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Human Molecular Genetics Advance Access published online on March 9, 2005
Human Molecular Genetics, doi:10.1093/hmg/ddi122
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© The Author 2005. Published by Oxford University Press. All rights reserved

Article

A cell-based screen for modulators of ataxin-1 phosphorylation

Michael D. Kaytor 1, Courtney E. Byam 1, Susan K Tousey 1, Samuel D. Stevens 2, Huda Y. Zoghbi 3, and Harry T. Orr 2*

1 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455.; Department of Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455.
2 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455.; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455.; Department of Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455.
3 Department of Molecular and Human Genetics, and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030.

* To whom correspondence should be addressed.
Harry T. Orr, E-mail: orrxx002{at}umn.edu


  Abstract

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a glutamine repeat within the SCA1-encoded protein ataxin-1. We have previously shown that serine 776 (S776) of both wild type and mutant ataxin-1 is phosphorylated in vivo and in vitro. Moreover, preventing phosphorylation of this residue by replacing it with alanine resulted in a mutant protein that was not pathogenic in spite of its nuclear localization. To further investigate pathways leading to phosphorylation of S776 of ataxin-1 we developed a cell-culture based assay to screen for modulators of S776 phosphorylation. In this assay, ataxin-1 expression was monitored by EGFP fluorescence in cell lines stably expressing an EGFP-ataxin-1 fusion protein. The phospho-S776 ataxin-1 specific antibody (PN1168) was used to assess ataxin-1 S776 phosphorylation. A library of 84 known kinase and phosphatase inhibitors was screened. Analysis of the list of drugs that modified S776 phosphorylation place many of the inhibited kinases into known cell signaling pathways. A pathway associated with calcium signaling resulted in phosphorylation of both wild type and mutant ataxin-1. Interestingly, inhibitors of the PI3K/Akt pathway predominantly diminished mutant ataxin-1 phosphorylation. These results provide new molecular tools to aid in elucidating the biological role of ataxin-1 phosphorylation and perhaps provide potential leads toward the development of a therapy for SCA1.


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