SH2 domain-mediated interaction of inhibitory protein tyrosine kinase Csk with protein tyrosine phosphatase-HSCF

The protein tyrosine kinase (PTK) Csk is a potent negative regulator of several signal transduction processes, as a consequence of its exquisite ability to inactivate Src-related PTKs. This function requires not only the kinase domain of Csk, but also its Src homology 3 (SH3) and SH2 regions. We showed previously that the Csk SH3 domain mediates highly specific associations with two members of the PEP family of nonreceptor protein tyrosine phosphatases (PTPs), PEP and PTP-PEST. In comparison, the Csk SH2 domain interacts with several tyrosine phosphorylated molecules, presumed to allow targetting of Csk to sites of Src family kinase activation. Herein, we attempted to understand better the regulation of Csk by identifying ligands for its SH2 domain. Using a modified yeast two-hybrid screen, we uncovered the fact that Csk associates with PTP-HSCF, the third member of the PEP family of PTPs. This association was documented not only in yeast cells but also in a heterologous mammalian cell system and in cytokine-dependent hemopoietic cells. Surprisingly, the Csk-PTP-HSCF interaction was found to be mediated by the Csk SH2 domain and two putative sites of tyrosine phosphorylation in the noncatalytic portion of PTP-HSCF. Transfection experiments indicated that Csk and PTP-HSCF synergized to inhibit signal transduction by Src family kinases and that this cooperativity was dependent on the domains mediating their association. Finally, we obtained evidence that PTP-HSCF inactivated Src-related PTKs by selectively dephosphorylating the positive regulatory tyrosine in their kinase domain. Taken together, these results demonstrate that part of the function of the Csk SH2 domain is to mediate an inducible association with a PTP, thereby engineering a more efficient inhibitory mechanism for Src-related PTKs. Coupled with previously published observations, these data also establish that Csk forms complexes with all three known members of the PEP family.     

Docking-based substrate recognition by the catalytic domain of a protein tyrosine kinase, C-terminal Src kinase (Csk)

Protein tyrosine kinases are key enzymes of mammalian signal transduction. Substrate specificity is a fundamental property that determines the specificity and fidelity of signaling by protein tyrosine kinases. However, how protein tyrosine kinases recognize the protein substrates is not well understood. C-terminal Src kinase (Csk) specifically phosphorylates Src family kinases on a C-terminal Tyr residue, which down-regulates their activities. We have previously determined that Csk recognizes Src using a substrate-docking site away from the active site. In the current study, we identified the docking determinants in Src recognized by the Csk substrate-docking site and demonstrated an interaction between the docking determinants of Src and the Csk substrate-docking site for this recognition. A similar mechanism was confirmed for Csk recognition of another Src family kinase, Yes. Although both Csk and MAP kinases used docking sites for substrate recognition, their docking sites consisted of different substructures in the catalytic domain. These results helped establish a docking-based substrate recognition mechanism for Csk. This model may provide a framework for understanding substrate recognition and specificity of other protein tyrosine kinases.     

Affinity purification of Csk protein tyrosine kinase based on its catalytic requirement for divalent metal cations

Protein tyrosine kinase Csk requires two Mg2+ ions for activity: one magnesium is part of the ATP-Mg complex, and the second free Mg2+ ion is required as an essential activator. Zn2+ can bind to this site to replace Mg2+, which inhibits Csk kinase activity. The binding is reversible and removal of Zn2+ results in an active Csk apoenzyme. In this communication, we report that this tight binding can be used as a mechanism for affinity purification of Csk. When bacterial cell lysate containing overexpressed GST-Csk was applied to a column of Zn2+-iminodiacetic acid immobilized to agarose, Csk was specifically retained by the column. Since the binding of Csk to Zn2+ is not affected by up to 200 mM NaCl, high ionic strength conditions were used in the purification procedure, minimizing nonspecific binding due to ionic interactions. Washing the column with 200 mM NaCl and 50 mM imidazole removed virtually all other proteins from the column while Csk remained bound. The retained Csk enzyme was eluted with 1 M imidazole. The 1 M imidazole-eluted fraction contained pure Csk that had a specific activity similar to the enzyme purified by a glutathione-agarose affinity column.     

