Okamoto et al, 2000;Li et al, 2001). Although apoptosis was detected in other regions of the embryo at E8.5, none was detectable in the linear hearts of either wildtype (Fig.…”

Caspase-mediated Specific Cleavage of Human Histone Deacetylase 4

“…The identification of caspase-3 as the likely protease involved in the degradation of HDAC4 is especially intriguing as this caspase has been implicated in the degradation of MEF2 family members (36,37). The smaller cleavage products resulting from caspase-mediated cleavage of HDAC4 at Asp-289 would encompass the MEF2 binding site as well as THE nuclear localization signal (both located in the N-terminal portion of HDAC4) and consequently might retain or even augment the ability to repress MEF2 (41,42).…”