Kinomics: methods for deciphering the kinome

Phosphorylation by protein kinases is the most widespread and well-studied signaling mechanism in eukaryotic cells. Phosphorylation can regulate almost every property of a protein and is involved in all fundamental cellular processes. Cataloging and understanding protein phosphorylation is no easy task: many kinases may be expressed in a cell, and one-third of all intracellular proteins may be phosphorylated, representing as many as 20,000 distinct phosphoprotein states. Defining the kinase complement of the human genome, the kinome, has provided an excellent starting point for understanding the scale of the problem. The kinome consists of 518 kinases, and every active protein kinase phosphorylates a distinct set of substrates in a regulated manner. Deciphering the complex network of phosphorylation-based signaling is necessary for a thorough and therapeutically applicable understanding of the functioning of a cell in physiological and pathological states. We review contemporary techniques for identifying physiological substrates of the protein kinases and studying phosphorylation in living cells.     

Rho-kinase in sea urchin eggs and embryos

The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.     

Genome-Wide Identification and Comprehensive Analyses of the Kinomes in Four Pathogenic Microsporidia Species

Microsporidia have attracted considerable attention because they infect a wide range of hosts, from invertebrates to vertebrates, and cause serious human diseases and major economic losses in the livestock industry. There are no prospective drugs to counteract this pathogen. Eukaryotic protein kinases (ePKs) play a central role in regulating many essential cellular processes and are therefore potential drug targets. In this study, a comprehensive summary and comparative analysis of the protein kinases in four microsporidia–Enterocytozoon bieneusi, Encephalitozoon cuniculi, Nosema bombycis and Nosema ceranae–was performed. The results show that there are 34 ePKs and 4 atypical protein kinases (aPKs) in E. bieneusi, 29 ePKs and 6 aPKs in E. cuniculi, 41 ePKs and 5 aPKs in N. bombycis, and 27 ePKs and 4 aPKs in N. ceranae. These data support the previous conclusion that the microsporidian kinome is the smallest eukaryotic kinome. Microsporidian kinomes contain only serine-threonine kinases and do not contain receptor-like and tyrosine kinases. Many of the kinases related to nutrient and energy signaling and the stress response have been lost in microsporidian kinomes. However, cell cycle-, development- and growth-related kinases, which are important to parasites, are well conserved. This reduction of the microsporidian kinome is in good agreement with genome compaction, but kinome density is negatively correlated with proteome size. Furthermore, the protein kinases in each microsporidian genome are under strong purifying selection pressure. No remarkable differences in kinase family classification, domain features, gain and/or loss, and selective pressure were observed in these four species. Although microsporidia adapt to different host types, the coevolution of microsporidia and their hosts was not clearly reflected in the protein kinases. Overall, this study enriches and updates the microsporidian protein kinase database and may provide valuable information and candidate targets for the design of treatments for pathogenic diseases.     

Comparative kinomics of Plasmodium organisms: unity in diversity

Phosphorylation by protein kinases is a very common and crucial process in many signal transduction pathways in eukaryotes. This review describes comparative protein kinase analysis of two apicomplexa Plasmodium falciparum (3D7 strain) and Plasmodium yoelii yoelii (17XNL strain) which are causative agents of malaria in human and African rat respectively. Sensitive bioinformatics techniques enable identification of 82 and 60 putative protein kinases in P. falciparum and P. yoelii yoelii respectively and these sequences could be classified into known subfamilies of protein kinases. The most populated kinase subfamilies in both the plasmodium species correspond to CAMK and CMGC groups. Analysis of domain architectures enables detection of uncommon domain organization in kinases of both the organisms such as kinase domain tethered to EF hands as well as PH domain. Components of MAPK signaling pathway is not well conserved in plasmodium organisms. Such observations suggest that plasmodium protein kinases are highly divergent from other eukaryotes. A transmembrane kinase with 6 membrane spanning segments in P. falciparum seems to have no orthologue in P. yoelii yoelii. 19 P. falciparum kinases have been found to cluster separately from P. yoelii yoelii kinases and hence these kinases are unique to P. falciparum genome. Only 28 orthologous pairs of kinases seem to be present between these two plasmodium organisms. Comparative kinome analysis of two plasmodium species has thus provided clues to the function of many protein kinases based upon their classification and domain organization and also implicate marked differences even between two plasmodium organisms.     

