Yeast Protein Kinase Research Articles

A novel role for the yeast protein kinase Dbf2p in vacuolar H+-ATPase function and sorbic acid stress tolerance

In Saccharomyces cerevisiae, the serine-threonine protein kinase activity of Dbf2p is required for tolerance to the weak organic acid sorbic acid. Here we show that Dbf2p is required for normal phosphorylation of the vacuolar H(+)-ATPase (V-ATPase) A and B subunits Vma1p and Vma2p. Loss of V-ATPase activity due to bafilomycin treatment or deletion of either VMA1 or VMA2 resulted in sorbic acid hypersensitivity and impaired vacuolar acidification, phenotypes also observed in both a kinase-inactive dbf2 mutant and cells completely lacking DBF2 (dbf2Delta). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2Delta cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.

The Arabidopsis Mitogen-Activated Protein Kinase Kinase MKK3 Is Upstream of Group C Mitogen-Activated Protein Kinases and Participates in Pathogen Signaling

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H(2)O(2), but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.

Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell‐cycle phase‐dependent degradation in yeast

Using co-immunoprecipitation combined with MS analysis, we identified the alpha' subunit of casein kinase 2 (CK2) as an interaction partner of the atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2 from Deltacka1 or Deltacka2 mutant extracts shows that Rio1p preferentially interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential and sufficient for this interaction. Six C-terminally located clustered serines were identified as the only CK2 sites present in Rio1p. Replacement of all six serine residues by aspartate, mimicking constitutive phosphorylation, stimulates Rio1p kinase activity about twofold in vitro compared with wild-type or the corresponding (S > A)(6) mutant proteins. Both mutant alleles (S > A)(6) or (S > D)(6) complement in vivo, however, growth of the RIO1 (S > A)(6) mutant is greatly retarded and shows a cell-cycle phenotype, whereas the behaviour of the RIO1 (S > D)(6) mutant is indistinguishable from wild-type. This suggests that phosphorylation by protein kinase CK2 leads to moderate activation of Rio1p in vivo and promotes cell proliferation. Physiological studies indicate that phosphorylation by CK2 renders the Rio1 protein kinase susceptible to proteolytic degradation at the G(1)/S transition in the cell-division cycle, whereas the non-phosphorylated version is resistant.

Structurally Unique Yeast and Mammalian Serine-Arginine Protein Kinases Catalyze Evolutionarily Conserved Phosphorylation Reactions

The mammalian serine-arginine (SR) protein, ASF/SF2, contains multiple contiguous RS dipeptides at the C terminus, and approximately 12 of these serines are processively phosphorylated by the SR protein kinase 1 (SRPK1). We have recently shown that a docking motif in ASF/SF2 specifically interacts with a groove in SRPK1, and this interaction is necessary for processive phosphorylation. We previously showed that SRPK1 and its yeast ortholog Sky1p maintain their active conformations using diverse structural strategies. Here we tested if the mechanism of ASF/SF2 phosphorylation by SRPK is evolutionarily conserved. We show that Sky1p forms a stable complex with its heterologous mammalian substrate ASF/SF2 and processively phosphorylates the same sites as SRPK1. We further show that Sky1p utilizes the same docking groove to bind yeast SR-like protein Gbp2p and phosphorylates all three serines present in a contiguous RS dipeptide stretch. However, the mechanism of Gbp2p phosphorylation appears to be non-processive. Thus, there are physical attributes of SR and SR-like substrates that dictate the mechanism of phosphorylation, whereas the ability to processively phosphorylate substrates is inherent to SR protein kinases.

DNA Sequences from Arabidopsis, Which Encode Protein Kinases and Function as Upstream Regulators of Snf1 in Yeast

Sucrose nonfermenting-1 (Snf1)-related protein kinase-1 (SnRK1) of plants is a global regulator of carbon metabolism through the modulation of enzyme activity and gene expression. It is structurally and functionally related to the yeast protein kinase, Snf1, and to mammalian AMP-activated protein kinase. Two DNA sequences from Arabidopsis thaliana, previously known only by their data base accession numbers of NM_ 125448.3 (protein ID NP_200863) and NM_114393.3 (protein ID NP_566876) each functionally complemented a Saccharomyces cerevisiae elm1 sak1 tos3 triple mutant. This indicates that the Arabidopsis proteins are able to substitute for one of the missing yeast upstream kinases, which are required for activity of Snf1. Both plant proteins were shown to phosphorylate a peptide with the amino acid sequence of the phosphorylation site in the T-loop of SnRK1 and by inference SnRK1 in Arabidopsis. The proteins encoded by NM_125448.3 and NM_114393.3 have been named AtSnAK1 and AtSnAK2 (Arabidopsis thaliana SnRK1-activating kinase), respectively. We believe this is the first time that upstream activators of SnRK1 have been described in any plant species.