Probing the communication between the regulatory and catalytic domains of a protein tyrosine kinase, Csk

Protein tyrosine kinases (PTKs) are important regulators of mammalian cell function and their own activities are tightly regulated. Underlying their tight regulation, all PTKs contain multiple regulatory domains in addition to a catalytic domain. C-terminal Src kinase (Csk) contains a catalytic domain and a regulatory region, consisting of an SH3 and an SH2 domain. In this study, we probed the communication between the regulatory and catalytic domains of Csk. First, kinetic characterization of SH3 and SH2 domain deletion mutants demonstrated that the SH3 and SH2 domains were crucial in maintaining the full activity of Csk, but were not directly involved in Csk recognition of its physiological substrate, Src. Second, highly conserved Trp188, corresponding to a key residue in domain-domain communication in other PTKs, was found to be important for maintaining the active structure of Csk by the presence of the regulatory region, but not required for Csk activation triggered by a phosphopeptide binding to the SH2 domain. Third, structural alignment indicated that the presence of the regulatory domains modulated the conformation of multiple substructures in the catalytic domain, some directly and others remotely. Mutagenic and kinetic studies supported this assignment. This report extended previous studies of Csk domain-domain communication, and provided a foundation for further detailed investigation of this communication.     

Role of tyrosine kinase Csk in G protein-coupled receptor- and receptor tyrosine kinase-induced fibroblast cell migration

Tyrosine kinase Csk is essential for mouse embryonic development. Csk knock-out mice died at early stages of embryogenesis (around embryonic day 10). The molecular mechanism for this defect is not completely understood. Here we report that Csk deficiency in mouse embryonic fibroblast cells blocked cell migration induced by lysophosphatidic acid through G protein-coupled receptors, by platelet-derived growth factor and epidermal growth factor through receptor tyrosine kinases, and by serum. Re-expression of Csk in these Csk-deficient cells rescued the migratory phenotype. Furthermore, deletion of Csk did not interfere with Rac activation and lamellipodia formation, but impaired the focal adhesions. Our data demonstrate a critical role for Csk in cell migration.     

Tyrosine analogues as alternative substrates for protein tyrosine kinase Csk: insights into substrate selectivity and catalytic mechanism

Protein tyrosine kinases are critical enzymes in cell signal transduction but relatively little is known about the molecular recognition of the tyrosine substrate by these enzymes. Details of tyrosine substrate specificity within the context of a short peptide were investigated for protein tyrosine kinase Csk. It was found that aryl ring functional group substitutions the size of methyl group or smaller were generally well tolerated by the protein tyrosine kinase Csk whereas larger groups caused a decline in substrate efficiency. Extension of the phenol from the peptide backbone by a single methylene was acceptable for phosphorylation whereas removal of a methylene nearly abolished reactivity. Only the L-tyrosine derivative was processed. A negative charge ortho to the phenol hydroxyl was incompatible with substrate reactivity, consistent with previous pH rate profiles which indicated the importance of the neutral phenol. Overall, these studies confirmed the interpretation of a previous linear free energy relationship analysis which suggested that the enzyme followed a dissociative transition state mechanism.     

Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.     

SRC tail phosphorylation is limited by structural changes in the regulatory tyrosine kinase Csk

Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.     

Obtaining a full-length encoding cDNA of Csk family tyrosine kinase from human lymphocytes

Tyrosine kinases of Csk family play important role in the cell growth regulation and normal cell differentiation and also can participate in the process of cancer genesis as oncoproteins. The main function of these tyrosine kinases is the phosphorylation of the Src family tyrosine kinases at their carboxyl terminus, which is the basis of their activity negative regulation. The disturbance of the csk gene expression leads to the increase of the Src tyrosine kinase activity. We have cloned a full-length encoding cDNA of the tyrosine kinase csk gene of human lymphocytes. 1.6-kilobase cDNA encodes the protein, which consists of 12 exons with conserved SH2 and SH3 domains. The homology between this protein and human Csk tyrosine kinase is 99%. A full-length DNA-copy of human lymphocytes RNA can be used for the analysis of csk gene structure in normal and pathologically changed human cells.     

Contribution of an active site cation-pi interaction to the spectroscopic properties and catalytic function of protein tyrosine kinase Csk

Csk is a soluble protein tyrosine kinase that phosphorylates and negatively regulates protein tyrosine kinases of the Src family. The spectral properties of the intrinsic Trp fluorescence of Csk and their underlying structural features were investigated in combination with urea denaturation and site-specific mutagenesis. It was found that W352 contributed approximately 35% of the total Trp fluorescence of Csk even though seven other Trp residues were present. The enhanced contribution by W352 to Csk fluorescence was due to an interaction between its indole ring and the positively charged guanidino group of R318. W352 is located on the peptide substrate binding P+1 loop, and R318 is located on the catalytic loop. The W352-R318 interaction, called a cation-pi interaction, uniquely couples the two loops in the active site. Mutations that disrupted this coupling resulted in varying levels of decreases in Csk activity, and consistent and significant increases in K(m) values for its physiological substrate, Src protein tyrosine kinase. These results indicated that structural coupling between the two loops by the cation-pi interaction played an important role in Csk substrate binding. Since both R318 and W352 are highly conserved among protein tyrosine kinases, this cation-pi interaction is likely a signature structural feature of most, if not all, PTKs. These studies elucidated the roles of two conserved signature residues in Csk and formed a baseline for further structure-function studies of Csk and other PTKs.     