Analysis of the protein kinome of Entamoeba histolytica

Protein kinases play important roles in almost all major signaling and regulatory pathways of eukaryotic organisms. Members in the family of protein kinases make up a substantial fraction of eukaryotic proteome. Analysis of the protein kinase repertoire (kinome) would help in the better understanding of the regulatory processes. In this article, we report the identification and analysis of the repertoire of protein kinases in the intracellular parasite Entamoeba histolytica. Using a combination of various sensitive sequence search methods and manual analysis, we have identified a set of 307 protein kinases in E. histolytica genome. We have classified these protein kinases into different subfamilies originally defined by Hanks and Hunter and studied these kinases further in the context of noncatalytic domains that are tethered to catalytic kinase domain. Compared to other eukaryotic organisms, protein kinases from E. histolytica vary in terms of their domain organization and displays features that may have a bearing in the unusual biology of this organism. Some of the parasitic kinases show high sequence similarity in the catalytic domain region with calmodulin/calcium dependent protein kinase subfamily. However, they are unlikely to act like typical calcium/calmodulin dependent kinases as they lack noncatalytic domains characteristic of such kinases in other organisms. Such kinases form the largest subfamily of kinases in E. histolytica. Interestingly, a PKA/PKG-like subfamily member is tethered to pleckstrin homology domain. Although potential cyclins and cyclin-dependent kinases could be identified in the genome the likely absence of other cell cycle proteins suggests unusual nature of cell cycle in E. histolytica. Some of the unusual features recognized in our analysis include the absence of MEK as a part of the Mitogen Activated Kinase signaling pathway and identification of transmembrane region containing Src kinase-like kinases. Sequences which could not be classified into known subfamilies of protein kinases have unusual domain architectures. Many such unclassified protein kinases are tethered to domains which are Cysteine-rich and to domains known to be involved in protein-protein interactions. Our kinome analysis of E. histolytica suggests that the organism possesses a complex protein phosphorylation network that involves many unusual kinases.     

Quantitation of a src-like tyrosine protein kinase during fertilization of the sea urchin egg

Fertilization of the sea urchin egg is known to involve an increase in overall protein tyrosine kinase activity which precede the first cell division. In order to determine the types of tyrosine kinases that are involved in fertilization, we have used immunological and other criteria to identify a c-src related protein kinase in eggs of the sea urchin L. variegatus. Using an immune complex assay, we have measured the level of this c-src related protein kinase during fertilization and early embryonic development. Fertilization results in a decrease in the c-src kinase detectable by this technique suggesting that c-src does not contribute to the fertilization induced increase in protein tyrosine kinase activity.     

A quantitative analysis of kinase inhibitor selectivity

Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.     

Kinases and Pseudokinases: Lessons from RAF

Protein kinases are thought to mediate their biological effects through their catalytic activity. The large number of pseudokinases in the kinome and an increasing appreciation that they have critical roles in signaling pathways, however, suggest that catalyzing protein phosphorylation may not be the only function of protein kinases. Using the principle of hydrophobic spine assembly, we interpret how kinases are capable of performing a dual function in signaling. Its first role is that of a signaling enzyme (classical kinases; canonical), while its second role is that of an allosteric activator of other kinases or as a scaffold protein for signaling in a manner that is independent of phosphoryl transfer (classical pseudokinases; noncanonical). As the hydrophobic spines are a conserved feature of the kinase domain itself, all kinases carry an inherent potential to play both roles in signaling. This review focuses on the recent lessons from the RAF kinases that effectively toggle between these roles and can be “frozen” by introducing mutations at their hydrophobic spines.     

