KinMutBase, a database of human disease-causing protein kinase mutations

KinMutBase (http://www.uta.fi/imt/bioinfo/KinMutBase/) is a registry of mutations in human protein kinases related to disorders. Kinases are essential cellular signaling molecules, in which mutations can lead to diseases, including immunodeficiencies, cancers and endocrine disorders. The first release of KinMutBase contained information for protein tyrosine kinases. The current release includes also serine/threonine protein kinases, as well as an update of the tyrosine kinases. There are 251 entries altogether, representing 337 families and 621 patients. Mutations appear both in conserved hallmark residues of the kinases as well as in non-homologous sites. The KinMutBase WWW pages provide plenty of information, namely mutation statistics and display, clickable sequences with mutations and changes to restriction enzyme patterns.     

Exploring Missense Mutations in Tyrosine Kinases Implicated with Neurodegeneration

Protein kinases are one of the largest families of evolutionarily related proteins and the third most common protein class of human genome. All the protein kinases share the same structural organization. They are made up of an extracellular domain, transmembrane domain and an intra cellular kinase domain. Missense mutations in these kinases have been studied extensively and correlated with various neurological disorders. Individual mutations in the kinase domain affect the functions of protein. The enhanced or reduced expression of protein leads to hyperactivation or inactivation of the signalling pathways, resulting in neurodegeneration. Here, we present extensive analyses of missense mutations in the tyrosine kinase focussing on the neurodegenerative diseases encompassing structure function relationship. This is envisaged to enhance our understanding about the neurodegeneration and possible therapeutic measures.     

ProKinO: an ontology for integrative analysis of protein kinases in cancer

Background

Protein kinases are a large and diverse family of enzymes that are genomically altered in many human cancers. Targeted cancer genome sequencing efforts have unveiled the mutational profiles of protein kinase genes from many different cancer types. While mutational data on protein kinases is currently catalogued in various databases, integration of mutation data with other forms of data on protein kinases such as sequence, structure, function and pathway is necessary to identify and characterize key cancer causing mutations. Integrative analysis of protein kinase data, however, is a challenge because of the disparate nature of protein kinase data sources and data formats.

Results

Here, we describe ProKinO, a protein kinase-specific ontology, which provides a controlled vocabulary of terms, their hierarchy, and relationships unifying sequence, structure, function, mutation and pathway information on protein kinases. The conceptual representation of such diverse forms of information in one place not only allows rapid discovery of significant information related to a specific protein kinase, but also enables large-scale integrative analysis of protein kinase data in ways not possible through other kinase-specific resources. We have performed several integrative analyses of ProKinO data and, as an example, found that a large number of somatic mutations (∼288 distinct mutations) associated with the haematopoietic neoplasm cancer type map to only 8 kinases in the human kinome. This is in contrast to glioma, where the mutations are spread over 82 distinct kinases. We also provide examples of how ontology-based data analysis can be used to generate testable hypotheses regarding cancer mutations.

Conclusion

We present an integrated framework for large-scale integrative analysis of protein kinase data. Navigation and analysis of ontology data can be performed using the ontology browser available at: http://vulcan.cs.uga.edu/prokino.     

Small molecule modulators of eukaryotic initiation factor 2α kinases,the key regulators of protein synthesis

Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.     

Mice lacking both mixed-lineage kinase genes Mlk1 and Mlk2 retain a wild type phenotype

The mitogen-activated protein kinase kinase kinases of the mixed-lineage kinase (MLK) family have been shown to activate the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway, and to regulate the other two principal MAPK cascades, p38 and extracellular signal-regulated kinase (ERK). Although there is growing evidence for their involvement in neuronal cell death leading to neurodegenerative disorders, little in vivo data is available for the members of this family of kinases. Here, we report that the inactivation of mouse Mlk1 and Mlk2 genes. Mlk1-/- and Mlk2-/- mice were found to be viable and healthy. Surprisingly, mice carrying the compound Mlk1/Mlk2 null mutations were also found to be viable, fertile and to have a normal life span. The nervous system, testis and kidney, the major sites of MLK1 and 2 expression, all appear normal, as do other organs where these kinases were found to be more weakly expressed. Surprisingly, developmental neuronal programmed cell death, another potential target for MLK family members, was also found to be unaffected. Our results suggest that there is extensive functional redundancy between MLK1/MLK2 and the other member of the family, MLK3, which is also not required for survival in mouse.     

