Human Molecular Genetics Advance Access originally published online on March 9, 2005
Human Molecular Genetics 2005 14(8):1095-1105; doi:10.1093/hmg/ddi122
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A cell-based screen for modulators of ataxin-1 phosphorylation
1Department of Laboratory Medicine and Pathology, 2Department of Biochemistry, Molecular Biology and Biophysics and 3Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA and 4Department of Molecular and Human Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA
* To whom correspondence should be addressed at: Institute of Human Genetics, University of Minnesota, Mayo Mail Code 206, 4-122 Moos Tower, 515 Delaware Street, SE Minneapolis, MN 55455, USA. Tel: +1 6126253647; Fax: +1 6126267031; Email: orrxx002{at}umn.edu
Received January 5, 2005; Accepted March 3, 2005
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, which was not pathogenic in spite of its nuclear localization. To further investigate pathways leading to S776 phosphorylation 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 enhanced green fluorescent protein (EGFP) fluorescence in cell lines stably expressing 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 places 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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