The Csk homologous kinase, Chk, binds tyrosine phosphorylated paxillin in human blastic T cells

In determining the role of Chk in T cell signaling, we have focused on its protein-protein interactions. We detected a tyrosine phosphoprotein that coimmunoprecipitated with Chk from pervanadate stimulated human blastic T cells. Subsequent Western blot analysis identified this tyrosine phosphoprotein as paxillin. Paxillin, a cytoskeletal protein involved in focal adhesions, was first identified as a v-Src substrate in transformed fibroblasts. Interestingly, Chk specifically bound tyrosine phosphorylated paxillin. Consistent with our in vivo data, Chk and paxillin were observed to localize in similar cellular regions prior to and following stimulation. Using GST fusion proteins, we determined that the Chk SH2 domain, not the SH3 domain, bound tyrosine phosphorylated paxillin. Specifically, paxillin bound to the FLVRES motif of the Chk SH2 domain. Using Far Western analysis, we revealed that the Chk SH2 domain directly associates with tyrosine phosphorylated paxillin. Finally, p52(Chk) expression in Csk-deficient mouse embryo fibroblasts decreased total phosphotyrosine levels of paxillin, implying a physiological role for Chk. These studies provide important insight into the role of Chk in tyrosine mediated signaling, as well as T cell physiology.     

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.     

Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets.

Chk/Hyl is a recently isolated non-receptor tyrosine kinase with greatest homology to a ubiquitous negative regulator of Src family kinases, Csk. To understand the significance of co-expression of Chk and Csk in platelets, we examined the subcellular localization of each protein. Chk, but not Csk, was completely translocated from the Triton X-100-soluble to the Triton X-100-insoluble cytoskeletal fraction within 10 s of thrombin stimulation. Chk and Lyn, but not Csk and c-Src, co-fractionated in the higher density lysate fractions of resting platelets, with Chk being found to localize close to CD36 (membrane glycoprotein IV)-anchored Lyn. The kinase activity of co-fractionated Lyn was suppressed 3-fold. In vitro phosphorylation assays showed that Chk suppressed Lyn activity by phosphorylating its C-terminal negative regulatory tyrosine. Upon stimulation of platelets with thrombin, the rapid and complete translocation of Chk away from Lyn caused concomitant activation of Lyn. This activation was accompanied by dephosphorylation of Lyn at its C-terminal negative regulatory tyrosine in cooperation with a protein tyrosine phosphatase. These results suggest that Chk, but not Csk, may function as a translocation-controlled negative regulator of CD36-anchored Lyn in thrombin-induced platelet activation.     

Marine sponge polyketide inhibitors of protein tyrosine kinase.

The marine polyketide natural product, halenaquinone, was shown to be an irreversible inhibitor of pp60v-src, the oncogenic protein tyrosine kinase encoded by the Rous sarcoma virus. This compound had an IC50 of approximately 1.5 microM against pp60v-src and also inhibited the ligand-stimulated kinase activity of the human epidermal growth factor receptor with an IC50 of approximately 19 microM. Halenaquinone blocked the proliferation of a number of cultured cell lines, including several transformed by oncogenic protein tyrosine kinases. Halenaquinol, xestoquinone, halenaquinol sulfate, and several simple synthetic quinone analogs were also shown to inhibit pp60v-src.     

Domain stabilities in protein kinase R (PKR): evidence for weak interdomain interactions

PKR (protein kinase R) is induced by interferon and is a key component of the innate immunity antiviral pathway. Upon binding dsRNA, PKR undergoes autophosphorylation reactions that activate the kinase, leading it to phosphorylate eIF2alpha, thus inhibiting protein synthesis in virally infected cells. PKR contains a dsRNA-binding domain (dsRBD) and a kinase domain. The dsRBD is composed of two tandem dsRNA-binding motifs. An autoinhibition model for PKR has been proposed, whereby dsRNA binding activates the enzyme by inducing a conformational change that relieves the latent enzyme of the inhibition that is mediated by the interaction of the dsRBD with the kinase. However, recent biophysical data support an open conformation for the latent enzyme, where activation is mediated by dimerization of PKR induced upon binding dsRNA. We have probed the importance of interdomain contacts by comparing the relative stabilities of isolated domains with the same domain in the context of the intact enzyme using equilibrium chemical denaturation experiments. The two dsRNA-binding motifs fold independently, with the C-terminal motif exhibiting greater stability. The kinase domain is stabilized by about 1.5 kcal/mol in the context of the holenzyme, and we detect low-affinity binding of the kinase and dsRBD constructs in solution, indicating that these domains interact weakly. Limited proteolysis measurements confirm the expected domain boundaries and reveal that the activation loop in the kinase is accessible to cleavage and unstructured. Autophosphorylation induces a conformation change that blocks proteolysis of the activation loop.     

