Showing papers on "Kinome published in 2008"

A quantitative analysis of kinase inhibitor selectivity.

Abstract: 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.

Type-II kinase inhibitor docking, screening, and profiling using modified structures of active kinase states.

Abstract: Type-II kinase inhibitors represent a class of chemicals that trap their target kinases in an inactive, so-called DFG-out state, occupying a hydrophobic pocket adjacent to the ATP binding site. These compounds are often more specific than those that target active DFG-in kinase conformations. Unfortunately, the discovery of novel type-II scaffolds presents a considerable challenge, partially because the lack of compatible kinase structures makes structure-based methods inapplicable. We present a computational protocol for converting multiple available DFG-in structures of various kinases (approximately 70% of mammalian structural kinome) into accurate and specific models of their type-II bound state. The models, described as deletion-of-loop Asp-Phe-Gly-in (DOLPHIN) kinase models, demonstrate exceptional performance in various inhibitor discovery applications, including compound pose prediction, screening, and in silico activity profiling. Given the abundance of the DFG-in structures, the presented approach opens possibilities for kinome-wide discovery of specific molecules targeting inactive kinase states.

Assessment of chemical coverage of kinome space and its implications for kinase drug discovery.

Abstract: More than 500 compounds chosen to represent kinase inhibitor space have been screened against a panel of over 200 protein kinases. Significant results include the identification of hits against new kinases including PIM1 and MPSK1, and the expansion of the inhibition profiles of several literature compounds. A detailed analysis of the data through the use of affinity fingerprints has produced findings with implications for biological target selection, the choice of tool compounds for target validation, and lead discovery and optimization. In a detailed examination of the tyrosine kinases, interesting relationships have been found between targets and compounds. Taken together, these results show how broad cross-profiling can provide important insights to assist kinase drug discovery.

Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis.

Abstract: An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H2O2-treated Arabido...

Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome

Abstract: Ser/Thr/Tyr kinases, which together comprise a major class of regulatory proteins in eukaryotes, were not believed to play an important role in prokaryotes until recently. However, our analysis of 626 prokaryotic genomes reveals that eukaryotic-like protein kinases (ELKs) are found in nearly two-thirds of the sequenced strains. We have identified 2697 ELKs, most of which are encoded by multicellular strains of the phyla Proteobacteria (Myxococcales), Actinobacteria, Cyanobacteria, and Chloroflexi, and 2 Acidobacteria and 1 Planctomycetes. Astonishingly, 7 myxobacterial strains together encode 892 ELKs, with 4 of the strains exhibiting a genomic ELK density similar to that observed in eukaryotes. Most myxobacterial ELKs show a modular organization in which the kinase domain is located at the N terminus. The C-terminal portion of the ELKs is highly diverse and often contains sequences with similarity to characterized domains, most of them involved in signaling mechanisms or in protein–protein interactions. However, many of these architectures are unique to the myxobacteria, an observation that suggests that this group exploits sophisticated and novel signal transduction systems. Phylogenetic reconstruction using the kinase domains revealed many orthologous sequence pairs and a huge number of gene duplications that probably occurred after speciation. Furthermore, studies of the microsynteny in the ELK-encoding regions reveal only low levels of synteny among Myxococcus xanthus, Plesiocystis pacifica, and Sorangium cellulosum. However, extensive similarities between M. xanthus, Stigmatella aurantiaca, and 3 Anaeromyxobacter strains were observed, indicating that they share regulatory pathways involving various ELKs.

Targeting the oncogene and kinome chaperone CDC37

Abstract: CDC37 is a molecular chaperone that physically stabilizes the catalytic domains found in protein kinases and is therefore a wide-spectrum regulator of protein phosphorylation. It is also an overexpressed oncoprotein that mediates carcinogenesis by stabilizing the compromised structures of mutant and/or overexpressed oncogenic kinases. Recent work shows that such dependency of malignant cells on increased CDC37 expression is a vulnerability that can be targeted in cancer by agents that deplete or inhibit CDC37. CDC37 is thus a candidate for broad-spectrum molecular cancer therapy.

Kinome-wide RNAi screen implicates at least 5 host hepatocyte kinases in Plasmodium sporozoite infection.