The RGG Domain of Npl3p Recruits Sky1p Through Docking Interactions

The SR protein kinase in yeast, Sky1p, phosphorylates yeast SR-like protein, Npl3p, at a single serine residue located at its C terminus. We report here the X-ray crystal structure of Sky1p bound to a substrate peptide and ADP. Surprisingly, an Npl3p-derived substrate peptide occupies a groove 20 Å away from the kinase active site. In vitro studies support the substrate-docking role of this groove. Mutagenesis and binding studies reveal that multiple degenerate short peptide motifs located within the RGG domain of Npl3p serve as the substrate docking motifs. However, a single docking motif is sufficient for its stable interaction with the kinase. Methylation of the docking motifs abolishes kinase binding and phosphorylation of Npl3p. Remarkably, removal of the docking groove in the kinase or the docking motifs of the substrate does not reduce the overall catalytic efficiency of the phosphorylation reaction in any significant manner. We suggest that docking interaction between Sky1p and Npl3p is essential for substrate recruitment and binding specificity.

Two Glucose-sensing Pathways Converge on Rgt1 to Regulate Expression of Glucose Transporter Genes in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae deploys two different types of glucose sensors on its cell surface that operate in distinct glucose signaling pathways: the glucose transporter-like Snf3 and Rgt2 proteins and the Gpr1 receptor that is coupled to Gpa2, a G-protein alpha subunit. The ultimate target of the Snf3/Rgt2 pathway is Rgt1, a transcription factor that regulates expression of HXT genes encoding glucose transporters. We have found that the cAMP-dependent protein kinase A (PKA), which is activated by the Gpr1/Gpa2 glucose-sensing pathway and by a glucose-sensing pathway that works through Ras1 and Ras2, catalyzes phosphorylation of Rgt1 and regulates its function. Rgt1 is phosphorylated in vitro by all three isoforms of PKA, and this requires several serine residues located in PKA consensus sequences within Rgt1. PKA and the consensus serine residues of Rgt1 are required for glucose-induced removal of Rgt1 from the HXT promoters and for induction of HXT expression. Conversely, overexpression of the TPK genes led to constitutive expression of the HXT genes. The PKA consensus phosphorylation sites of Rgt1 are required for an intramolecular interaction that is thought to regulate its DNA binding activity. Thus, two different glucose signal transduction pathways converge on Rgt1 to regulate expression of glucose transporters.

Shortcut to PKA in Yeast

Cells usually activate the cyclic AMP (cAMP, or adenosine 3′,5′-monophosphate)-dependent protein kinase through G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors, which activate G proteins, which in turn control the activity of adenylyl cyclase, the enzyme that makes cAMP. Peeters et al ., however, report that in yeast, and just maybe in mammalian cells as well, there appears to be a more direct route to activation of the cAMP-dependent protein kinase (PKA). Peeters et al . studied the roles of Krh1 (kelch-repeat homologue 1, also called Gpb2) and Krh2 (also called Gpb1). Krh1 and Krh2 associate with the yeast G protein α subunit Gpa2, which appears not to interact with canonical G protein β-γ subunits. Interestingly, the Krh1 and Krh2 proteins have the seven-bladed β-propeller structure characteristic of G β proteins. Deletion of Krh1 and Krh2 resulted in a phenotype indicative of high PKA activity, but there was no associated increase in the abundance of cAMP in the cells. Furthermore deletion of Krh1 and Krh2 suppressed the poor growth of cells lacking the yeast gene encoding adenylyl cyclase, as long as a minimal amount of cAMP was present in the culture. Thus, Krh1 and Krh2 appeared to act independently of cAMP. Nevertheless, the effects of the Krh proteins depended on the yeast PKA enzymes, and cells lacking the three genes encoding PKA catalytic subunits or the BCY1 regulatory subunit gene failed to respond to loss of Krh1 and Krh2. The authors used two-hybrid methods and in vitro binding assays to show that Krh1 bound to Gpa2, but also to the PKA holoenzyme through direct interaction with the catalytic subunit. A two-hybrid reporter assay showed that interaction of the regulatory and catalytic subunits appeared to decrease in vivo when function of Krh1 and Krh2 was lost. The authors suggest that these effects may be relevant in mammals as well because interaction between mouse catalytic and regulatory subunits, substituted in this yeast two-hybrid assay, was also decreased when Krh1 and Krh2 were not present. Overall, the results suggest a mechanism in which PKA can be controlled without participation of adenylyl cyclase: A sugar-sensing receptor on the yeast cell surface activates the Gpa2 alpha subunit, which then relieves the direct inhibition of PKA by Krh1 and Krh2. T. Peeters, W. Louwet, R. Geladé, D. Nauwelaers, J. M. Thevelein, M. Versele, Kelch-repeat proteins interacting with the G α protein Gpa2 bypass adenylate cyclase for direct regulation of protein kinase A in yeast. Proc. Natl. Acad. Sci. U.S.A. 103 , 13034-13039 (2006). [Abstract] [Full Text]

