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 the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: 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 interaction and transcription factor binding data 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.

Strain construction, materials, and Net1 mutagenesisAll strains used are in the W303 background (can1-100, leu2-3, his3-11, trp1-1, ura3-1, ade2-1) except where noted in the strain table (Supplementary Table 1). A strain expressing a stable form of Clb2 lacking both the KEN and destruction boxes (Clb2C 2 DK 100 )HA3 was used in over-expression experiments with Clb2 (1).Net1 mutant constructs were created as previously described (2). Briefly, a wild type NET1-myc9 epitope tagged construct was cloned into a modified pRS304 vector containing 300bp upstream of the ATG translation start site using NcoI and EagI. Sitedirected mutagenesis of Serine/Threonine to Alanine was carried out using QuikChange Site-Directed mutagenesis kit from Stratagene (La Jolla, CA). The indicated Serine/Threonine were mutated to Alanine in 169,212,231,252,259,356,362,384,385,497,611,676), 169,212,231,252,259),212,252), 166,212,252,297,304) where * indicates that residue 62 was mutated to ensure complete elimination of all Cdk consensus sites even though it was not determined to be phosphorylated in vivo. Mutagenesis was confirmed using restriction digests followed by Antibodies specifically reactive against the phosphopeptides were positively selected on a resin derivatized with the phosphopeptide immunogen and negatively selected by passage through a resin derivatized with the unphosphorylated version of the peptide. Antiphosphopeptide B (α-PP-B) was used in all experiments described since it generated the strongest signal against Phospho-Net1 ( Fig. 2A). Cell Growth and Synchronization ProceduresCells were grown in yeast extract-peptone (YP) or in yeast minimal (YM) media containing 2% glucose (YPD,YMD), 2% raffinose (YPR,YMR) or 2% galactose (YPG,YMG) as carbon source. Where appropriate, minimal media were supplemented with leucine, histidine, tryptophan, uracil, and adenine to complement auxotrophies.Synchronization of cells in G1 phase was achieved with α-factor added at 10 µg/ml for BAR1 cells and 0.1 µg/ml for bar1∆ cells for at least 3 hrs at 25°C. Cells were judged to be arrested when greater than 90% of cells displayed the elongated "shmoo" phenotype.Cells were released from α factor by filtration through a 0.2µm filter followed by a wash with 150 ml of YP, then resuspended in the desired volume at a density of 1 O.D. 600 /ml.For elutriation, cells were grown overnight in YP containing 2% raffinose and 2%galactose and harvested at log phase. Elutriation was performed as described (3-5) for the collection of small, unbudded G1 cells; contamination with budded cells was measured to be no more than 2%. For galactose induction experiments, cells were grown overnight in either YMR or YPR until an O.D. 600 of 1.0 was reached, then induced with 2% galactose followed by time point collection. Cell Extract Preparation and Western BlottingCells were grown to an O.D. 600 of 1.0, and for every time point 2 ml of culture was collected and TCA added to a final concentration of 20%. Cells were collected by centrifugation and washed with 2...