Global Analysis of Protein Phosphorylation in Yeast

Abstract

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. Over 4000 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.

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