ATM Phosphorylates and Activates the Transcription Factor MEF2D for Neuronal Survival in Response to DNA Damage

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by many clinical manifestations including neurodegeneration, most notably in the cerebellum resulting in gait ataxia and cancer predisposition. The mutated gene responsible for A-T is ATM (ataxia telangiectasia mutated), which encodes a kinase that activates multiple signal transduction pathways in response to DNA damage. Defects in survival signaling and the DNA damage response in neurons may cause the neurodegenerative pathology of A-T, but ATM substrates in the central nervous system are as yet unclear. Using protein sequence analysis, I have identified four potential ATM consensus phosphorylation motifs and one ATM -interaction motif in the neuronal pro-survival transcription factor, myocyte enhancer factor 2D (MEF2D). MEF2 represents a family of MADS (MCM1-agamous-deficiens- serum response factor) domain-containing transcription factors and plays a critical role in the nervous system regulating neurogenesis, synaptic plasticity and neuronal survival. There are four members of the MEF2 family, MEF2A to -D. MEF2A and -D predominate in the cerebellum, which is most affected in A-T. The activity of MEF2 proteins is governed in part by phosphorylation. Using in vitro immunocomplex kinase assays, I found that ATM phosphorylates MEF2A, -C and -D. DNA damaging agents that induce double-strand breaks increased phosphorylation of MEF2A and -D in cerebellar granule cell neurons. MEF2D phosphorylation was detectable in Atm wild-type cells but not in Atm-deficient cells. GAL4-dependent luciferase reporter gene assays revealed that ATM activates MEF2A and -D activity, but attenuates MEF2C activity. In addition, MEF2-dependent luciferase reporter gene assays showed that ATM increases endogenous MEF2 activity, and the potentiation of endogenous MEF2 activity is abolished either by RNA interference targeting ATM or by a small molecule inhibitor of ATM, KU-55933. Analysis by site- directed mutagenesis indicated that MEF2D is phosphorylated by ATMa t four ATM consensus phosphorylation sites : Thr²⁵⁹, Ser²⁷⁵, Ser²⁹⁴ and Ser³¹⁴. Knockdown of endogenous MEF2D expression by a short- hairpin RNA (shRNA) increased cellular sensitivity to etoposide-induced neuronal cell death. Interestingly, substitution of endogenous MEF2D expression with an shRNA- resistant phosphomimetic MEF2D mutant protected primary neurons from cell death after DNA damage, whereas an shRNA -resistant nonphosphorylatable MEF2D mutant did not. Coimmunoprecipitation studies indicated that ATM and MEF2D form a complex following DNA damage. Collectively, these results suggest that ATM associates with MEF2D and activates its activity via phosphorylation, thus promoting neuronal survival in response to DNA damage