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

KinMutBase (http://www.uta.fi/laitokset/imt/KinMut Base.html) is a registry of mutations in human protein kinases related to disorders. Kinases are essential cellular signalling molecules, in which mutations can lead into diseases including, e.g., immunodeficiencies, cancers and endocrine disorders. The first release of KinMutBase contains information for nine protein tyrosine kinases. There are altogether 170 entries representing 273 families and 403 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, restriction enzyme patterns and online submission.     

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.     

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.     

Clinical Targeting of Mutated and Wild-Type Protein Tyrosine Kinases in Cancer

Clinical therapies for cancer have evolved from toxic, nontargeted agents to manageable, highly targeted therapies. Protein tyrosine kinases are a family of signaling molecules implicated in nearly every cancer type and are the foundation for the development of modern targeted agents. Recent genomic analyses have identified activating mutations, translocations, and amplifications of tyrosine kinases. Selective targeting of these genetically altered tyrosine kinases has resulted in significant clinical advances, including increased patient survival. This indicates that altered protein tyrosine kinases are the main drivers of many different cancers. However, lost during analyses of genetic lesions are the contributions of activated, wild-type kinases on tumor-dependent pathways. New approaches in phosphoproteomic technologies have identified several wild-type tyrosine kinase activation states, suggesting that non-genetically altered kinases can be essential “nodes” for signal transduction. Here, we summarize the evidence supporting the common mechanisms of protein tyrosine kinase activation in cancer and provide a personal perspective on the kinases BCR-ABL and BTK, as well as nonmutated kinase targets in prostate cancer, through our work. We outline the mechanisms of tyrosine kinase activation in the absence of direct mutation and discuss whether non-genetically altered tyrosine kinases or their associated downstream signaling pathways can be effectively targeted.     

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.     

O-methylated catechins from tea leaves inhibit multiple protein kinases in mast cells

Tea contains a variety of bioactive compounds. In this study, we show that two O-methylated catechins, (-)-epigallocatechin-3-O-(3-O-methyl) gallate and (-)-epigallocatechin-3-O-(4-O-methyl) gallate, inhibit in vivo mast cell-dependent allergic reactions more potently than their nonmethylated form, (-)-epigallocatechin-3-O-gallate. Consistent with this, these O-methylated catechins inhibit IgE/Ag-induced activation of mouse mast cells: histamine release, leukotriene release, and cytokine production and secretion were all inhibited. As a molecular basis for the catechin-mediated inhibition of mast cell activation, Lyn, Syk, and Bruton's tyrosine kinase, the protein tyrosine kinases, known to be critical for early activation events, are shown to be inhibited by the O-methylated catechins. In vitro kinase assays using purified proteins show that the O-methylated catechins can directly inhibit the above protein tyrosine kinases. These catechins inhibit IgE/Ag-induced calcium response as well as the activation of downstream serine/threonine kinases such as Akt and c-Jun N-terminal kinase. These observations for the first time have revealed the molecular mechanisms of antiallergic effects of tea-derived catechins.     

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.     

The Tyrosine Kinase Inhibitor Genistein Increases Endogenous Dopamine Release from Normal and Weaver Mutant Mouse Striatal Slices

Abstract: Protein tyrosine kinases that are known to have major roles in the control of cell growth and transformation are abundant and have numerous phosphoprotein substrates in the adult CNS. Although less well characterized than serine/threonine kinases, tyrosine kinases are also concentrated in the synapse. The effect of genistein, a selective inhibitor of tyrosine kinase activity, was examined on the in vitro release of endogenous dopamine (DA) from superfused mouse striatal slices. Fractional release of DA was significantly increased over basal release levels by genistein (100 and 200 µ M ). The effect was concentration dependent and rapidly reversible on washout of the kinase inhibitor. No significant change from basal release levels was observed with two structural analogues of genistein that do not inhibit tyrosine kinase activity at the same concentration. We have previously described alterations in basal and evoked DA release from the striatum of the weaver ( wv/wv ) mutant mouse, and genotypic differences in fractional release were also observed with genistein stimulation. The total evoked release was 25–50% greater from the wv/wv striatum. These results suggest a modulatory role for tyrosine kinase activity in neurotransmitter release and perhaps an alteration of kinase-regulated mechanisms in the DA-deficient wv/wv striatum.     

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.     

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix

Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation.     

Dual role of the tyrosine activation motif of the Ig-alpha protein during signal transduction via the B cell antigen receptor.

