Dissertation/Thesis Abstract

ATM phosphorylates and activates the transcription factor MEF2D for neuronal survival in response to DNA damage
by Chan, Shing Fai, Ph.D., University of California, San Diego, 2009, 109; 3359980
Abstract (Summary)

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 ATM at 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 phospho-mimetic MEF2D mutant protected primary neurons from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant did not. Co-immunoprecipitation 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.

Indexing (document details)
Advisor: Lipton, Stuart A.
Commitee: Gleeson, Joseph, Masliah, Eliezer, Pasquale, Elena, Walter, Gernot
School: University of California, San Diego
Department: Molecular Pathology
School Location: United States -- California
Source: DAI-B 70/06, Dissertation Abstracts International
Subjects: Neurosciences
Keywords: ATM, Ataxia-telangiectasia, DNA damage, MEF2, Neuronal survival
Publication Number: 3359980
ISBN: 978-1-109-23088-8
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