Global Analysis of Protein Phosphorylation in Yeast

Jason Ptacek1*, Geeta Devgan2*, Gregory Michaud3*, Heng Zhu2, Xiaowei Zhu2, Joseph Fasolo2, Hong Guo3, Ghil Jona2, Ashton Breitkreutz4, Richelle Sopko4, Rhonda R. McCartney6, Martin C. Schmidt6, Najma Rachidi7, Soo-Jung Lee8, Angie S. Mah9, Lihao Meng3, Michael J.R. Stark7, David F. Stern8, Claudio De Virgilio10, Michael Tyers4, Brenda Andrews4,5, Mark Gerstein2, Barry Schweitzer3, Paul F. Predki3, and Michael Snyder1,2

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Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and for less than 160 phosphoproteins is the phosphorylating kinase known. Using proteome chip technology, we determined the in vitro substrates recognized by the majority of yeast protein kinases. 4,192 phosphorylation events involving 1,325 different proteins were identified; these substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four CDKs that vary only in their cyclin subunits. While many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, suggesting new roles for a number of kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed a number of novel regulatory modules. Our phosphorylation results have been assembled into a first generation phosphorylation map for yeast. Since many yeast proteins and pathways are conserved, these results provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

1Department of Molecular Biophysics & Biochemistry and 2Molecular, Cellular & Developmental Biology. Yale University. New Haven, CT 06511, USA
3Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008, USA
4Department of Medical Genetics and Microbiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario M55 1A8, Canada
5Banting & Best Department of Medical Research, University of Toronto, Rm. 4285, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M55 1A8, Canada
6Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine Pittsburgh, PA 15261, USA
7Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom
8Department of Pathology, Yale University School of Medicine, P.O. Box 208023 310 Cedar St. BML 342, New Haven, CT 06520-8023, USA
9Department of Biology, California Institute of Technology, Pasadena, CA 91125, USA
10Department of Microbiology and Molecular Medicine, CMU, University of Geneva,1211 Geneva, Switzerland