Abstract: Plasmodium sporozoites, the causative agent of malaria, are injected into their vertebrate host through the bite of an infected Anopheles mosquito, homing to the liver where they invade hepatocytes to proliferate and develop into merozoites that, upon reaching the bloodstream, give rise to the clinical phase of infection. To investigate how host cell signal transduction pathways affect hepatocyte infection, we used RNAi to systematically test the entire kinome and associated genes in human Huh7 hepatoma cells for their potential roles during infection by P. berghei sporozoites. The three-phase screen covered 727 genes, which were tested with a total of 2,307 individual siRNAs using an automated microscopy assay to quantify infection rates and qRT-PCR to assess silencing levels. Five protein kinases thereby emerged as top hits, all of which caused significant reductions in infection when silenced by RNAi. Follow-up validation experiments on one of these hits, PKCς (PKCzeta), confirmed the physiological relevance of our findings by reproducing the inhibitory effect on P. berghei infection in adult mice treated systemically with liposome-formulated PKCς-targeting siRNAs. Additional cell-based analyses using a pseudo-substrate inhibitor of PKCς added further RNAi-independent support, indicating a role for host PKCς on the invasion of hepatocytes by sporozoites. This study represents the first comprehensive, functional genomics-driven identification of novel host factors involved in Plasmodium sporozoite infection.

Kinome siRNA Screen Identifies Regulators of Ciliogenesis and Hedgehog Signal Transduction

Abstract: Disruption or improper activation of the Hedgehog (Hh) pathway is associated with developmental abnormalities and cancer Although characterized in Drosophila, the mechanisms that mediate the Hh signal downstream of the Smoothened (Smo) seven-transmembrane protein in vertebrates remain poorly understood In particular, the Fused (Fu) kinase, which mediates Hh signaling in flies, is dispensable in mammals To identify kinases that positively regulate the Hh pathway in mammals, we screened a mouse kinome small interfering RNA library and validated nine candidates that modulate Hh signaling Among these candidates, Nek1 and Prkra did not directly function in the Hh pathway but exerted their effects on Hh signaling indirectly through a primary role in ciliogenesis In contrast, another kinase, Cdc2l1, directly participated in the Hh pathway Cdc2l1 was necessary and sufficient for activation of the Hh pathway, functioning downstream of Smo and upstream of the Glioma-associated (Gli) transcription factors More specifically, Cdc2l1 interacted with the negative regulator Suppressor of Fused (Sufu) and relieved its inhibition on Gli, thus providing a mechanism for how Cdc2l1 might play a role in Hh signaling Finally, with zebrafish as model organism, we showed that Cdc2l1 activated the Hh pathway in vivo We propose that Cdc2l1 is a previously unrecognized member of the Hh signal transduction cascade

Chapter 6 Mechanisms Regulating the Susceptibility of Hematopoietic Malignancies to Glucocorticoid‐Induced Apoptosis

Abstract: Glucocorticoids (GCs) are commonly used in the treatment of hematopoietic malignancies owing to their ability to induce apoptosis of these cancerous cells. Whereas some types of lymphoma and leukemia respond well to this drug, others are resistant. Also, GC‐resistance gradually develops upon repeated treatments ultimately leading to refractory relapsed disease. Understanding the mechanisms regulating GC‐induced apoptosis is therefore uttermost important for designing novel treatment strategies that overcome GC‐resistance. This review discusses updated data describing the complex regulation of the cell's susceptibility to apoptosis triggered by GCs. We address both the genomic and nongenomic effects involved in promoting the apoptotic signals as well as the resistance mechanisms opposing these signals. Eventually we address potential strategies of clinical relevance that sensitize GC‐resistant lymphoma and leukemia cells to this drug. The major target is the nongenomic signal transduction machinery where the interplay between protein kinases determines the cell fate. Shifting the balance of the kinome towards a state where Glycogen synthase kinase 3α (GSK3α) is kept active, favors an apoptotic response. Accumulating data show that it is possible to therapeutically modulate GC‐resistance in patients, thereby improving the response to GC therapy.

Platelet Protein Interactions Map, Signaling Components, and Phosphorylation Groundstate

Abstract: Objectives— Assembly of a comprehensive proteome and transcriptome database of human platelets, derivation of a model of the platelet-specific interactome, and generation of a functional interaction map of platelet phosphorylations and kinases. Methods and Results— Interactions are derived from literature-curated data from HPRD and yeast two hybrid (Y2H) and mapped to platelet-specific expression data (SAGE or proteome). From this a cell-type specific model of platelet proteins and protein–protein interactions is derived. The obtained inventory of platelet-specific proteins includes key domains, protein GO annotations, and receptors. Collected interactions point to new platelet signaling components, actin remodeling processes, and pharmacological targets and offer incentives for further studies (eg, on the IPP complex). Integration of platelet-specific phosphoproteins and the characterization of the platelet kinase repertoire sketch a first outline of kinase signaling in human platelets. Conclusions— A first view of the platelet interactome, platelet phosphorylation, and platelet kinome is available from the in silico data.

