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J. Biol. Chem., Vol. 281, Issue 33, 23686-23697, August 18, 2006

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Huntingtin Phosphorylation Sites Mapped by Mass Spectrometry

MODULATION OF CLEAVAGE AND TOXICITY^*

Birgit Schilling, Juliette Gafni, Cameron Torcassi, Xin Cong, Richard H. Row, Michelle A. LaFevre-Bernt, Michael P. Cusack, Tamara Ratovitski, Ricky Hirschhorn^¶, Christopher A. Ross^||, Bradford W. Gibson^**, and Lisa M. Ellerby¹

From the The Buck Institute for Age Research, Novato, California 94945, the Division of Neurobiology, Department of Psychiatry, ^||Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, ^¶Hood College, Frederick, Maryland 21701, and the ^**Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143

Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.

Received for publication, December 19, 2005 , and in revised form, May 23, 2006.

^* This work was supported by National Institutes of Health Grant NS40251A and HighQ (to L. M. E. and B. W. G.), National Institutes of Health Postdoctoral Fellowship F32 NS043937 (to J. G.), National Institutes of Health Grants NS 16375 and 38144, and the Huntington's Disease Society of America and Hereditary Disease Foundation (to C. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S5 and Figs. S1–S10.

¹ To whom the correspondence should be addressed: 8001 Redwood Blvd., Novato, CA 94945. Tel.: 415-209-2088; Fax: 415-209-2230; E-mail: lellerby{at}buckinstitute.org.

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