The B cell antigen receptor (BCR) is a multimeric protein complex consisting of the ligand binding immunoglobulin molecule and the Ig-alpha/beta heterodimer that mediates intracellular signalling by coupling the receptor to protein tyrosine kinases (PTKs). Transfection of the Ig-alpha deficient myeloma cell line J558L microns with expression vectors coding for mutated Ig-alpha allowed us to test the function of the tyrosines in the cytoplasmic region of Ig-alpha in the context of the BCR. Furthermore we expressed Ig-alpha mutations as chimeric CD8-Ig-alpha molecules on K46 B lymphoma cells and tested their signalling capacity in terms of PTK activation and release of calcium. We show here that the conserved tyrosine residues in the cytoplasmic portion of Ig-alpha have a dual role. First, they are required for efficient activation of PTKs during signal induction and second, one of them is subject to phosphorylation by activated src-related PTKs. Phosphorylation on tyrosine in the cytoplasmic portion of Ig-alpha is discussed as a possible mechanism to couple the BCR to SH2 domain-carrying molecules.     

Tyrosine kinase mutations in human cancer

A subset of tyrosine kinases are activated by mutations which contribute to the malignant transformation, growth, and metastasis of human cancers. Mutations change the expression, conformation and/or stability of tyrosine kinases, often leading to constitutive activation of the signaling pathways the kinases regulate. Given that tyrosine kinases are key members of signaling cascades, mutations have multiple effects on various cellular proteins. This review will focus on four kinases (EGFR, c-Met, c-Kit, and PI3-kinase) known to be mutated in human cancer. It will discuss the effects that these mutations have on the biology of tumors, and how our understanding of the structure and function of kinases and their mutations is currently being used to design targeted treatments.     

Kinase pathways in chemoattractant-induced degranulation of neutrophils: the role of p38 mitogen-activated protein kinase activated by Src family kinases

The aim of the present study was to investigate the role of tyrosine phosphorylation pathways in fMLP-induced exocytosis of the different secretory compartments (primary and secondary granules, as well as secretory vesicles) of neutrophils. Genistein, a broad specificity tyrosine kinase inhibitor, blocked the exocytosis of primary and secondary granules, but had only a marginal effect on the release of secretory vesicles. Genistein also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK), raising the possibility that inhibition of ERK and/or p38 MAPK might be responsible for the effect of the drug on the degranulation response. Indeed, SB203580, an inhibitor of p38 MAPK, decreased the release of primary and secondary granules, but not that of secretory vesicles. However, blocking the ERK pathway with PD98059 had no effect on any of the exocytic responses tested. PP1, an inhibitor of Src family kinases, also attenuated the release of primary and secondary granules, and neutrophils from mice deficient in the Src family kinases Hck, Fgr, and Lyn were also defective in secondary granule release. Furthermore, activation of p38 MAPK was blocked by both PP1 and the hck-/-fgr-/-lyn-/- mutation. Taken together, our data indicate that fMLP-induced degranulation of primary and secondary granules of neutrophils is mediated by p38 MAPK activated via Src family tyrosine kinases. Although piceatannol, a reportedly selective inhibitor of Syk, also prevented degranulation and activation of p38 MAPK, no fMLP-induced phosphorylation of Syk could be observed, raising doubts about the specificity of the inhibitor.     

Phytoestrogen action in Leydig cells of Biłgoraj ganders (Anser anser)

The mechanism of phytoestrogen action in gonadal cells of ganders has not been elucidated. The aim of the study was to investigate in Bi?goraj ganders the possibility of phytoestrogen action via estrogen or androgen receptors or via protein tyrosine kinase pathways in Leydig cells. Genistein and daidzein (5 and 50 microM) as well as equol (50 microM) inhibited testosterone (T) secretion by incubated Leydig cells (1x10(5)/ml; 20 h; 37 degrees C). The effects of hydroxytamoxifen (estrogen receptor inhibitor) and cyproterone acetate (androgen receptor antagonist) on phytoestrogen inhibition of T release by Leydig cells were not observed. Lavendustin A (protein tyrosine kinases inhibitor) did not change T production. The influence of phytoestrogens seems not to be conducted via estrogen and androgen receptors or protein tyrosine kinases system in these cells, but further studies are required to completely examine the mechanism of phytoestrogens action in testes of ganders.     

PhosphoBase, a database of phosphorylation sites: release 2.0.