WNK Kinases and Renal Sodium Transport in Health and Disease. An Integrated View

Abstract: The with no lysine (WNK) kinases comprise a novel branch of the human kinome that plays a central role in regulating renal sodium, potassium, and chloride transport, and, therefore, blood pressure. Mutations of two WNK kinases, WNK1 and WNK4, cause familial hyperkalemic hypertension (Gordon’s syndrome or Type II pseudohypoaldosteronism), a rare monogenic disease. Many aspects of WNK action have been elucidated during the past seven years. WNKs are all expressed along a short segment of renal distal tubule, where they modulate the activity of a wide variety of transport proteins. These diverse effects, however, make it difficult to describe an integrated model of WNK function within the kidney. Recently, work in vivo and in vitro has begun to clarify this picture. The present review emphasizes recent insights into mechanism by which WNK kinases interact to modulate sodium and potassium transport along the aldosterone-sensitive distal nephron. We describe a potential mechanism by which WNK4 mutations convert the action of WNK4 from inhibiting renal sodium chloride retention to stimulating it, thereby affecting both blood pressure and potassium balance. An explanation for how WNK kinases can alter the effects of aldosterone from primarily kaliuretic to primarily sodium chloride retentive, according to physiological need, is also described.

Analysis of the Yeast Kinome Reveals a Network of Regulated Protein Localization during Filamentous Growth

Abstract: The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling modules, wherein yeast cells form interconnected and elongated chains. Because standard strains of yeast are nonfilamentous, we constructed a unique set of 125 kinase-yellow fluorescent protein chimeras in the filamentous Sigma1278b strain for this study. In total, we identified six cytoplasmic kinases (Bcy1p, Fus3p, Ksp1p, Kss1p, Sks1p, and Tpk2p) that localize predominantly to the nucleus during filamentous growth. These kinases form part of an interdependent, localization-based regulatory network: deletion of each individual kinase, or loss of kinase activity, disrupts the nuclear translocation of at least two other kinases. In particular, this study highlights a previously unknown function for the kinase Ksp1p, indicating the essentiality of its nuclear translocation during yeast filamentous growth. Thus, the localization of Ksp1p and the other kinases identified here is tightly controlled during filamentous growth, representing an overlooked regulatory component of this stress response.

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

Abstract: 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 proteinphosphorylation 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.

Analysis of the protein kinome of Entamoeba histolytica.

Abstract: Protein kinases play important roles in almost all major gnaling 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 rticle,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 cyclindependent 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.

Molecular basis for specificity in the druggable kinome

Abstract: MOTIVATION Rational design of kinase inhibitors remains a challenge partly because there is no clear delineation of the molecular features that direct the pharmacological impact towards clinically relevant targets. Standard factors governing ligand affinity, such as potential for intermolecular hydrophobic interactions or for intermolecular hydrogen bonding do not provide good markers to assess cross reactivity. Thus, a core question in the informatics of drug design is what type of molecular similarity among targets promotes promiscuity and what type of molecular difference governs specificity. This work answers the question for a sizable screened sample of the human pharmacokinome including targets with unreported structure. RESULTS We show that drug design aimed at promoting pairwise interactions between ligand and kinase target actually fosters promiscuity because of the high conservation of the partner groups on or around the ATP-binding site of the kinase. Alternatively, we focus on a structural marker that may be reliably determined from sequence and measures dehydration propensities mostly localized on the loopy regions of kinases. Based on this marker, we construct a sequence-based kinase classifier that enables the accurate prediction of pharmacological differences. Our indicator is a microenvironmental descriptor that quantifies the propensity for water exclusion around preformed polar pairs. The results suggest that targeting polar dehydration patterns heralds a new generation of drugs that enable a tighter control of specificity than designs aimed at promoting ligand-kinase pairwise interactions. AVAILABILITY The predictor of polar hot spots for dehydration propensity, or solvent-accessible hydrogen bonds in soluble proteins, named YAPView, may be freely downloaded from the University of Chicago website http://protlib.uchicago.edu/dloads.html. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Identification of novel substrates for Cdk5 and new targets for Cdk5 inhibitors using high-density protein microarrays.

