Predicting kinase substrates using conservation of local motif density.

MOTIVATION: Protein kinases represent critical links in cell signaling. A central problem in computational biology is to systematically identify their substrates. RESULTS: This study introduces a new method to predict kinase substrates by extracting evolutionary information from multiple sequence alignments in a manner that is tolerant to degenerate motif positioning. Given a known consensus, the new method (ConDens) compares the observed density of matches to a null model of evolution and does not require labeled training data. We confirmed that ConDens has improved performance compared with several existing methods in the field. Further, we show that it is generalizable and can predict interesting substrates for several important eukaryotic kinases where training data is not available. AVAILABILITY AND IMPLEMENTATION: ConDens can be found at http://www.moseslab.csb.utoronto.ca/andyl/. CONTACT: alan.moses@utoronto.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Global mapping of protein phosphorylation events identifies Ste20, Sch9 and the cell-cycle regulatory kinases Cdc28/Pho85 as mediators of fatty acid starvation responses in Saccharomyces cerevisiae.

Synthesis, degradation, and metabolism of fatty acids are strictly coordinated to meet the nutritional and energetic needs of cells and organisms. In the absence of exogenous fatty acids, proliferation and growth of the yeast Saccharomyces cerevisiae depends on endogenous synthesis of fatty acids, which is catalysed by fatty acid synthase. In the present study, we have used quantitative proteomics to examine the cellular response to inhibition of fatty acid synthesis in Saccharomyces cerevisiae. We have identified approximately 2000 phosphorylation sites of which more than 400 have been identified as being regulated in a temporal manner in response to inhibition of fatty acid synthesis by cerulenin. By bioinformatic analysis of these phosphorylation events, we have identified the cell cycle kinases Cdc28 and Pho85, the PAK kinase Ste20 as well as the protein kinase Sch9 as central mediators of the cellular response to inhibition of fatty acid synthesis.

A phosphorylation-independent role for the yeast cyclin-dependent kinase activating kinase Cak1.

Cdc28 is the main cyclin-dependent kinase (CDK) directing the cell cycle in the budding yeast Saccharomyces cerevisiae. Besides cyclin binding, Cdc28 requires phosphorylation by the Cak1 kinase to achieve full activity. We have previously isolated carboxy-terminal cdc28(CST) mutants that are temperature sensitive and exhibit high chromosome instability. Both phenotypes are suppressed by high copy Cak1 in a manner that is independent of its catalytic activity and conversely, combination of cdc28(CST) and cak1 mutations results in synthetic lethality. Altogether, these results suggest that for the Cdc28 complexes to remain stable and active, an interaction with Cak1 is needed via the carboxyl terminus of Cdc28. We report two-hybrid assay data that support this model, and results that indicate that actively growing yeast cells require an optimum Cdc28:Cak1 ratio. While Cak1 is constitutively active and expressed, dividing cells tightly regulate Cak1 protein levels to ensure presence of adequate levels of Cdc28 CDK activity.

Sister chromatid cohesion role for CDC28-CDK in Saccharomyces cerevisiae.

High-fidelity chromosome segregation requires that the sister chromatids produced during S phase also become paired during S phase. Ctf7p (Eco1p) is required to establish sister chromatid pairing specifically during DNA replication. However, Ctf7p also becomes active during G2/M in response to DNA damage. Ctf7p is a phosphoprotein and an in vitro target of Cdc28p cyclin-dependent kinase (CDK), suggesting one possible mechanism for regulating the essential function of Ctf7p. Here, we report a novel synthetic lethal interaction between ctf7 and cdc28. However, neither elevated CDC28 levels nor CDC28 Cak1p-bypass alleles rescue ctf7 cell phenotypes. Moreover, cells expressing Ctf7p mutated at all full- and partial-consensus CDK-phosphorylation sites exhibit robust cell growth. These and other results reveal that Ctf7p regulation is more complicated than previously envisioned and suggest that CDK acts in sister chromatid cohesion parallel to Ctf7p reactions.

Identification of Clb2 residues required for Swe1 regulation of Clb2-Cdc28 in Saccharomyces cerevisiae.