Yeast holoenzyme of protein kinase CK2 requires both β and β′ regulatory subunits for its activity

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (alpha and/or alpha') subunits and two regulatory beta and beta' subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of alphaalpha'betabeta', alpha2betabeta', alpha'2betabeta' and a free alpha'-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory beta subunit cannot substitute other beta' subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the beta or beta' regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic alpha subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2beta/beta' subunits in the regulation of holoenzyme assembly and phosphotransferase activity.

Eukaryotic release factor 1 phosphorylation by CK2 protein kinase is dynamic but has little effect on the efficiency of translation termination in Saccharomyces cerevisiae.

Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Delta mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.

Yeast protein kinase Ptk2 localizes at the plasma membrane and phosphorylates in vitro the C-terminal peptide of the H +-ATPase

Glucose triggers posttranslational modifications that increase the activity of the Saccharomyces cerevisiae plasma membrane H +-ATPase (Pma1). Glucose activation of yeast H +-ATPase results from the change in two kinetic parameters: an increase in the affinity of the enzyme for ATP, depending on Ser 899, and an increase in the V max involving Thr 912. Our previous studies suggested that Ptk2 mediates the Ser 899-dependent part of the activation. In this study we find that Ptk2 localized to the plasma membrane in a Triton X-100 insoluble fraction. In vitro phosphorylation assays using a recombinant GST-fusion protein comprising 30 C-terminal amino acids of Pma1 suggest that Ser 899 is phosphorylated by Ptk2. Furthermore, we show that the Ptk2 carboxyl terminus is essential for glucose-dependent Pma1 activation and for the phosphorylation of Ser 899.

Protein kinases associated with the yeast phosphoproteome.

BackgroundProtein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned.ResultsWe used Predikin in combination with other bioinformatic tools, to predict which of 116 unique protein kinases in yeast phosphorylates each experimentally determined site in the phosphoproteome. The prediction was based on the match between the phosphorylated 7-residue sequence and the predicted substrate specificity of each kinase, with the highest weight applied to the residues or positions that contribute most to the substrate specificity. We estimated the reliability of the predictions by performing a parallel prediction on phosphopeptides for which the kinase has been experimentally determined.ConclusionThe results reveal that the functions of the protein kinases and their predicted phosphoprotein substrates are often correlated, for example in endocytosis, cytokinesis, transcription, replication, carbohydrate metabolism and stress response. The predictions link phosphoproteins of unknown function with protein kinases with known functions and vice versa, suggesting functions for the uncharacterized proteins. The study indicates that the phosphoproteins and the associated protein kinases represented in our dataset have housekeeping cellular roles; certain kinases are not represented because they may only be activated during specific cellular responses. Our results demonstrate the utility of our previously reported protein kinase substrate prediction approach (Predikin) as a tool for establishing links between kinases and phosphoproteins that can subsequently be tested experimentally.

Cellular localization and phosphorylation of Hrb1p is independent of Sky1p

Protein phosphorylation plays a major role in regulating cellular functions. We have previously demonstrated that Sky1p, the SR protein kinase of the budding yeast Saccharomyces cerevisiae, is a regulator of polyamine transport and ion homeostasis. Since its kinase activity was demonstrated essential for fulfilling these roles, we assumed that Sky1p function via substrates phosphorylation. Using an in vitro phosphorylation assay, we have identified Hrb1p as a putative Sky1p substrate. However, phosphorylation analysis in WT and sky1Δ cells and localization studies disproved Hrb1p as a true Sky1p substrate, although a segment of the RS domain is required for determining its subcellular localization. Furthermore, we demonstrate that Hrb1p and additional putative Sky1p substrates, identified by computational approach, are not involved in mediating the spermine tolerant phenotype of sky1Δ cells.

