Ntk: a Csk-related protein-tyrosine kinase expressed in brain and T lymphocytes.
ABSTRACT: The activity of Src-related protein-tyrosine kinases is repressed by the phosphorylation of a conserved carboxyl-terminal tyrosine by another cytoplasmic protein-tyrosine kinase termed p50csk. In this study, we characterize Ntk, a protein-tyrosine kinase bearing striking similarities to p50csk. Like p50csk, Ntk possesses Src homology 3 and Src homology 2 domains and lacks the consensus tyrosine phosphorylation and myristoylation sites found in members of the Src family. Expression of ntk transcripts was maximal in brain, and was observed at significant levels in thymus and spleen. ntk RNA levels were dramatically reduced upon mitogenic stimulation of normal T lymphocytes and were minimal in transformed T-cell populations. Firm evidence that Ntk is a Csk-related enzyme was provided by the observation that it phosphorylated a Src-related polypeptide on the inhibitory carboxyl-terminal tyrosine. These findings indicate that Ntk is a Csk-related enzyme that may play an inhibitory role in the control of T-cell proliferation.
Project description:We used the polymerase chain reaction with degenerate oligonucleotide primers to search for Csk-related kinases. A cDNA coding for a Csk-like protein-tyrosine kinase was cloned from mouse brain and was designated ctk, for csk-type protein-tyrosine kinase. The 1.9-kb ctk mRNA was found to be expressed predominantly in brain and capable of encoding a 52-kDa protein-tyrosine kinase. The amino acid sequence of Ctk was found to possess 53% identity with mouse Csk, shared all the predicted structural features of Csk, and was capable of phosphorylating the carboxyl-terminal conserved tyrosine of Src family members. Our results thereby indicate that ctk represents a gene that defines a family of structurally and functionally related Csk-like protein-tyrosine kinases.
Project description:Fibrinogen binding to activated integrin induces outside-in signaling that results in stable platelet aggregates and clot retraction. How integrin ?IIb?3 is discouraged from spontaneous activation is not known. We have recently shown that junctional adhesion molecule-A (JAM-A) renders protection from thrombosis by suppressing integrin outside-in signaling. In this study, we show that JAM-A associates with integrin ?IIb?3 in resting platelets and dissociates upon platelet activation by agonists. We also show that integrin-associated JAM-A is tyrosine phosphorylated and is rapidly dephosphorylated upon platelet activation. C-terminal Src kinase (Csk) binds to tyrosine phosphorylated JAM-A through its Src homology 2 domain. Thus, JAM-A recruits Csk to the integrin-c-Src complex in resting platelets. Csk, in turn, keeps integrin-associated c-Src in an inactive state by phosphorylating Y(529) in its regulatory domain. Absence of JAM-A results in impaired c-SrcY(529) phosphorylation and augmentation of outside-in signaling-dependent c-Src activation. Our results strongly suggest that tyrosine-phosphorylated JAM-A is a Csk-binding protein and functions as an endogenous inhibitor of integrin signaling. JAM-A recruits Csk to the integrin-c-Src complex, where Csk negatively regulates c-Src activation, thereby suppressing the initiation of outside-in signaling. Upon agonist stimulation, JAM-A is dephosphorylated on the tyrosine, allowing the dissociation of Csk from the integrin complex, and thus facilitating outside-in signaling.
Project description:The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We have determined the crystal structure of a complex between the kinase domains of Csk and c-Src at 2.9 A resolution, revealing that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate binding site used by most tyrosine kinases to recognize substrates is destabilized in Csk by a deletion in the activation loop.
Project description:The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.