Assessing the Subcellular Dynamics of Alpha-synuclein Using Photoactivation Microscopy

Alpha-synuclein (aSyn) is implicated in Parkinson’s disease and several other neurodegenerative disorders. To date, the function and intracellular dynamics of aSyn are still unclear. Here, we tracked the dynamics of aSyn using photoactivatable green fluorescent protein as a reporter. We found that the availability of the aSyn N terminus modulates its shuttling into the nucleus. Interestingly, familial aSyn mutations altered the dynamics at which the protein distributes throughout the cell. Both the A30P and A53T aSyn mutations increase the speed at which the protein moves between the nucleus and cytoplasm, respectively. We also found that specific kinases potentiate the shuttling of aSyn between nucleus and cytoplasm. A mutant aSyn form that blocks S129 phosphorylation, S129A, results in the formation of cytoplasmic inclusions, suggesting phosphorylation modulates aggregation in addition to modulating aSyn intracellular dynamics. Finally, we found that the molecular chaperone HSP70 accelerates the entry of aSyn into the nuclear compartment.     

Phosphorylation of microtubule-associated protein tau by AMPK-related kinases

Microtubule-associated protein tau is abnormally hyperphosphorylated in the intracellular filamentous inclusions seen in neurodegenerative disorders with dementia, such as Alzheimer's disease and other tauopathies. Microtubule-associated protein/microtubule-affinity regulating kinases (MARKs) have previously been identified as kinases which phosphorylate KxGS motifs in the tandem repeats of tau. They are members of the 5'-AMP-activated protein kinase (AMPK)-related kinases in the Ca(2+)/calmodulin-dependent protein kinase group. In this study, we examined the ability of AMPK-related kinases, brain-specific kinases 1 and 2, maternal embryonic leucine-zipper kinase, MARK1, and salt-inducible kinase (SIK), to phosphorylate tau. We found that they phosphorylated S262 and S356 in KxGS motifs in the repeats of tau, thus resulting in immunoreactivity with antibody 12E8. MARK1 and SIK most effectively phosphorylated tau, and their down-regulation resulted in a reduction of 12E8-labelling. BX 795, an inhibitor of MARK1 and SIK, reduced 12E8-immunolabelling of tau in rat cortical neurons. These findings reveal a significant contribution of AMPK-related kinases to the phosphorylation of tau at S262/S356.     

A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi

Two independent quorum-sensing systems control the expression of bioluminescence (lux) in the marine bacterium Vibrio harveyi. Each system is composed of an autoinducer (AI-1 or AI-2) and its cognate sensor (LuxN or LuxQ). The sensors are two-component hybrid kinases, containing both sensor kinase domains and response regulator domains. Sensory information from the two systems is relayed by a phosphotransfer mechanism to a shared integrator protein called LuxO. LuxO is a member of the response regulator class of the two-component family of signal transduction proteins, and LuxO acts negatively to control luminescence. In this report, missense and in frame deletion mutations were constructed in luxO that encoded proteins mimicking either the phosphorylated or the unphosphorylated form, and these mutations were introduced into the V. harveyi chromosome at the luxO locus. Phenotypical analyses of the resulting mutant V. harveyi strains indicate that the phosphorylated form of LuxO is the repressor, and that the unphosphorylated form of the protein is inactive. Analysis of the lux phenotypes of V. harveyi strains containing single and double luxN and luxQ mutations indicate that LuxN and LuxQ have two activities on LuxO. They act as LuxO protein kinases at low cell density in the absence of autoinducers, and they switch to LuxO protein phosphatases at high cell density in the presence of autoinducers. Furthermore, the timing and potency of inputs from the two systems into regulation of quorum sensing are different.     

Phosphorylation of microtubule-associated protein tau by isoforms of c-Jun N-terminal kinase (JNK)

Microtubule-associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases commonly referred to as tauopathies. Hyperphosphorylation of tau at most sites appears to precede filament assembly. Many of the hyperphosphorylated sites are serine/threonine-proline sequences. Here we show that c-Jun N-terminal kinases JNK1, JNK2 and JNK3 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Of the three protein kinases, JNK2 phosphorylated the most sites in tau, followed by JNK3 and JNK1. Phosphorylation by JNK isoforms resulted in a greatly reduced ability of tau to promote microtubule assembly. These findings extend the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.     

Targeting phosphatases as the next generation of disease modifying therapeutics for Parkinson’s disease