Abstract: Cyclin-dependent kinase (Cdk) 5 is a serine/threonine kinase that plays an important role during CNS development and its dysregulation is causally involved in the process of neuronal degeneration. To date more than 20 Cdk5 substrates have been identified and the number of Cdk5 substrates is still increasing. The different cellular functions of Cdk5 and its substrates are not completely known at present. High-throughput protein microarray technology is a powerful tool to identify a large number of potential kinase substrates in parallel under the same experimental conditions. Using Protoarray protein microarrays we identified protein phosphatase 1, regulatory subunit 14A (PPP1R14A) as a novel substrate of Cdk5/p25. Phosphorylation was confirmed in two secondary assays. Our findings may contribute to the elucidation of the physiological function of Cdk5 in synaptic signalling. Functional Kinome Arrays were validated in a second set of experiments to characterize the selectivity of the Cdk5 inhibitor indolinone A. This lead to the identification of two additional kinases that are targeted by this compound and may provide a deeper understanding of its neuroprotective mode of action. However, several false negative results possibly due to a denatured or inactive conformation of the arrayed proteins, sound a note of caution when using protein array techniques.

Targeting the unactivated conformations of protein kinases for small molecule drug discovery.

Abstract: Background: The number of drugs in active clinical development or on the market that target the unactivated conformational states of protein kinases is growing and represents a significant portion of kinase research at biopharmaceutical companies. These non-classical kinase inhibitors have a mode of action which may overcome some of the liabilities of classical ATP-site inhibitors that substantially overlap the space that ATP occupies in the activated kinase. Objective: This review will discuss state-of-the-art methods of inhibiting protein kinases by targeting the unactivated conformations of the enzyme with small molecules directed to the ATP binding region. Methods: Biochemical and structural biology publications and public domain crystal structures were evaluated to identify key concepts in drug discovery for unactivated protein kinase inhibitors that target the ATP binding region. Conclusion: The potential for enhanced selectivity, potency and duration of pharmacological action may allow non-classica...

Supplementary Materials for Kinome siRNA Screen Identifies Regulators of Ciliogenesis and Hedgehog Signal Transduction

Abstract: Cdc2l1is a component of the Hh signaling pathway and opposes the activity of the negative regulator Sufu. Modulating Hedgehog Signals Signaling initiated by Hedgehog (Hh) ligands is critical to tissue patterning in both vertebrates and invertebrates; however, the specific downstream mechanisms are distinct. For instance, Fused, a kinase crucial to fly Hh signaling, does not appear to be essential to Hh signaling in mammals. Evangelista et al. screened a mouse kinome small interfering RNA library and used stringent criteria to validate candidates for genes encoding proteins that enhance Hh signaling. Two of these, Nek1 and Prkra, appeared to regulate Hh signaling indirectly by affecting primary cilia formation. The kinase Cdc2l1, however, interacted with the negative regulator of Hh signaling Suppressor of Fused, thereby relieving its inhibition of the Glioma-associated family of transcription factors, suggesting that Cdc2l1 plays a direct role in the Hh signal transduction cascade in vertebrates. Disruption or improper activation of the Hedgehog (Hh) pathway is associated with developmental abnormalities and cancer. Although characterized in Drosophila, the mechanisms that mediate the Hh signal downstream of the Smoothened (Smo) seven-transmembrane protein in vertebrates remain poorly understood. In particular, the Fused (Fu) kinase, which mediates Hh signaling in flies, is dispensable in mammals. To identify kinases that positively regulate the Hh pathway in mammals, we screened a mouse kinome small interfering RNA library and validated nine candidates that modulate Hh signaling. Among these candidates, Nek1 and Prkra did not directly function in the Hh pathway but exerted their effects on Hh signaling indirectly through a primary role in ciliogenesis. In contrast, another kinase, Cdc2l1, directly participated in the Hh pathway. Cdc2l1 was necessary and sufficient for activation of the Hh pathway, functioning downstream of Smo and upstream of the Glioma-associated (Gli) transcription factors. More specifically, Cdc2l1 interacted with the negative regulator Suppressor of Fused (Sufu) and relieved its inhibition on Gli, thus providing a mechanism for how Cdc2l1 might play a role in Hh signaling. Finally, with zebrafish as model organism, we showed that Cdc2l1 activated the Hh pathway in vivo. We propose that Cdc2l1 is a previously unrecognized member of the Hh signal transduction cascade.

