A computational approach for the classification of protein tyrosine kinases

Protein tyrosine kinases (PTKs) play a central role in the modulation of a wide variety of cellular events such as differentiation, proliferation and metabolism, and their unregulated activation can lead to various diseases including cancer and diabetes. PTKs represent a diverse family of proteins including both receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (NRTKs). Due to the diversity and important cellular roles of PTKs, accurate classification methods are required to better understand and differentiate different PTKs. In addition, PTKs have become important targets for drugs, providing a further need to develop novel methods to accurately classify this set of important biological molecules. Here, we introduce a novel statistical model for the classification of PTKs that is based on their structural features. The approach allows for both the recognition of PTKs and the classification of RTKs into their subfamilies. This novel approach had an overall accuracy of 98.5% for the identification of PTKs, and 99.3% for the classification of RTKs.     

Kinases and glutathione transferases: selective and sensitive targeting

Kinases, representing almost 500 proteins in the human genome, are responsible for catalyzing the phosphorylation reaction of amino acid residues at their targets. As the largest family of kinases, the protein tyrosine kinases (PTKs) have roles in controlling the essential cellular activities, and their deregulation is generally related to pathologic conditions. The recent efforts on identifying their signal transducer or mediator role in cellular signaling revealed the interaction of PTKs with numerous enzymes of different classes, such as Ser/Thr kinases (STKs), glutathione transferases (GSTs), and receptor tyrosine kinases (RTKs). In either regulation or enhancing the signaling, PTKs are determined in close interaction with these enzymes, under specific cellular conditions, such as oxidative stress and inflammation. In this concept, intensive research on thiol metabolizing enzymes recently showed their involvement in the physiologic functions in cellular signaling besides their well known traditional role in antioxidant defense. The shared signaling components between PTK and GST family enzymes will be discussed in depth in this research review to evaluate the results of recent studies important in drug targeting for therapeutic intervention, such as cell viability, migration, differentiation and proliferation.     

Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth.

Protein tyrosine kinases (PTKs) have major roles in signal transduction and growth control. There are several lines of evidence implicating PTKs in the regulation of axon growth, and this has led to the suggestion that they are centrally involved in the transduction of neuronal growth signals. To test this idea, we assayed the effect of the compounds genistein and lavendustin, specific inhibitors of PTKs, on neurite growth. We find that genistein greatly reduces phosphotyrosine in neurons, as expected from its action on other cells. Surprisingly, administration of genistein or lavendustin potentiated substrate-induced neurite growth in at least several different neuronal types. Stimulation of neurite growth by genistein was abolished by vanadate, providing additional evidence that inhibition of PTKs is responsible for this effect. The potentiation of growth is rather general, in that it occurs on several different extracellular matrix substrates and on two different cell adhesion molecules. Both the initiation of neurite growth and the rate of neurite elongation appear to be potentiated. Our results do not provide evidence for models of substrate-induced signal transduction that involve PTKs as a positive and necessary step, but suggest that such kinases play a regulatory role in neurite elongation.     

Ligands and signaling through receptor-type tyrosine phosphatases

Virtually every aspect of cellular proliferation and differentiation is regulated by changes in tyrosine phosphorylation. Tyrosine phosphorylation, in turn, is controlled by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). PTKs are often transmembrane proteins (receptor PTKs) whose enzymatic activities and signaling functions are tightly regulated by the binding of specific ligands. A variety of transmembrane PTPs has also been identified; these proteins are called receptor PTPs (RPTPs), but in most cases their roles as receptors are very poorly understood. This review discusses the evidence that RPTPs are actually receptors for extrinsic ligands, and the extent to which interactions with putative ligands are known or suspected to cause changes in enzymatic activity. Finally, some of the RPTP substrates believed to be physiologically important are described. The evidence gathered to date suggests that models derived from studies of receptor PTKs may be too simple to account for the diversity and complexity of mechanisms through which ligand binding controls RPTP function.     

Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth

Protein tyrosine kinases (PTKs) have major roles in signal transduction and growth control. There are several lines of evidence implicating PTKs in the regulation of axon growth, and this has led to the suggestion that they are centrally involved in the transduction of neuronal growth signals. To test this idea, we assayed the effect of the compounds genistein and lavendustin, specific inhibitors of PTKs, on neurite growth. We find that genistein greatly reduces phosphotyrosine in neurons, as expected from its action on other cells. Surprisingly, administration of genistein or lavendustin potentiated substrate-induced neurite growth in at least several different neuronal types. Stimulation of neurite growth by genistein was abolished by vanadate, providing additional evidence that inhibition of PTKs is responsible for this effect. The potentiation of growth is rather general, in that it occurs on several different extracellular matrix substrates and on two different cell adhesion molecules. Both the initiation of neurite growth and the rate of neurite elongation appear to be potentiated. Our results do not provide evidence for models of substrate-induced signal transduction that involve PTKs asa positive and necessary step, but suggest that such kinases play aregulatory role in neurite elongation. © 1992 John Wiley & Sons, Inc.     

