Li M, Linseman D A, Allen M P, Meintzer M K, Wang X, Laessig T, Wierman M E, Heidenreich K A
Department of Pharmacology, University of Colorado Health Sciences Center and the Denver Veterans Affairs Medical Center, Denver, Colorado 80262, USA.
J Neurosci. 2001 Sep 1;21(17):6544-52. doi: 10.1523/JNEUROSCI.21-17-06544.2001.
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.
肌细胞增强因子2（MEF2）蛋白是骨骼肌、心肌和平滑肌发育过程中基因表达的重要调节因子。MEF2蛋白也存在于大脑中，最近被认为与神经元的存活和分化有关。在本研究中，我们研究了在培养的小脑颗粒神经元凋亡过程中调节MEF2活性的细胞机制，小脑颗粒神经元是研究去极化依赖性神经元存活的一种成熟的体外模型。通过免疫印迹分析在小脑颗粒神经元中检测到所有四种MEF2亚型（A、B、C和D）。内源性MEF2A和MEF2D在凋亡诱导时被磷酸化，而MEF2B或MEF2C未被磷酸化。凋亡过程中被磷酸化的假定位点在功能上与先前报道的增强MEF2转录的位点不同。MEF2A和MEF2D磷酸化增加后，DNA结合减少、转录活性降低，并通过半胱天冬酶依赖性切割产生含有N端DNA结合结构域和C端反式激活结构域的片段。MEF2A高度同源的N端（1 - 131个氨基酸）的表达拮抗了MEF2D组成型活性突变体（MEF2D - VP16）的转录活性和促存活作用。我们得出结论，MEF2A和MEF2D是有丝分裂后小脑颗粒神经元中具有高转录活性的促存活因子。当这些神经元通过降低细胞外钾离子浓度被诱导发生凋亡时，MEF2A和MEF2D被磷酸化，随后DNA结合减少，并通过半胱天冬酶敏感途径切割成缺乏反式激活结构域的N端片段。MEF2D和MEF2A的降解以及具有潜在显性失活转录因子作用的MEF2片段的产生导致凋亡性细胞死亡。