PhosphoBase contains information about phosphorylated residues in proteins and data about peptide phosphorylation by a variety of protein kinases. The data are collected from literature and compiled into a common format. The current release of PhosphoBase (October 1998, version 2.0) comprises 414 phosphoprotein entries covering 1052 phosphorylatable serine, threonine and tyrosine residues. The kinetic data from peptide phosphorylation assays for approximately 330 oligopeptides is also included. The database entries are cross-referenced to the corresponding records in the Swiss-Prot protein database and literature references are linked to MedLine records. PhosphoBase is available via the WWW at http://www.cbs.dtu. dk/databases/PhosphoBase/     

Regulation of phosphatidylserine synthesis in Jurkat T cell clones: caffeine bypasses CD3/TCR-induced protein tyrosine kinases and calcium signals

Phosphatidylserine synthesis as measured by the incorporation of [(3)H]serine into phosphatidylserine (PtdSer) through the serine-base exchange enzyme system (serine-BEES) is markedly inhibited in Jurkat cells treated with caffeine. The caffeine-induced inhibition was compared to that observed in cells treated with either CD3 mAb or thapsigargin. While CD3- and thapsigargin-induced inhibition was related to the release of Ca(2+) from the endoplasmic reticulum (ER), a process that deprives the serine-BEES of its major cofactor, caffeine modified PtdSer synthesis in the absence of decreased Ca(2+) content of ER. Using Jurkat clones differing by the expression of cell surface markers or protein tyrosine kinases implicated in the CD3/TCR signal transmission pathway, we have shown that CD3 mAb-induced inhibition of PtdSer synthesis necessitates the expression of both the CD3/TCR and the protein tyrosine phosphatase CD45 at the cell surface as well as the presence of p56(lck) and ZAP-70 protein tyrosine kinases. By contrast, thapsigargin, a blocker of the Ca(2+)-ATPase of the ER, known for its Ca(2+) releasing properties, inhibited PtdSer synthesis in all the Jurkat clones tested, indicating that this compound bypasses the CD3/TCR-induced signals. Despite its lack of effect on Ca(2+) release from ER and on protein tyrosine phosphorylations, caffeine inhibited PtdSer synthesis in all the Jurkat clones. The use of several cAMP-inducing drugs and of others xanthine derivatives indicated that caffeine modify PtdSer synthesis either by a direct action on the serine-BEES or by a modification of the structure of the phospholipids used as substrate by the enzyme.     

The host phosphoinositide 5-phosphatase SHIP2 regulates dissemination of vaccinia virus

After fusing with the plasma membrane, enveloped poxvirus virions form actin-filled membranous protrusions, called tails, beneath themselves and move toward adjacent uninfected cells. While much is known about the host and viral proteins that mediate formation of actin tails, much less is known about the factors controlling release. We found that the phosphoinositide 5-phosphatase SHIP2 localizes to actin tails. Localization requires phosphotyrosine, Abl and Src family tyrosine kinases, and neural Wiskott-Aldrich syndrome protein (N-WASP) but not the Arp2/Arp3 complex or actin. Cells lacking SHIP2 have normal actin tails but release more virus. Moreover, cells infected with viral strains with mutations in the release inhibitor A34 release more virus but recruit less SHIP2 to tails. Thus, the inhibitory effects of A34 on virus release are mediated by SHIP2. Together, these data suggest that SHIP2 and A34 may act as gatekeepers to regulate dissemination of poxviruses when environmental conditions are conducive.     

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.     

A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators.

In a screen of mouse erythroleukemia cDNA expression libraries with anti-phosphotyrosine antibodies, designed to isolate tyrosine kinase coding sequences, we identified several cDNAs encoding proteins identical or very similar to known protein-tyrosine kinases. However, two frequently isolated cDNAs, clk and nek, encode proteins which are most closely related to protein kinases involved in regulating progression through the cell cycle, and contain motifs generally considered diagnostic of protein-serine/threonine kinases. The clk gene product contains a C-terminal cdc2-like kinase domain, most similar to the FUS3 catalytic domain. The Clk protein, expressed in bacteria, becomes efficiently phosphorylated in vitro on tyrosine as well as serine/threonine, and phosphorylates the exogenous substrate poly(glu, tyr) on tyrosine. Direct biochemical evidence indicates that both protein-tyrosine and protein-serine/threonine kinase activities are intrinsic to the Clk catalytic domain. These results suggest the existence of a novel class of protein kinases, with an unusual substrate specificity, which may be involved in cell cycle control.