Identification of a novel mutation in ZAP70 and prenatal diagnosis in a Turkish family with severe combined immunodeficiency disorder

Protein tyrosine kinases (PTKs) play an important role in T cell development and activation. In vitro and in vivo defects, resulting in variable deficiencies in thymic development and in T cell antigen receptor (TCR) signal transduction, in PTKs have been shown. ZAP70, one of those PTKs, is a 70-kDa tyrosine phosphoprotein and associates with the ζ chain and undergoes tyrosine phosphorylation following TCR stimulation. It is expressed in T and natural killer (NK) cells. Several mutations were shown to lead to an autosomal recessive form of severe combined immunodeficiency disease (SCID).     

Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization

Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core.     

Structure-based drug design and AutoDock study of potential protein tyrosine kinase inhibitors

Different classes of compounds were investigated for their binding affinities into different protein tyrosine kinases (PTKs) employing a novel flexible ligand docking approach by using AutoDock 3.05 and 4. These compounds include many flavin analogs, which were developed in our group with varying degrees of cytotoxic activity (comparable or moderately superior to cisplatin and ara-c), and database selected analogs. They were docked onto twelve different families of PTKs retrieved from the Protein Data Bank. These proteins are representatives of plausible models of interactions with chemotherapeutic agents. A comparative study of the intact co-crystallized ligands of various types of PTKs was carried out. Results revealed that the new class of 5-deazapteridine and steroid hybrid compounds VIa,b, and d, and the vertical-type bispyridodipyrimidine with n-hexyl chain junction between its N-10 and N-10 atoms Xa, exhibited non-selective PTK binding capacities, with the lowest (Gb). On the other hand, 2-amino benzoic acid analog IIa, phenoxypyrido [3, 4-d]pyrimidine derivative IVc, tyrosine containing tripeptide Vd, and the one from Sumisho data base 831 are proposed to have selective PTK binding affinities to certain classes of tyrosine kinases, namely, HGFR (c-met), ZAP-70, insulin receptor kinase, EGFR, respectively. All These compounds of highest affinities were docked within the binding sites of PTKs with reasonable RMSD and 1-5 hydrogen bonds.     

JAK protein tyrosine kinases: their role in cytokine signalling

Protein tyrosine kinases (PTKs) are integral components of the cellular machinery that mediates the transduction and/or processing of many extra- and intracellular signals. Members of the JAK family of intracellular PTKs (JAK1, JAK2 and TYK2) are characterized by the possession of a PTK-related domain and five additional homology domains, in addition to a classical PTK domain. An important breakthrough in the understanding of JAK kinases function(s) has come from the recent observations that many cytokine receptors compensate for their lack of a PTK domain by utilizing members of the JAK family for signal transduction.     

Protein tyrosine kinases as new potential targets against human schistosomiasis

In spite of the numerous efforts made to control their transmission, parasite schistosomes still represent a serious public health concern and a major economic problem in many developing countries. Praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis and the only one that is available for mass chemotherapy. However, its widespread use and its inefficacy on juvenile parasites raise fears that schistosomes will develop drug resistance, and make the development of alternative drugs highly desirable. Protein tyrosine kinases (PTKs) are key molecules that control cell differentiation and proliferation and they already represent important targets for molecular cancer therapy. The recent characterization in Schistosoma mansoni of several cytosolic and receptor PTKs, with properties similar but also divergent from their vertebrate counterparts, opens new perspectives for the development of novel strategies in chemotherapy of schistosomiasis, which could be based on the use of parasite-specific tyrosine phosphorylation inhibitors.     