Project description:SFKs (Src family kinases) contribute importantly to platelet function in haemostasis. SFK activity is controlled by Csk (C-terminal Src kinase), which phosphorylates a C-terminal tyrosine residue on SFKs, resulting in inhibition of SFK activity. Csk is recruited to sites of SFK activity by tyrosine-phosphorylated Csk-binding proteins. Paxillin, a multidomain adaptor protein, has been shown to act as a Csk-binding protein and to inhibit Src activity during growth factor signalling. Human platelets express Hic-5, a member of the paxillin family; however, its ability to act as a Csk-binding protein has not been characterized. We sought to identify and characterize the ability of paxillin family members to act as Csk-binding proteins during platelet activation. We found that murine and human platelets differ in the complement of paxillin family members expressed. Human platelets express Hic-5, whereas murine platelets express paxillin and leupaxin in addition to Hic-5. In aggregating human platelets, Hic-5 was tyrosine phosphorylated and recruited Csk via its SH2 domains. In aggregating murine platelets, however, Csk bound preferentially to paxillin, even though both paxillin and Hic-5 were abundantly present and became tyrosine phosphorylated. The SFK Lyn, but not Src or Fyn, was associated with paxillin family members in resting and aggregated human and murine platelets. Lyn, however, was phosphorylated on its C-terminal inhibitory tyrosine residue only following platelet aggregation, which was coincident with recruitment of Csk to paxillin and/or Hic-5 in a manner dependent on prior alpha(IIb)beta3 engagement. These observations support the notion that Hic-5 and paxillin function as negative feedback regulators of SFKs in aggregated platelets and that, when both are present, paxillin is preferentially used.
Project description:SRC kinase is activated in castration resistant prostate cancer (CRPC), phosphorylates the androgen receptor (AR), and causes its ligand-independent activation as a transcription factor. However, activating SRC mutations are exceedingly rare in human tumors, and mechanisms of ectopic SRC activation therefore remain largely unknown. Performing a functional genomics screen, we found that downregulation of SRC inhibitory kinase CSK is sufficient to overcome growth arrest induced by depriving human prostate cancer cells of androgen. CSK knockdown led to ectopic SRC activation, increased AR signaling, and resistance to anti-androgens. Consistent with the in vitro observations, stable knockdown of CSK conferred castration resistance in mouse xenograft models, while sensitivity to the tyrosine kinase inhibitor dasatinib was retained. Finally, CSK was found downregulated in a distinct subset of CRPCs marked by AR amplification and ETS2 deletion but lacking PTEN and RB1 mutations. These results identify CSK downregulation as a principal driver of SRC activation and castration resistance and validate SRC as a drug target in a molecularly defined subclass of CRPCs.
Project description:Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the ?B/?C loop of the SH2 domain.
Project description:BACKGROUND:C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined. METHODS:We determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells. RESULTS:Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain. CONCLUSIONS:SFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells.
Project description:Csk and Src protein tyrosine kinases are structurally homologous but use opposite regulatory strategies. The isolated catalytic domain of Csk is intrinsically inactive and is activated by interactions with the regulatory Src homology 3 (SH3) and SH2 domains, while the isolated catalytic domain of Src is intrinsically active and is suppressed by interactions with the regulatory SH3 and SH2 domains. The structural basis for why one isolated catalytic domain is intrinsically active while the other is inactive is not clear. In this study, we identified structural elements in the N-terminal lobe of the catalytic domain that render the Src catalytic domain active. These structural elements include the alpha-helix C region, a beta turn between the beta4 and beta5 strands, and an Arg residue at the beginning of the catalytic domain. These three motifs interact with one another to activate the Src catalytic domain, but the equivalent motifs in Csk directly interact with the regulatory domains that are important for Csk activation. The Src motifs can be grafted to the Csk catalytic domain to obtain an active Csk catalytic domain. These results, together with available Src and Csk tertiary structures, reveal an important structural switch that determines the kinase activity of a catalytic domain and dictates the regulatory strategy of a kinase.
Project description:The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2?M sodium bromide, 0.1?M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best crystals diffracted to ?1.6?Å resolution. The crystals belonged to space group P2, with unit-cell parameters a=25.8, b=34.6, c=63.2?Å, ?=99.4°.