Analysis of the Human Kinome Using Methods Including Fold Recognition Reveals Two Novel Kinases

Abstract: Author(s): Briedis, Kristine M; Starr, Ayelet; Bourne, Philip E | Abstract: BackgroundProtein sequence similarity is a commonly used criterion for inferring the unknown function of a protein from a protein of known function. However, proteins can diverge significantly over time such that sequence similarity is difficult, if not impossible, to find. In some cases, a structural similarity remains over long evolutionary time scales and once detected can be used to predict function.Methodology/principal findingsHere we employed a high-throughput approach to assign structural and functional annotation to the human proteome, focusing on the collection of human protein kinases, the human kinome. We compared human protein sequences to a library of domains from known structures using WU-BLAST, PSI-BLAST, and 123D. This approach utilized both sequence comparison and fold recognition methods. The resulting set of potential protein kinases was cross-checked against previously identified human protein kinases, and analyzed for conserved kinase motifs.Conclusions/significanceWe demonstrate that our structure-based method can be used to identify both typical and atypical human protein kinases. We also identify two potentially novel kinases that contain an interesting combination of kinase and acyl-CoA dehydrogenase domains.

In silico drug profiling of the human kinome based on a molecular marker for cross reactivity.

Abstract: Protein kinases are paradigmatic targets in molecular cancer therapy. Affinity profiles of kinase drug inhibitors are of considerable interest to assess and modulate clinical drug impact. In the initial stages of discovery, a thorough experimental screening of lead compound libraries becomes critically limited by the size of the kinase sample. This work introduces a computational screening approach which provides a tool for extensive screening that uses experimentally obtained small-scale profiles as input data and makes predictions for a larger kinase set. These predictions result from a propagation of the reduced profile, exploiting a structural comparison of kinases based on a feature-similarity matrix. The comparison focuses on a molecular marker for specificity and promiscuity of kinase inhibitors. Our approach enables the computational high-throughput screening of entire libraries of compounds to search for suitable leads, mapping their inhibitory impact on a sizable sample of the human kinome. Our in silico tool is validated by contrasting predictions against reported high-throughput screening experiments.

[The kinome and glucocorticoid-induced apoptosis].

Abstract: In this perspective, I discuss the complex interplay between GC signaling and the kinome that ultimately determines the cell fate after GC treatment. Apoptosis ensues when the cell express sufficient levels of GR and Bim together with a kinome favoring GSK3 activation. Protein kinases that prevent Bim up-regulation and/or inhibit GSK3, confer GC-resistance on the cell. GC-resistance may be overcome in T and B lymphoid malignancies by inhibiting the JNK, Src, PI3K, Akt or mTOR survival pathways. Both staurosporine and rapamycin have recently been proved efficient to sensitize resistant T and B malignant cells to GC-induced apoptosis. This is a proof-of-principle that it is possible to improve GC therapy by altering the cell's kinome.

The distribution and evolution of protein kinase and phosphatase families in the three superkingdoms of life

Abstract: Protein phosphorylation and dephosphorylation plays a critical role in the regulation of many important cellular processes. The protein families responsible for this, the kinases and phosphatases, have been the focus of enormous amounts of research. However, our knowledge of these families is in many respects still incomplete, as prior studies have oftentimes focused only on humans and other higher eukaryotes. The advent of the genome sequencing era now allows us to examine these protein families on a more global scale. I present here a study of protein kinase and phosphatase families in 115 completely sequenced genomes. This is an important contribution towards understanding not only which families are present in different lineages, but also how the evolution of these families relates to each other. In chapter 2, I define the human kinome using a method called iGAP. This method combines sequence similarity and fold recognition methods to annotate proteins. I searched the human proteome for members of the eukaryotic protein kinase-like superfamily and identified two novel putative kinases. In subsequent chapters, I extend this focus to include phosphatases and other genomes in the Eukarya, Bacteria and Archaea superkingdoms. Chapter 3 is centered on phosphatases. I built profile hidden Markov models of known phosphatase families and searched 115 complete proteomes for the presence or absence of these families. I define which genomes and lineages contain particular families and discuss what we can learn about the evolution of the phosphatase families. In chapter 4, I present a similar study of the kinases. I built models for microbial and eukaryotic kinase families and searched the same 115 proteomes for the presence or absence of the kinase families. I report here the results and discuss the evolutionary implications, incorporating past sequence and structure-based research of the evolution of the protein kinase-like superfamily. Chapter 5 compares and contrasts the evolutionary patterns of protein kinase and phosphatase families that target either the same substrate or each other. I report the presence or absence of these families in the aforementioned species. I then compare the phylogenetic profiles of these families and discuss how the evolution of each family relates to the other

Tony Hunter: Kinase king

Abstract: Before 1979, kinases were only thought to stick phosphates on two of the twenty amino acids: serine and threonine. But then Tony Hunter discovered that tyrosine could also be phosphorylated (1, 2), thereby uncovering an entirely new mechanism of protein regulation in cells. Since then, Hunter has worked on all sorts of protein phosphorylation events and the kinases that deliver them. Indeed, he has been instrumental in deducing the human kinome (3, 4).