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

Michael D. Kaytor, Courtney E. Byam, Susan K. Tousey, Samuel D. Stevens, Huda Y. Zoghbi, Harry T. Orr

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the exansion 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.

Original languageEnglish (US)
Pages (from-to)1095-1105
Number of pages11
JournalHuman molecular genetics
Volume14
Issue number8
DOIs
StatePublished - Apr 15 2005

Bibliographical note

Funding Information:
This work was supported by NIH grants NS045667 and NS050133 to H.T.O.

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