Imprint of evolutionary conservation and protein structure variation on the binding function of protein tyrosine kinases

MOTIVATION: According to the models of divergent molecular evolution, the evolvability of new protein function may depend on the induction of new phenotypic traits by a small number of mutations of the binding site residues. Evolutionary relationships between protein kinases are often employed to infer inhibitor binding profiles from sequence analysis. However, protein kinases binding profiles may display inhibitor selectivity within a given kinase subfamily, while exhibiting cross-activity between kinases that are phylogenetically remote from the prime target. The emerging insights into kinase function and evolution combined with a rapidly growing number of publically available crystal structures of protein kinases complexes have motivated structural bioinformatics analysis of sequence-structure relationships in determining the binding function of protein tyrosine kinases. RESULTS: In silico profiling of Imatinib mesylate and PD-173955 kinase inhibitors with protein tyrosine kinases is conducted on kinome scale by using evolutionary analysis and fingerprinting inhibitor-protein interactions with the panel of all publically available protein tyrosine kinases crystal structures. We have found that sequence plasticity of the binding site residues alone may not be sufficient to enable protein tyrosine kinases to readily evolve novel binding activities with inhibitors. While evolutionary signal derived solely from the tyrosine kinase sequence conservation can not be readily translated into the ligand binding phenotype, the proposed structural bioinformatics analysis can discriminate a functionally relevant kinase binding signal from a simple phylogenetic relationship. The results of this work reveal that protein conformational diversity is intimately linked with sequence plasticity of the binding site residues in achieving functional adaptability of protein kinases towards specific drug binding. This study offers a plausible molecular rationale to the experimental binding profiles of the studied kinase inhibitors and provides a theoretical basis for constructing functionally relevant kinase binding trees.     

The Ste20 group kinases as regulators of MAP kinase cascades

Ste20p (sterile 20 protein) is a putative yeast mitogen-activated protein kinase kinase kinase kinase (MAP4K) involved in the mating pathway. Its homologs in mammals, Drosophila, Caenorhabditis elegans and other organisms make up a large emerging group of protein kinases including 28 members in human. The Ste20 group kinases are further divided into the p21-activated kinase (PAK) and germinal center kinase (GCK) families. They are characterized by the presence of a conserved kinase domain and a noncatalytic region of great structural diversity that enables the kinases to interact with various signaling molecules and regulatory proteins of the cytoskeleton. This review describes the phylogenetic relationships of the Ste20 group kinases based on discussions with many researchers in this field. With the newly established phylogenetic relationships, crucial arguments can be advanced regarding the functions of these kinases as upstream activators of the MAPK pathways and possible activity as MAP4Ks. Their involvement in apoptosis, morphogenesis and cytoskeletal rearrangements is also discussed.     

Linking the kinome and phosphorylome--a comprehensive review of approaches to find kinase targets

Protein phosphorylation is associated with most cell signaling and developmental processes in eukaryotes. Despite the vast extent of the phosphoproteome within the cell, connecting specific kinases with relevant targets remains a significant experimental frontier. The challenge of linking kinases and their substrates reflects the complexity of kinase function. For example, kinases tend to exert their biological effects through supernumerary, redundant phosphorylation, often on multiple protein complex components. Although these types of phosphorylation events are biologically significant, those kinases responsible are often difficult to identify. Recent methods for global analysis of protein phosphorylation promise to substantially accelerate efforts to map the dynamic phosphorylome. Here, we review both conventional methods to identify kinase targets and more comprehensive genomic and proteomic approaches to connect the kinome and phosphorylome.     

Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics

The identification of dynamic protein phosphorylation events is critical for understanding kinase/phosphatase‐regulated signaling pathways. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Here we present a method to study the global kinome and phosphoproteome in tandem in a model photosynthetic organism, the alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry‐based label‐free proteomics. A dual enrichment strategy targets intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide‐based phosphorylation enrichment. To increase depth of coverage, both data‐dependent and data‐independent (via SWATH, Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) mass spectrometric acquisitions were performed to obtain a more than 50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative phosphoproteomic dataset yielded 2250 phosphopeptides and 1314 localized phosphosites with excellent reproducibility across biological replicates (90% of quantified sites with coefficient of variation below 11%). This approach enables simultaneous investigation of kinases and phosphorylation events at the global level to facilitate understanding of kinase networks and their influence in cell signaling events.     

Comprehensive analysis of kinase inhibitor selectivity

We tested the interaction of 72 kinase inhibitors with 442 kinases covering >80% of the human catalytic protein kinome. Our data show that, as a class, type II inhibitors are more selective than type I inhibitors, but that there are important exceptions to this trend. The data further illustrate that selective inhibitors have been developed against the majority of kinases targeted by the compounds tested. Analysis of the interaction patterns reveals a class of 'group-selective' inhibitors broadly active against a single subfamily of kinases, but selective outside that subfamily. The data set suggests compounds to use as tools to study kinases for which no dedicated inhibitors exist. It also provides a foundation for further exploring kinase inhibitor biology and toxicity, as well as for studying the structural basis of the observed interaction patterns. Our findings will help to realize the direct enabling potential of genomics for drug development and basic research about cellular signaling.     

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.     

Kinome analysis of receptor-induced phosphorylation in human natural killer cells

Background

Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.

Results

A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated.

Conclusions

The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.     

Evidence that Src-type tyrosine kinase activity is necessary for initiation of calcium release at fertilization in sea urchin eggs

The initiation of Ca(2+) release from internal stores in the egg is a hallmark of egg activation. In sea urchins, PLCgamma activity is necessary for the production of IP(3), which leads to the initial rise in Ca(2+). To examine the possible function of a tyrosine kinase in activating PLCgamma at fertilization, sea urchin eggs were treated with the specific Src kinase inhibitor PP1 or microinjected with recombinant Src-family SH2-domain proteins, which act as dominant interfering inhibitors of Src-family kinase function. Both modes of inhibiting Src-family kinases resulted in a specific and dose-dependent delay in the onset of Ca(2+) release from the endoplasmic reticulum at fertilization. The rise in cytoplasmic pH at fertilization also was inhibited by microinjection of Src-family SH2-domain proteins. Further, an antibody directed against Src-type kinases recognized a protein of ca. M(r) 57K that was enriched in the membrane fraction of eggs. The kinase activity of this protein was stimulated rapidly and transiently at fertilization, as measured by autophosphorylation and by phosphorylation of an exogenous substrate. Together, these data indicate that a Src-type tyrosine kinase is necessary for the initiation of Ca(2+) release from the egg ER at fertilization and identify a Src-type p57 protein as a candidate in the signaling pathway leading to this Ca(2+) release.     

SAD-A and AMPK kinases: The “yin and yang” regulators of mTORC1 signaling in pancreatic β cells

SAD-A kinase is a member of the AMPK-related family of kinases, which are under the control of LKB1 kinase. In the human kinome, SAD-A is most closely related to AMPK, a key energy sensor and master regulator of metabolism. In contrast to AMPK, little is known about the physiological function of the SAD-A kinase in metabolism. Recent studies using knockout mice have revealed a striking role of the SAD-A kinase in regulating dynamic functions of islet β cells, from glucose-stimulated insulin secretion (GSIS), islet β-cell size and mass, to GLP-1 response as the first tissue-specific effector of mTORC1 signaling. These studies suggest that SAD-A and AMPK kinase may function as the positive and negative regulators of mTORC1 signaling in islet β cells. Importantly, these findings have implicated SAD-A kinase as a novel drug target for the treatment of type 2 diabetes.