Cloning of the doding cDNA sequence of the gene for Csk tyrosine kinase from human lymphocytes

Tyrosine kinases of the Csk family play an important role in cell growth regulation and normal cell differentiation. They are also involved in carcinogenesis as oncoproteins. The main function of these tyrosine kinases is phosphorylation of tyrosine kinases of the Src family at their C-terminal regions to negatively regulate their activity. Disturbance of csk expression increases the Src tyrosine kinase activity. The full-length coding sequence of the csk cDNA was cloned from human lymphocytes. The 1624-bp cDNA consists of 12 exons and encodes a protein that has conserved SH2 and SH3 domains and is similar to human Csk tyrosine kinase by 99%. The full-length cDNA can be used to analyze the csk structure in normal or illdefined human cells.     

Interaction of Bruton's tyrosine kinase and protein kinase Ctheta in platelets. Cross-talk between tyrosine and serine/threonine kinases.

The nonreceptor Bruton's tyrosine kinase (Btk) has been previously shown to associate physically and functionally with members of the protein kinase C (PKC) family of serine/threonine kinases in a variety of cell types. Here we show evidence for a novel interaction between Btk and PKCtheta; in platelets activated through the adhesion receptors GP Ib-V-IX and GP VI. Alboaggregin A, a snake venom component capable of activating both receptors in combination, leads to tyrosine phosphorylation of Btk downstream of Src family kinases. Inhibition of Btk by the selective antagonist LFM-A13 causes a reduction in calcium entry, although secretion of 5-hydroxytryptamine is potentiated. Btk is also phosphorylated on threonine residues in a PKC-dependent manner and associates with PKCtheta; upon platelet activation by either alboaggregin A or activation of GP Ib-V-IX alone by von Willebrand factor/ristocetin. PKCtheta; in turn becomes tyrosine-phosphorylated in a manner dependent upon Src family and Btk kinase activity. Inhibition of Btk activity by LFM-A13 leads to enhancement of PKCtheta; activity, whereas nonselective inhibition of PKC activity by bisindolylmaleimide I leads to reduction in Btk activity. We propose a reciprocal feedback interaction between Btk and PKCtheta; in platelets, in which PKCtheta; positively modulates activity of Btk, which in turn feeds back negatively upon PKCtheta;.     

Focal adhesion kinase: an integrin-linked protein tyrosine kinase

Recent evidence has shown that binding of integrins to components of the extracellular matrix serves to initiate cellular signals, resulting in changes in cytoskeletal architecture and activation of intracellular signalling pathways. Here we review the evidence that protein tyrosine kinases, including focal-adhesion-associated kinases, play a central role in the generation of some of these molecular signals.     

Effect of cyclic AMP-dependent protein kinase on insulin receptor tyrosine kinase activity.

To explain the insulin resistance induced by catecholamines, we studied the tyrosine kinase activity of insulin receptors in a state characterized by elevated noradrenaline concentrations in vivo, i.e. cold-acclimation. Insulin receptors were partially purified from brown adipose tissue of 3-week- or 48 h-cold-acclimated mice. Insulin-stimulated receptor autophosphorylation and tyrosine kinase activity of insulin receptors prepared from cold-acclimated mice were decreased. Since the effect of noradrenaline is mediated by cyclic AMP and cyclic AMP-dependent protein kinase, we tested the effect of the purified catalytic subunit of this enzyme on insulin receptors purified by wheat-germ agglutinin chromatography. The catalytic subunit had no effect on basal phosphorylation, but completely inhibited the insulin-stimulated receptor phosphorylation. Similarly, receptor kinase activity towards exogenous substrates such as histone or a tyrosine-containing copolymer was abolished. This inhibitory effect was observed with receptors prepared from brown adipose tissue, isolated hepatocytes and skeletal muscle. The same results were obtained on epidermal-growth-factor receptors. Further, the catalytic subunit exerted a comparable effect on the phosphorylation of highly purified insulin receptors. To explain this inhibition, we were able to rule out the following phenomena: a change in insulin binding, a change in the Km of the enzyme for ATP, activation of a phosphatase activity present in the insulin-receptor preparation, depletion of ATP, and phosphorylation of a serine residue of the receptor. These results suggest that the alteration in the insulin-receptor tyrosine kinase activity induced by cyclic AMP-dependent protein kinase could contribute to the insulin resistance produced by catecholamines.