Rck2 Is Required for Reprogramming of Ribosomes during Oxidative Stress

Rck2 is a mitogen-activated protein kinase-activated protein kinase in yeast implicated in translational regulation. rck2Delta mutants are mildly sensitive to oxidative stress, a condition that causes dissociation of actively translating ribosomes (polysomes). In rck2Delta cells, polysomes are lost to an even higher degree than in the wild-type upon stress. Cells overexpressing the catalytically inactive rck2-kd allele are highly sensitive to oxidative stress. In such cells, dissociation of polysomes upon stress was instead greatly delayed. The protein synthesis rate decreased to a similar degree as in wild-type cells, however, indicating that in rck2-kd cells, the polysome complexes were inactive. Array analyses of total and polysome-associated mRNAs revealed major deregulation of the translational machinery in rck2 mutant cells. This involves transcripts for cytosolic ribosomal proteins and for processing and assembly of ribosomes. In rck2Delta cells, weakly transcribed mRNAs associate more avidly with polysomes than in wild-type cells, whereas the opposite holds true for rck2-kd cells. This is consistent with perturbed regulation of translation elongation, which is predicted to alter the ratio between mRNAs with and without strong entry sites at ribosomes. We infer that imbalances in the translational apparatus are a major reason for the inability of these cells to respond to stress.

The identification of a functional interaction between PKC and topoisomerase II

Topoisomerase II plays an essential role in the segregation of chromosomes during cell division. It is also a major component of the nuclear matrix. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between the catalytic domain of the yeast protein kinase 1 enzyme (Pkc1) and the N-terminal domain of the S. cerevisiae topoisomerase II. The S. cerevisiae Pkc1 is the homologue of the mammalian calcium dependent PKC.

Combining chemical genetics and proteomics to identify protein kinase substrates

Phosphorylation is a ubiquitous protein modification important for regulating nearly every aspect of cellular biology. Protein kinases are highly conserved and constitute one of the largest gene families. Identifying the substrates of a kinase is essential for understanding its cellular role, but doing so remains a difficult task. We have developed a high-throughput method to identify substrates of yeast protein kinases that employs a collection of yeast strains each expressing a single epitope-tagged protein and a chemical genetic strategy that permits kinase reactions to be performed in native, whole-cell extracts. Using this method, we screened 4,250 strains expressing epitope-tagged proteins and identified 24 candidate substrates of the Pho85-Pcl1 cyclin-dependent kinase, including the known substrate Rvs167. The power of this method to identify true kinase substrates is strongly supported by functional overlap and colocalization of candidate substrates and the kinase, as well as by the specificity of Pho85-Pcl1 for some of the substrates compared with another Pho85-cyclin kinase complex. This method is readily adaptable to other yeast kinases.

Global analysis of protein phosphorylation in yeast

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.

Identification of VCP/p97, Carboxyl Terminus of Hsp70-interacting Protein (CHIP), and Amphiphysin II Interaction Partners Using Membrane-based Human Proteome Arrays

Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.

The Spatial Organization of Lipid Synthesis in the Yeast Saccharomyces cerevisiae Derived from Large Scale Green Fluorescent Protein Tagging and High Resolution Microscopy

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins ( approximately 9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.

Systematic Deletion Analysis of Fission Yeast Protein Kinases

Eukaryotic protein kinases are key molecules mediating signal transduction that play a pivotal role in the regulation of various biological processes, including cell cycle progression, cellular morphogenesis, development, and cellular response to environmental changes. A total of 106 eukaryotic protein kinase catalytic-domain-containing proteins have been found in the entire fission yeast genome, 44% (or 64%) of which possess orthologues (or nearest homologues) in humans, based on sequence similarity within catalytic domains. Systematic deletion analysis of all putative protein kinase-encoding genes have revealed that 17 out of 106 were essential for viability, including three previously uncharacterized putative protein kinases. Although the remaining 89 protein kinase mutants were able to form colonies under optimal growth conditions, 46% of the mutants exhibited hypersensitivity to at least 1 of the 17 different stress factors tested. Phenotypic assessment of these mutants allowed us to arrange kinases into functional groups. Based on the results of this assay, we propose also the existence of four major signaling pathways that are involved in the response to 17 stresses tested. Microarray analysis demonstrated a significant correlation between the expression signature and growth phenotype of kinase mutants tested. Our complete microarray data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/kinome.