Coevolution of the domains of cytoplasmic tyrosine kinases

Many signaling molecules are multidomain proteins that have other domains in addition to the catalytic kinase domain. Protein tyrosine kinases almost without exception contain Src homology 2 (SH2) and/or SH3 domains that can interact with other signaling proteins. Here, we studied evolution of the tyrosine kinases containing SH2 and/or SH3 and kinase domains. The three domains seem to have duplicated together, since the phylogenetic analysis using parsimony gave almost identical evolutionary trees for the separate domains and the multidomain complexes. The congruence analysis of the sequences for the separate domains also suggested that the domains have coevolved. There are several reasons for the domains to appear in a cluster. Kinases are regulated in many ways, and the presence of SH2 and SH3 domains at proper positions is crucial. Because all three domains can recognize different parts of ligands and substrates, their evolution has been interconnected. The reasons for the clustering and coevolution of the three domains in protein tyrosine kinases (PTKs) are discussed.     

ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain.

Protein-tyrosine kinases (PTKs) play an integral role in T cell activation. Stimulation of the T cell antigen receptor (TCR) results in tyrosine phosphorylation of a number of cellular substrates. One of these is the TCR zeta chain, which can mediate the transduction of extracellular stimuli into cellular effector functions. We have recently identified a 70 kd tyrosine phosphoprotein (ZAP-70) that associates with zeta and undergoes tyrosine phosphorylation following TCR stimulation. Here we report the isolation of a cDNA clone encoding ZAP-70. ZAP-70 represents a novel PTK and is expressed in T and natural killer cells. Moreover, tyrosine phosphorylation and association of ZAP-70 with zeta require the presence of src family PTKs and provide a potential mechanism by which the src family PTKs and ZAP-70 may interact to mediate TCR signal transduction.     

Redox control of catalytic activities of membrane-associated protein tyrosine kinases

Protein tyrosine kinases (PTKs) play key roles in starting the signal transduction network for cellular development and functions. A number of both receptor-type and non-receptor-type PTKs, which are normally at a resting state, are initially activated in association with functions of the cell membrane and membrane rafts. Results of recent studies have suggested that these membrane-associated mechanisms for activation of PTKs consist of the two steps that are under redox control. The first step is activation of cell surface receptors through chemical crosslinkage or aggregation of receptors and membrane rafts, which leads to production of reactive oxygen species (ROS) as second messengers of intracellular signal transduction. The second step involves chemical modification of PTKs at the highly conserved cysteine in the MXXCW motif as a global switch for starting the tyrosine phosphorylation-dependent local switch for activation of the catalytic activity of the enzyme.     

Comparative characterization of receptor and non-receptor associated protein tyrosine kinases

By using poly(Glu: Tyr, 4:1) as an exogenous substrate, the characteristics of insulin receptor associated protein tyrosine kinase (PTK) from rabbit skeletal muscle has been compared with a growth factor-independent non-receptor PTK partially purified from rat lung particulate fraction. The two PTKs phosphorylated poly(Glu: Tyr; 4:1) very effectively with apparent Km values of 0.3 mg/ml for insulin receptor PTK and 0.8 mg/ml for lung PTK. ATP was the preferred phosphoryl donor for both PTKs (Km = 150 microM); however, in the case of lung PTK, GTP was able to partially replace ATP. ATP analogues, AMP-PNP and ATP-gamma-S inhibited the activities of both enzymes. Receptor PTK was more active in the presence of Mn2+ whereas the lung PTK did not discriminate between Mg2+ or Mn2+ for enzyme activity. Para-hydroxymercurobenzoate (pHMB), a SH-group blocking agent, inhibited the activities of both PTKs, suggesting the requirement of SH-groups for enzymatic activities. Both enzymes were inhibited by fluorosulfonylbenzoyl 5'-adenosine (FSBA). NaCl also inhibited both kinases, however, lung PTK was more sensitive to inhibition. In addition, the lung PTK was not retained on a wheat germ agglutinin (WGA)-agarose column, suggesting that the lung enzyme is either not a glycoprotein or that the carbohydrate moieties present, if any, have no affinity for WGA. Furthermore, the lung PTK appears to be immunologically distinct from both insulin receptor and pp60Src, since it was not immunoprecipitated by antibodies to either pp60Src or insulin receptor. These data indicate that only a few but significant differences exist in the characteristics of receptor and non-receptor associated PTKs.     

Protein tyrosine kinases: autoregulation and small-molecule inhibition

Receptor and non-receptor protein tyrosine kinases (PTKs) are essential enzymes in cellular signaling processes that regulate cell growth, differentiation, migration and metabolism. The kinase activity of PTKs is tightly controlled through steric, autoregulatory mechanisms, as well as by the action of protein tyrosine phosphatases. Recent structural studies have revealed several modes of autoregulation governing the catalytic state of these enzymes. Aberrant catalytic activity of many PTKs, via mutation or overexpression, plays an important role in numerous pathological conditions, including cancer. Structural studies of the Abl tyrosine kinase domain in complex with the small-molecule inhibitor STI571 provide a molecular basis for understanding the specificity determinants of this highly successful drug used in the treatment of chronic myeloid leukemia.     