Wee1 kinases regulate the cell cycle through inhibitory phosphorylation of cyclin-dependent kinases (CDKs). Eukaryotic cells express multiple CDKs, each having a kinase subunit (Cdk) and a regulatory "cyclin" subunit that function at different stages of the cell cycle to regulate distinct processes. The cyclin imparts specificity to CDK-substrate interactions and also determines whether a particular CDK is subject to Wee1 regulation. Saccharomyces Wee1 (Swe1) inhibits Cdc28 (Cdk1) associated with the mitotic cyclin, Clb2, but not with the G(1) (Cln1, -2, and -3) or the S-phase (Clb5 and -6) cyclins. Here, we show that this specificity depends on two amino acids associated with a conserved "hydrophobic patch" (HP) motif on the cyclin surface, which mediates specificity of CDK-substrate interactions. Mutation of Clb2 residues N260 and K270 largely abrogates Clb2-Cdc28 regulation by Swe1, and reciprocal mutation of the corresponding residues in Clb5 can subject Clb5-Cdc28 to regulation by Swe1. Swe1 phosphorylation by Clb2-Cdc28, which is thought to activate Swe1 kinase, depends on N260 and K270, suggesting that specific regulation of Clb2-Cdc28 by Swe1 derives from the specific ability of Clb2 to target Swe1 for activating phosphorylation. The stable association of Swe1 with Clb2-Cdc28 also depends on these residues, suggesting that Swe1 may competitively inhibit Clb2-Cdc28 interactions with substrates, in addition to its well-known function as a regulator of CDK activity through tyrosine phosphorylation.

Discrimination between stable and dynamic components of protein complexes by means of quantitative proteomics.

To discriminate between stable and dynamic protein-protein interactions, we propose a strategy in which cells with and without tagged bait are differentially labeled with stable isotope and combined prior to complex purification. Mass-spectrometric analysis of the purified complexes identifies stable and dynamic components as those derived exclusively from the tagged cells and those from both cells, respectively. We successfully applied this strategy to analyze two yeast protein complexes, eIF2B-eIF2 and cyclin-Cdc28.

Prediction of cyclin-dependent kinase phosphorylation substrates.

Protein phosphorylation, mediated by a family of enzymes called cyclin-dependent kinases (Cdks), plays a central role in the cell-division cycle of eukaryotes. Phosphorylation by Cdks directs the cell cycle by modifying the function of regulators of key processes such as DNA replication and mitotic progression. Here, we present a novel computational procedure to predict substrates of the cyclin-dependent kinase Cdc28 (Cdk1) in the Saccharomyces cerevisiae. Currently, most computational phosphorylation site prediction procedures focus solely on local sequence characteristics. In the present procedure, we model Cdk substrates based on both local and global characteristics of the substrates. Thus, we define the local sequence motifs that represent the Cdc28 phosphorylation sites and subsequently model clustering of these motifs within the protein sequences. This restraint reflects the observation that many known Cdk substrates contain multiple clustered phosphorylation sites. The present strategy defines a subset of the proteome that is highly enriched for Cdk substrates, as validated by comparing it to a set of bona fide, published, experimentally characterized Cdk substrates which was to our knowledge, comprehensive at the time of writing. To corroborate our model, we compared its predictions with three experimentally independent Cdk proteomic datasets and found significant overlap. Finally, we directly detected in vivo phosphorylation at Cdk motifs for selected putative substrates using mass spectrometry.

[Molecular dynamics modeling of the substitution of serine for the conservative glycine in the G loop in the yeast cdc28-srm mutant using the crystalline lattice of human kinase CDK2].

Two-nanosecond molecular dynamics modeling of the crystalline lattice of an active complex of kinase pT160-CDK2/cyclin A/ATP-Mg2+ substrate has been performed. The results of modeling indicated that the structures of the nonmutant CDK2 complex and mutant CDK2 complex, which involves the G 16S-CD K2 substitution corresponding to that of yeast, markedly differ, the differences in structural conformations being particularly well pronounced in those regions that play a key role in the functioning of kinase. Based on the results of computations, structural elements are considered that may affect the kinase activity and regulatory phosphorylation, and the binding of protein kinase to cyclins and substrates.

Excess mannose limits the growth of phosphomannose isomerase PMI40 deletion strain of Saccharomyces cerevisiae.

Phosphomannose isomerase (PMI40) catalyzes the conversion between fructose 6-phosphate and mannose 6-phosphate and thus connects glycolysis, i.e. energy production and GDP-mannose biosynthesis or cell wall synthesis in Saccharomyces cerevisiae. After PMI40 deletion (pmi(-)) the cells were viable only if fed with extracellular mannose and glucose. In an attempt to force the GDP-mannose synthesis in the pmi(-) strain by increasing the extracellular mannose concentrations, the cells showed significantly reduced growth rates without any alterations in the intracellular GDP-mannose levels. To reveal the mechanisms resulting in reduced growth rates, we measured genome-wide gene expression levels, several metabolite concentrations, and selected in vitro enzyme activities in central metabolic pathways. The increasing of the initial mannose concentration led to an increase in the mannose 6-phosphate concentration, which inhibited the activity of the second enzyme in glycolysis, i.e. phosphoglucose isomerase converting glucose 6-phosphate to fructose 6-phosphate. As a result of this limitation, the flux through glycolysis was decreased as was the median expression of the genes involved in glycolysis. The expression levels of RAP1, a transcription factor involved in the regulation of the mRNA levels of several enzymes in glycolysis, as well as those of cell cycle regulators CDC28 and CLN3, decreased concomitantly with the growth rates and expression of many genes encoding for enzymes in glycolysis.

