Identification of yeast kinase substrates using protein chips.
Jason Ptacek (1), Geeta Devgan (2), Heng Zhu (2), Xiaowei Zhu (3), Greg Michaud (4), Barry Schweitzer (4), Paul Predki (4), Michael Snyder (2)
(1) Mol. Biophysics and Biochem, Yale University, P.O. Box 208103, New Haven, CT, 06520, USA;
(2) Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520;
(3) Department of Compuational Biology and Bioinformatics, Yale University, P.O. Box 208103, New Haven, CT 06520;
(4) Protometrix, Inc., 688 East Main St., Branford, CT 06405
Protein chips are a versatile technology well suited for identifying protein-protein interactions, lipid-protein interactions, posttranslational modifications, and enzymatic activities, all at the proteome level. We have used newly developed protein chip technology to identify in vitro substrates of over half of the yeast kinases. S. cerevisiae contains ~6000 protein-coding genes, of which approximately 75% have been cloned and express full-length protein when fused to a GST-His6x N-terminal tag. The proteins are expressed and purified in a 96-well format and then printed at high-spatial density onto slides for use in a variety of assays, including kinase assays. Initial analysis of 34 kinases identified over 800 potential substrates, 450 of which were specific to only one kinase. For example, Gin4p phosphorylated its one known substrate, Shs1p, in addition to 4 other proteins. A portion of the novel substrates are being verified in vivo by deleting the corresponding kinase in the TAP-tag collection and assaying for a loss of phosphorylation. In conclusion, the use of protein chip technology will allow us to define a significant portion of the global signaling network of yeast and learn new roles of important regulators.
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