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Sci. Signal., 16 February 2010
Vol. 3, Issue 109, p. ra12
[DOI: 10.1126/scisignal.2000482]


Deciphering Protein Kinase Specificity Through Large-Scale Analysis of Yeast Phosphorylation Site Motifs

Janine Mok1*, Philip M. Kim2{dagger}, Hugo Y. K. Lam3, Stacy Piccirillo1, Xiuqiong Zhou1, Grace R. Jeschke4, Douglas L. Sheridan4{ddagger}, Sirlester A. Parker4, Ved Desai4, Miri Jwa5, Elisabetta Cameroni6§, Hengyao Niu7, Matthew Good8, Attila Remenyi8||, Jia-Lin Nianhan Ma9, Yi-Jun Sheu10, Holly E. Sassi11, Richelle Sopko11, Clarence S. M. Chan5, Claudio De Virgilio6, Nancy M. Hollingsworth7, Wendell A. Lim12, David F. Stern9, Bruce Stillman10, Brenda J. Andrews11, Mark B. Gerstein2,3, Michael Snyder1,2#, and Benjamin E. Turk4#

1 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA.
2 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
3 Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT 06520, USA.
4 Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
5 Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
6 Department of Medicine, Division of Biochemistry, University of Fribourg, CH-1700 Fribourg, Switzerland.
7 Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA.
8 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA.
9 Department of Pathology, School of Medicine, Yale University, New Haven, CT 06520, USA.
10 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
11 Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1.
12 Eötvös Loránd University, Department of Biochemistry and Howard Hughes Medical Institute, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary.

* Present address: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, Palo Alto, CA 94304, USA.

{dagger} Present address: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1.

{ddagger} Present address: Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, CT 06410, USA.

§ Present address: Institute for Research in Biomedicine, CH-6500 Bellinzona, Switzerland.

|| Present address: Eötvös Loránd University, Department of Biochemistry, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary.

Present address: Department of Genetics, Stanford University, Palo Alto, CA 94305, USA.

Abstract: Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae. By correlating these motifs with kinase primary sequence, we uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P–3 arginine specificity among members of the CMGC [CDK (cyclin-dependent kinase), MAPK (mitogen-activated protein kinase), GSK (glycogen synthase kinase), and CDK-like] group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo and identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of previously unknown kinase substrates across eukaryotes.

# To whom correspondence should be addressed. E-mail: ben.turk{at} (B.E.T.); mpsnyder{at} (M.S.).

Citation: J. Mok, P. M. Kim, H. Y. K. Lam, S. Piccirillo, X. Zhou, G. R. Jeschke, D. L. Sheridan, S. A. Parker, V. Desai, M. Jwa, E. Cameroni, H. Niu, M. Good, A. Remenyi, J.-L. N. Ma, Y.-J. Sheu, H. E. Sassi, R. Sopko, C. S. M. Chan, C. De Virgilio, N. M. Hollingsworth, W. A. Lim, D. F. Stern, B. Stillman, B. J. Andrews, M. B. Gerstein, M. Snyder, B. E. Turk, Deciphering Protein Kinase Specificity Through Large-Scale Analysis of Yeast Phosphorylation Site Motifs. Sci. Signal. 3, ra12 (2010).

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