Genome-wide studies of protein-protein interaction.

Recent large-scale studies of protein complexes in yeast have demonstrated that the wide majority of proteins exist in the cell as parts of multicomponent assemblies, mostly novel and of unknown function. The structural and functional analysis of these complexes should be a priority for structural biologists in coming years. In silico methods such as docking simulations, which may contribute to this analysis, are being tested in the CAPRI community-wide experiment, which assesses blind predictions of the structure of protein-protein complexes.

An essential function of yeast cyclin-dependent kinase Cdc28 maintains chromosome stability.

Multiple surveillance pathways maintain genomic integrity in yeast during mitosis. Although the cyclin-dependent kinase Cdc28 is a well established regulator of mitotic progression, evidence for a direct role in mitotic surveillance has been lacking. We have now implicated a conserved sequence in the Cdc28 carboxyl terminus in maintaining chromosome stability through mitosis. Six temperature-sensitive mutants were isolated via random mutagenesis of 13 carboxyl-terminal residues. These mutants identify a Cdc28 domain necessary for proper mitotic arrest in the face of kinetochore defects or microtubule inhibitors. These chromosome stability-defective cdc28(CST) mutants inappropriately continue mitosis when the mitotic spindle is disrupted at 23 degrees C, display high rates of spontaneous chromosome loss at 30 degrees C, and suffer catastrophic aneuploidy at 35 degrees C. A dosage suppression screen identified Cak1, a kinase known to phosphorylate and activate Cdc28, as a specific high copy suppressor of cdc28(CST) temperature sensitivity and chromosome instability. Suppression is independent of the kinase activity of Cak1, suggesting that Cak1 may bind to the carboxyl terminus to serve a non-catalytic role in assembly and/or stabilization of active Cdc28 complexes. Significantly, these studies implicate Cdc28 and Cak1 in an essential surveillance function required to maintain genetic stability through mitosis.

CAK1 promotes meiosis and spore formation in Saccharomyces cerevisiae in a CDC28-independent fashion.

CAK1 encodes a protein kinase in Saccharomyces cerevisiae whose sole essential mitotic role is to activate the Cdc28p cyclin-dependent kinase by phosphorylation of threonine-169 in its activation loop. SMK1 encodes a sporulation-specific mitogen-activated protein (MAP) kinase homolog that is required to regulate the postmeiotic events of spore wall assembly. CAK1 was previously identified as a multicopy suppressor of a weakened smk1 mutant and shown to be required for spore wall assembly. Here we show that Smk1p, like other MAP kinases, is phosphorylated in its activation loop and that Smk1p is not activated in a cak1 missense mutant. Strains harboring a hyperactivated allele of CDC28 that is CAK1 independent and that lacks threonine-169 still require CAK1 to activate Smk1p. The data indicate that Cak1p functions upstream of Smk1p by activating a protein kinase other than Cdc28p. We also found that mutants lacking CAK1 are blocked early in meiotic development, as they show substantial delays in premeiotic DNA synthesis and defects in the expression of sporulation-specific genes, including IME1. The early meiotic role of Cak1p, like the postmeiotic role in the Smk1p pathway, is CDC28 independent. The data indicate that Cak1p activates multiple steps in meiotic development through multiple protein kinase targets.

Enhanced cell polarity in mutants of the budding yeast cyclin-dependent kinase Cdc28p.

The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.

A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex.

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

Mutation at the CK2 phosphorylation site on Cdc28 affects kinase activity and cell size in Saccharomyces cerevisiae.

We have recently reported that protein kinase CK2 phosphorylates both in vivo and in vitro residue serine-46 of the cell cycle regulating protein Cdc28 of budding yeast Saccharomyces cerevisiae, confirming a previous observation that the same site is phosphorylated in Cdc2/Cdk1, the human homolog of Cdc28. In addition, S. cerevisiae in which serine-46 of Cdc28 has been mutated to alanine show a decrease of 33% in both cell volume and protein content, providing the genetic evidence that CK2 is involved in the regulation of budding yeast cell division cycle, and suggesting that this regulation may be brought about in G1 phase of the mammalian cell cycle. Here, we extended this observation reporting that the mutation of serine-46 of Cdc28 to glutamic acid doubles, at least in vitro, the H1-kinase activity of the Cdc28/cyclin A complex. Since this mutation has only little effects on the cell size of the cells, we hypothesize multiple roles of yeast CK2 in regulating the G1 transition in budding yeast.