Phosphorylation is a key post-translational modification for cellular signaling, and abnormalities in this process are observed in several neurodegenerative disorders. Among these disorders, Parkinson’s disease (PD) is particularly intriguing as there are both genetic causes of disease that involve phosphorylation, and pathological hallmarks of disease composed of a hyperphosphorylated protein. Two of the major genes linked to PD are themselves kinases – leucine rich repeat kinase 2 (LRRK2) and phosphatase and tensin induced homolog kinase 1 (PINK1). Mutations in LRRK2 lead to its increased kinase activity and dominantly inherited PD, while mutations in PINK1 lead to loss of function and recessive PD. A third genetic linkage to disease is α-synuclein, a protein that is heavily phosphorylated in Lewy bodies and Lewy neurites, the pathological hallmarks of PD. The phosphorylation of α-synuclein at various residues influences its aggregation, either positively or negatively, thereby impacting its central role in disease pathogenesis. Given these associations of phosphorylation with PD, modulation of this modification is an attractive therapeutic strategy. The kinases that act in these disease relevant pathways have been the primary target for such approaches. But, the development of kinase inhibitors has been complicated by the necessary specificity to retain safety, the redundancy of kinases leading to lack of efficacy, and the difficulties in overcoming the blood–brain barrier. The field of modulating phosphatases has the potential to overcome some of these issues and provide the next generation of therapeutic targets for PD. In this review, we address the phosphorylation pathways involved in PD, the kinases and issues related to their inhibition, and the evolving field of the phosphatases relevant in PD and how they may be targeted pharmacologically.     

Protein kinases: Signaling molecules controlling ovarian functions

The present focus survey represents a review of current knowledge concerning involvement of protein kinases in control of basic ovarian functions in mammals. Ovarian cells produce a number of protein kinases, whose expression depends on type of cells, their state and action of hormones and other protein kinases. A number of protein kinases are involved in control of ovarian cell proliferation, apoptosis, oocyte maturation, hormone release, reception and response to hormones, as well as in mediating action of hormones on these ovarian functions. Protein kinases and their regulators could be used for characterization, prediction and control of ovarian folliculogenesis and atresia, corpus luteum functions, oocyte maturation, fertility, release of hormones, response of ovarian structures to hormonal regulators, as well as for treatment of some reproductive disorders.     

Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1

Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2 small middle dotMEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding alpha-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding alpha-helix G in the interaction of ERK2 with MEK1/2.     

Cancer‐associated mutations are preferentially distributed in protein kinase functional sites

Protein kinases are a superfamily involved in many crucial cellular processes, including signal transmission and regulation of cell cycle. As a consequence of this role, kinases have been reported to be associated with many types of cancer and are considered as potential therapeutic targets. We analyzed the distribution of pathogenic somatic point mutations (drivers) in the protein kinase superfamily with respect to their location in the protein, such as in structural, evolutionary, and functionally relevant regions. We find these driver mutations are more clearly associated with key protein features than other somatic mutations (passengers) that have not been directly linked to tumor progression. This observation fits well with the expected implication of the alterations in protein kinase function in cancer pathogenicity. To explain the relevance of the detected association of cancer driver mutations at the molecular level in the human kinome, we compare these with genetically inherited mutations (SNPs). We find that the subset of nonsynonymous SNPs that are associated to disease, but sufficiently mild to the point of being widespread in the population, tend to avoid those key protein regions, where they could be more detrimental for protein function. This tendency contrasts with the one detected for cancer associated‐driver‐mutations, which seems to be more directly implicated in the alteration of protein function. The detailed analysis of protein kinase groups and a number of relevant examples, confirm the relation between cancer associated‐driver‐mutations and key regions for protein kinase structure and function. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Tyrosine phosphatases as a superfamily of tumor suppressors in colorectal cancer

Phosphorylation and dephosphorylation processes catalyzed by numerous kinases and phosphorylases are essential for cell homeostasis and may lead to disturbances in a variety of vital cellular pathways, such as cell proliferation and differentiation, and thus to complex diseases including cancer. As over 80 % of all oncogenes encode protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), which can reverse the effects of tyrosine kinases, are very important tumor suppressors. Alterations in tyrosine kinase and phosphatase genes including point mutations, changes in epigenetic regulation, as well as chromosomal aberrations involving regions critical to these genes, are frequently observed in a variety of cancers. Colorectal cancer (CRC) is one of the most common cancers in humans. CRCs occur in a familial (about 15 % of all cases), hereditary (about 5%) and sporadic (almost 75-80 %) form. As genetic-environmental interrelations play an important role in the susceptibility to sporadic forms of CRCs, many studies are focused on genetic alterations in such tumors. Mutational analysis of the tyrosine phosphatome in CRCs has identified somatic mutations in PTPRG, PTPRT, PTPN3, PTPN13 and PTPN14. The majority of these mutations result in a loss of protein function. Also, alterations in the expression of these genes, such as decreased expression of PTPRR, PTPRO, PTPRG and PTPRD, mediated by epigenetic mechanisms have been observed in a variety of tumors. Since cancer is a social and global problem, there will be a growing number of studies on alterations in the candidate cancer genes, including protein kinases and phosphatases, to determine the origin, biology and potential pathways for targeted anticancer therapy.     

