Abstract
Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes1,2,3. 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 the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology4, the in vitro substrates recognized by most yeast protein kinases5: we identified over 4,000 phosphorylation events involving 1,325 different proteins. 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 cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein–protein interaction6,7,8,9,10 and transcription factor binding data11,12 revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.
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Acknowledgements
We thank J. Tang for help with the initial Cdc28 preparations, and D. Gelperin, J. Mok and K. Wise for comments on the manuscript. M.S. was funded by grants from the NIH; J.P., G.D. and J.F. were funded by NIH predoctoral fellowships, and B.A. and M.T. were funded by grants from the Canadian Institutes of Health Research. M.J.R.S. was funded by a project grant from the Wellcome Trust, UK.Author Contributions Assay development was performed by H.Z., J.P., G.D., G.M. and M.S. Proteome chips were prepared by G.M., B.S. and P.F.P. at Invitrogen. G.J. contributed transcription factors for the arrays. Kinase assays were performed by J.P., G.D., H.Z. and M.S. Most kinases were prepared by J.P. and G.D. Additional kinases were provided by A.B., R.S., R.R.M., M.C.S., N.R., S.J.L., A.S.M., M.J.R.S., D.F.S, C.D.V., M.T. and B.A. Data analysis was performed by X.Z., J.P. and G.D. Consensus mapping was by H.G. In vitro solution validations were performed by G.M. and L.M. In vivo substrate validations were performed by J.P., G.D. and J.F. Most assays and analyses were performed in the laboratory of M.S. with contributions from M.G.
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G.M., L.M., B.S. and P.F.P. are employed by Invitrogen. M.S. has financial interests in Invitrogen.
Supplementary information
Supplementary Tables
Supplementary Table 1 shows kinases and their targets that share same MIPS functionary categories. Supplementary Table 2 lists all the known kinase-substrate phosphorylation events found in our study. (PDF 138 kb)
Supplementary Figures
Supplementary Figure 1 shows the distribution of phosphorylation events. Number of kinases for each substrate (top). Number of substrates for each kinase (bottom). Supplementary Figure 2 details solution validation of yeast proteome identified protien kinase substrates. (PDF 194 kb)
Supplementary Data 1
List of kinases analysed by our protein chip assay. (PDF 3 kb)
Supplementary Data 2
This file summarizes the substrate report for all the kinases tested. * This file was incorrectly uploaded as Supplementary Data 3 at time of publication. This was corrected on 03 February 2006. (XLS 950 kb)
Supplementary Data 3
All 8 regulatory modules including phosphorylation links, protein-protein interaction and transcriptional regulation. * This file was incorrectly uploaded as Supplementary Data 2 at time of publication. This was corrected on 03 February 2006. (XLS 1292 kb)
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Ptacek, J., Devgan, G., Michaud, G. et al. Global analysis of protein phosphorylation in yeast. Nature 438, 679–684 (2005). https://doi.org/10.1038/nature04187
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DOI: https://doi.org/10.1038/nature04187
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