Protein tyrosine phosphatases as novel targets in breast cancer therapy

Breast cancer is linked to hyperactivation of protein tyrosine kinases (PTKs), and recent studies have unveiled that selective tyrosine dephosphorylation by protein tyrosine phosphatases (PTPs) of specific substrates, including PTKs, may activate or inactivate oncogenic pathways in human breast cancer cell growth-related processes. Here, we review the current knowledge on the involvement of PTPs in breast cancer, as major regulators of breast cancer therapy-targeted PTKs, such as HER1/EGFR, HER2/Neu, and Src. The functional interplay between PTKs and PTK-activating or -inactivating PTPs, and its implications in novel breast cancer therapies based on targeting of specific PTPs, are discussed.     

Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd,a UDP-glucose dehydrogenase

Autophosphorylation of protein-tyrosine kinases (PTKs) involved in exopolysaccharide and capsular polysaccharide biosynthesis and transport has been observed in a number of Gram-negative and Gram-positive bacteria. However, besides their own phosphorylation, little is known about other substrates targeted by these protein-modifying enzymes. Here, we present evidence that the protein-tyrosine kinase Wzc of Escherichia coli is able to phosphorylate an endogenous enzyme, UDP-glucose dehydrogenase (Ugd), which participates in the synthesis of the exopolysaccharide colanic acid. The process of phosphorylation of Ugd by Wzc was shown to be stimulated by previous autophosphorylation of Wzc on tyrosine 569. The phosphorylation of Ugd was demonstrated to actually occur on tyrosine and result in a significant increase of its dehydrogenase activity. In addition, the phosphotyrosine-protein phosphatase Wzb, which is known to effectively dephosphorylate Wzc, exhibited only a low effect, if any, on the dephosphorylation of Ugd. These data were related to the recent observation that two other UDP-glucose dehydrogenases have been also shown to be phosphorylated by a PTK in the Gram-positive bacterium Bacillus subtilis. Comparative analysis of the activities of PTKs from Gram-negative and Gram-positive bacteria showed that they are regulated by different mechanisms that involve, respectively, either the autophosphorylation of kinases or their interaction with a membrane protein activator.     

Biochemical characteristics of cytosolic and particulate forms of protein tyrosine kinases from N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma

Summary Protein tyrosine kinase (PTK) activities in methyl nitrosourea (MNU)-induced rat mammary carcinoma has been investigated by using poly (glu: tyr; 4 : 1) as an exogenous substrate. The PTK activity of the mammary carcinoma was almost equally distributed between the particulate and soluble (cytosolic) fractions at 110,000 × g. The activity of the particulate enzyme was stimulated by non-ionic detergent Triton X-100 by about 2-fold whereas the detergent had no effect on the cytosolic form. More than 60% of the particulate enzyme could be solubilized by 5% Triton X-100. Although, both particulate and cytosolic PTKs catalyzed the phosphorylation of several tyrosine containing synthetic substrates to various degrees, poly (glu: tyr; 4 : 1) was the best substrate (apparent Km, 0.7 mg/ml). Both forms of enzymes utilized ATP as the phosphoryl group donor, with an apparent Km of 40 µM. Among various divalent cations tested, Co², Mn² and Mg² were able to fulfill the divalent cation requirement of both forms of the PTKs. All these cations exerted biphasic effects on the kinase activities, however, Mg² was the most potent cation. Agents such as epidermal growth factor, insulin and platelet derived growth factor which stimulate their respective receptor-PTK activities were without effect on the PTK activities of mammary carcinoma. On the other hand, though heparin and quercetin inhibited both enzyme activities in a concentration dependent manner, the particulate form was more sensitive to inhibition than the cytosolic form. These data indicate that MNU-induced rat mammary carcinoma expresses both particulate and cytosolic forms of PTKs and that there are significant differences in the properties of the two forms of PTKs. Differential effects of some agents on mammary carcinoma PTKs suggest that these enzymes may be acutely regulated in vivo and could play an important role in mammary carcinogenesis.Abbreviations PTK Protein Tyrosine Kinase - EGF Epidermal Growth Factor - PDGF Platelet Derived Growth Factor