Protein kinases and ovarian functions

The present focus survey represents a short review of current knowledge concerning involvement of protein kinases in control of basic ovarian functions. Ovarian cells produce a number of protein kinases, whose expression depends on type of cells, their state and action of hormones and other protein kinases. A number of protein kinases are involved in control of ovarian cell proliferation, apoptosis, oocyte maturation, hormone release, reception and response to hormones, as well as in mediating action of hormones on these ovarian functions. Complexity of interrelationships between different protein kinase‐dependent signaling pathways occurs. Protein kinases and their regulators could be used for characterization, prediction and control of ovarian folliculogenesis and atresia, Corpus luteum functions, oocyte maturation, fertility, release of hormones, response of ovarian structures to hormonal regulators, as well as for treatment of some reproductive disorders. The present data demonstrate importance of protein kinases in control of basic ovarian function and potential usage of protein kinases for characterization, prediction and control of these functions. J. Cell. Physiol. 226: 37–45, 2010. © 2010 Wiley‐Liss, Inc.     

Regulation of vascular endothelial cell barrier function and cytoskeleton structure by protein phosphatases of the PPP family

Reversible phosphorylation of cytoskeletal and cytoskeleton-associated proteins is a significant element of endothelial barrier function regulation. Therefore, understanding the mechanisms of phosphorylation/dephosphorylation of endothelial cell cytoskeletal proteins is vital to the treatment of severe lung disorders such as high permeability pulmonary edema. In vivo, there is a controlled balance between the activities of protein kinases and phosphatases. Due to various external or internal signals, this balance may be shifted. The actual balances at a given time alter the phosphorylation level of certain proteins with appropriate physiological consequences. The latest information about the structure and regulation of different types of Ser/Thr protein phosphatases participating in the regulation of endothelial cytoskeletal organization and barrier function will be reviewed here.     

Familial hypercholesterolaemia

Familial hypercholesterolaemia (FH), defined as the heritable occurrence of severe hypercholesterolaemia with cholesterol deposits in tendons and premature heart disease, is caused by at least four genes in sterol and lipoprotein pathways and displays varying gene-dose effects. The genes are the low-density lipoprotein (LDL) receptor, apolipoprotein (apo) B, proprotein convertase subtilisin/kexin 9, and the autosomal recessive hypercholesterolaemia (ARH) adaptor protein. All of these disorders have in common defective clearance of LDL within a complex system of lipid and lipoprotein metabolism and regulation. Normal cellular cholesterol and lipoprotein metabolism is reviewed before describing the disorders, their metabolic derangements and their clinical effects. FH is classified as two simplified phenotypes of disease according to the severity of the metabolic derangement. The dominantly inherited heterozygous phenotype comprises defects in the LDL receptor, apoB100, and neural apoptosis regulatory cleavage protein. The homozygous phenotype is co-dominant in defects of the LDL receptor, and occurs also as the ARH of adapter protein mutations. Defective binding of apoB100 does not result in a significant gene dose effect, but enhances the severity of heterozygotes for LDL receptor mutations. The genetic diagnosis of FH has provided greater accuracy in definition and detection of disease and exposes information about migration of populations. All of these disorders pose a high risk of atherosclerosis, especially in the homozygous phenotype. Studies of influences on the phenotype and responses to treatment are also discussed in the context of the metabolic derangements.     

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.     

AGC protein kinases: From structural mechanism of regulation to allosteric drug development for the treatment of human diseases

The group of AGC protein kinases includes more than 60 protein kinases in the human genome, classified into 14 families: PDK1, AKT/PKB, SGK, PKA, PKG, PKC, PKN/PRK, RSK, NDR, MAST, YANK, DMPK, GRK and SGK494. This group is also widely represented in other eukaryotes, including causative organisms of human infectious diseases. AGC kinases are involved in diverse cellular functions and are potential targets for the treatment of human diseases such as cancer, diabetes, obesity, neurological disorders, inflammation and viral infections. Small molecule inhibitors of AGC kinases may also have potential as novel therapeutic approaches against infectious organisms. Fundamental in the regulation of many AGC kinases is a regulatory site termed the “PIF-pocket” that serves as a docking site for substrates of PDK1. This site is also essential to the mechanism of activation of AGC kinases by phosphorylation and is involved in the allosteric regulation of N-terminal domains of several AGC kinases, such as PKN/PRKs and atypical PKCs. In addition, the C-terminal tail and its interaction with the PIF-pocket are involved in the dimerization of the DMPK family of kinases and may explain the molecular mechanism of allosteric activation of GRKs by GPCR substrates. In this review, we briefly introduce the AGC kinases and their known roles in physiology and disease and the discovery of the PIF-pocket as a regulatory site in AGC kinases. Finally, we summarize the current status and future therapeutic potential of small molecules directed to the PIF-pocket; these molecules can allosterically activate or inhibit the kinase as well as act as substrate-selective inhibitors. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).