Phosphorylation of Cdc28 and regulation of cell size by the protein kinase CKII in Saccharomyces cerevisiae.

The CDK (cyclin-dependent kinase) family of enzymes is required for the G(1)-to-S-phase and G(2)-to-M-phase transitions during the cell-division cycle of eukaryotes. We have shown previously that the protein kinase CKII catalyses the phosphorylation of Ser-39 in Cdc2 during the G(1) phase of the HeLa cell-division cycle [Russo, Vandenberg, Yu, Bae, Franza and Marshak (1992) J. Biol. Chem. 267, 20317-20325]. To identify a functional role for this phosphorylation, we have studied the homologous enzymes in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of Cdc2, Cdc28, contains a consensus CKII site (Ser-46), which is homologous with that of human Cdc2. Using in vitro kinase assays, metabolic labelling, peptide mapping and phosphoamino acid analysis, we demonstrate that this site is phosphorylated in Cdc28 in vivo as well in vitro. In addition, S. cerevisiae cells in which Ser-46 has been mutated to alanine show a decrease in both cell volume and protein content of 33%, and this effect is most pronounced in the stationary phase. Because cell size in S. cerevisiae is regulated primarily at the G(1) stage, we suggest that CKII contributes to the regulation of the cell cycle in budding yeast by phosphorylation of Cdc28 as a checkpoint for G(1) progression.

Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells.

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage.

A yeast heat shock transcription factor (Hsf1) mutant is defective in both Hsc82/Hsp82 synthesis and spindle pole body duplication.

Cdc28 is a cyclin-dependent protein kinase of Saccharomyces cerevisiae that is required for the G1/S and G2/M transitions of the cell division cycle. All previously described cdc28 mutants aside from cdc28-1N arrest division specifically in the G1 phase. cdc28-1N arrests division in G2/mitosis. We show here that the cdc28-109 mutant exhibits a mixed cell division arrest at 37 degrees C with cells in both the G1 and G2 phases. In order to identify proteins that functionally interact with Cdc28, we isolated mutants that are colethal with cdc28-109 at its permissive temperature. We describe here our phenotypic analysis of two such mutants, hsf1-82 and ydj1-10, that affect the heat shock transcription factor and a yeast dnaj-like protein chaperone, respectively. hsf1-82 and ydj1-10 temperature-sensitive mutants arrest the cell division cycle at several stages. However, one predominant class of cells in both mutants was arrested with a large bud and a single vertex of microtubules. Electron microscopic analysis of such hsf1-82 cells showed that they contained an unduplicated spindle pole body with an enlarged half-bridge. Two-dimensional gel electrophoresis of total cell proteins revealed that the hsf1-82 cells were specifically defective in the expression of the Hsc82 and Hsp82 proteins. Furthermore, the hsf1-82 mutation was suppressed by the HSC82 gene on a multicopy plasmid that restored Hsc82 protein to high levels in these cells. These results show that Hsf1 is required for spindle pole body duplication at 37 degrees C.

Gin4 of S. cerevisiae is a bud neck protein that interacts with the Cdc28 complex.

BACKGROUND: A number of proteins make up the Cdc28 complex in Saccharomyces cerevisiae, and regulate CDK activity. The cell cycle regulator Nik1 (Hsl1) is a protein kinase that interacts with the Cdc28 complex. The growth inhibitor Gin4 is structurally similar to Nik1 and may play a redundant role in the regulation of the cell cycle. We investigated the functions of Gin4 with respect to those of Nik1. RESULTS: GIN4 was not essential for growth, and cells deficient in the GIN4 gene displayed no obvious defects in cell cycle regulation. The delta(gin)4 delta(nik)1 strain was temperature sensitive and showed an abnormal cell shape and FACS profile at permissive temperatures. GFP-fused Gin4 was localized at the bud-neck from late G1 to the M phase. Over-production of the C-terminal portion of Gin4 was toxic for cell growth, and this domain was required for the bud-neck localization of Gin4-GFP. High copy expression of Gin4-GFP disturbed the bud-neck localization of Gin4 in the abnormally elongated cells. Cytokinesis was defective in the delta(gin)4 cdc28 double mutants. The GST-Gin4 fusion protein physically associates with the Cdc28 complex. CONCLUSIONS: Gin4 is a bud-neck protein. GIN4 and NIK1 have distinct but partially overlapping functions. The major function of GIN4 is to ensure proper mitotic progression and cytokinesis.

Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28.

In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves. We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation.