Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein kinase ZAP-70 is involved in T-cell activation, and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs), which are present in three of the subunits of the T-cell receptor. We have studied the tandem SH2 (tSH2) domains of ZAP-70, by both X-ray and NMR. Here, we present the crystal structure of the apoprotein, i.e., the tSH2 domain in the absence of ITAM. Comparison with the previously reported complex structure reveals that binding to the ITAM peptide induces surprisingly large movements between the two SH2 domains and within the actual binding sites. The conformation of the ITAM-free protein is partly governed by a hydrophobic cluster between the linker region and the C-terminal SH2 domain. Our data suggest that the two SH2 domains are able to undergo large interdomain movements. The proposed relative flexibility of the SH2 domains is further supported by the finding that no NMR signals could be detected for the two helices connecting the SH2 domains; these are likely to be broadened beyond detection due to conformational exchange. It is likely that this conformational reorientation induced by ITAM binding is the main signaling event activating the kinase domain in ZAP-70. Another NMR observation was that the N-terminal SH2 domain could bind tetrapeptides derived from the ITAM sequence, apparently without the need to interact with the C-terminal domain. In contrast, the C-terminal domain has little affinity for tetrapeptides. The opposite situation is true for binding to plain phosphotyrosine, where the C-terminal domain has a higher affinity. Distinct features in the crystal structure, showing the interdependence of both domains, explain these binding data.
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PMID:Crystal structure and NMR studies of the apo SH2 domains of ZAP-70: two bikes rather than a tandem. 1245 Mar 81

Spleen tyrosine kinase (Syk), a nonreceptor protein kinase initially found to be expressed only in hemopoietic cells, has now been shown to be expressed in nonhemopoietic cells and to mediate signaling of various cytokines. Whether Syk plays any role in TNF signaling was investigated. Treatment of Jurkat T cells with TNF activated Syk kinase but not ZAP70, another member of Syk kinase family, and the optimum activation occurred at 10 s and with 1 nM TNF. TNF also activated Syk in myeloid and epithelial cells. TNF-induced Syk activation was abolished by piceatannol (Syk-selective inhibitor), which led to the suppression of TNF-induced activation of c- JNK, p38 MAPK, and p44/p42 MAPK. Jurkat cells that did not express Syk (JCaM1, JCaM1/lck) showed lack of TNF-induced Syk, JNK, p38 MAPK, and p44/p42 MAPK activation, as well as TNF-induced IkappaBalpha phosphorylation, IkappaBalpha degradation, and NF-kappaB activation. TNF-induced NF-kappaB activation was enhanced by overexpression of Syk by Syk-cDNA and suppressed when Syk expression was down-regulated by expression of Syk-small interfering RNA (siRNA-Syk). The apoptotic effects of TNF were reduced by up-regulation of NF-kappaB by Syk-cDNA, and enhanced by down-regulation of NF-kappaB by siRNA-Syk. Immunoprecipitation of cells with Syk Abs showed TNF-dependent association of Syk with both TNFR1 and TNFR2; this association was enhanced by up-regulation of Syk expression with Syk-cDNA and suppressed by down-regulation of Syk using siRNA-Syk. Overall, our results demonstrate that Syk activation plays an essential role in TNF-induced activation of JNK, p38 MAPK, p44/p42 MAPK, NF-kappaB, and apoptosis.
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PMID:TNF activates Syk protein tyrosine kinase leading to TNF-induced MAPK activation, NF-kappaB activation, and apoptosis. 1524 Jun 95

Chronic lymphocytic leukaemia (CLL) is a unique malignancy where quiescent B cells accumulate in the peripheral blood. Since clinical outcomes in CLL are very heterogeneous, it is of utmost importance to correctly assess the disease prognosis in each individual case. Recently, it has been shown that high ZAP-70 [Zeta-chain (T-cell receptor) associated protein kinase (70 kDa)] expression level strongly correlates with lack of IgV(H) mutations and poor prognosis in B-CLL. As CLL malignant cells are arrested in G(0), we investigated whether Dipeptidyl Peptidase 2 (DPP2), a serine protease that plays a key role in keeping cells in the quiescent state, is involved in cell-cycle control in CLL. We have previously shown that specific inhibition of DPP2 results in apoptosis of normal lymphocytes. In this study, cell apoptosis experiments were conducted in 38 patients with B-CLL. Two distinct subsets of B-CLL were identified, susceptible and resistant to DPP2-inhibition-induced apoptosis. If resistant to apoptosis (42.1%), the CLL cells have higher expression of ZAP-70 and exhibit a worse prognosis, such as shorter treatment-free time period. Thus, resistance vs. susceptibility to DPP2-inhibiton induced apoptosis can be employed as a novel prognostic factor in CLL.
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PMID:Differential control of G0 programme in chronic lymphocytic leukaemia: a novel prognostic factor. 1568 54

After specific activation, CD8+ cytotoxic T lymphocytes (CTLs) enter a refractory state termed activation-induced nonresponsiveness (AINR) that is characterized by the inability of T cells to respond to a secondary stimulus. Here, we show that T cell receptor triggering results in rapid degradation of the src-family protein kinase lck through a mechanism that is proteasome- and lysosome-independent, sensitive to cysteine protease inhibitors, and distinct from the pathways involved in degradation of ZAP-70 kinase or zeta-chain of the CD3 complex. Pharmacologic blockade of lck degradation, as well as transfection of refractory cells with an lck expression vector, increased responsiveness of CTLs to repeated antigenic challenge. The development or maintenance of AINR was not affected by exogenously added IL-2, whereas IL-15 or IFN-alpha restored both lck expression and responsiveness of preactivated CTLs. Our results suggest that lck degradation plays an important role in the development of AINR in human CTLs and that this condition can be reverted by pharmacologic agents or lymphokines that prevent lck degradation or induce its expression.
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PMID:Regulation of lck degradation and refractory state in CD8+ cytotoxic T lymphocytes. 1595 29

The protein kinase ZAP-70 is involved in T-cell activation and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs). We have studied the regulatory phosphorylation sites in the tryptic fragment containing amino acids 485-496 (ALGADDSYYTAR). The four possible peptides with phosphorylation at none, one, or both of the Y-492 and Y-493 tyrosines were specifically synthesized and analyzed by (1)H/(13)C-NMR at 600 MHz using a capillary HPLC-NMR microprobe. Unambiguous discrimination of the peptides was possible via effect of chemical shifts of phosphorylation on the aromatic tyrosine protons. With the microprobe and the detection volume of 1.5 microl, it was possible to perform structure elucidation with the very small amounts available for the various peptides. For the syringe injection, 15 microg of the analyte were used (corresponding to ca 2 mg in classical 5-mm tubes). Capillary HPLC-NMR spectra were recorded in the stopped-flow mode from less than 400 ng of each peptide, using 1D and 2D techniques ((1)H,(1)H-COSY-90, (1)H/(13)C-HSQC, and (1)H/(13)C-HMBC).
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PMID:Determination of regulatory phosphorylation sites in nanogram amounts of a synthetic fragment of ZAP-70 using microprobe NMR and on-line coupled capillary HPLC-NMR. 1604 44

The interactions between peptide/MHC complexes and their cognate TCR are essential for various T cell responses. However, the relationship between the avidity of TCR ligand and the subsequent intracellular signaling through the TCR is still unclear. To investigate the effects of TCR ligand avidity on TCR-mediated signaling, we established L cells expressing HLA-DR4 molecules covalently linked with agonistic peptide (high-affinity ligand) or altered peptide ligand (APL; low-affinity ligand) at various densities as APC for a cognate human CD4(+) T cell clone. Using this system, we demonstrated that the T cell clone stimulated with APL/HLA-DR4 complexes presented at an excessive density provoked the up-regulation of CD69, IL-2 production and proliferation, but no detectable phosphorylation of ZAP-70/LAT/SLP-76. Furthermore, in contrast to the high-affinity stimulation, the low-affinity stimulation evoked delayed and sustained activation of the B-Raf/extracellular signal-regulated kinase (ERK) pathway without Raf-1 activation. The strength and duration of B-Raf/ERK activations closely correlated with the density of the TCR ligand. A knockdown approach confirmed that B-Raf activation was indispensable for the APL-induced T cell responses. These observations suggest that the differences in TCR-peptide/MHC interactions reflect the strength and duration of B-Raf/Raf-1/ERK activation in the human CD4(+) T cells.
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PMID:TCR ligand avidity determines the mode of B-Raf/Raf-1/ERK activation leading to the activation of human CD4+ T cell clone. 1679 76

Lack of immunoglobulin heavy chain genes (IgV(H)) mutation in patients with chronic lymphocytic leukemia (CLL) is associated with rapid disease progression and shorter survival. The zeta-chain (T-cell receptor) associated protein kinase 70 kDa (ZAP-70) has been reported to be a surrogate marker for IgV(H) mutation status, and its expression in leukemic cells correlates with unmutated IgV(H). However, ZAP-70 detection by flow cytometry varies significantly dependant on the antibodies used, the method of performing the assay, and the condition of the cells in the specimen. The clinical value of ZAP-70 testing when samples are shipped under poorly controlled conditions is not known. Furthermore, testing in a research environment may differ from testing in a routine clinical laboratory. We validated an assay for ZAP-70 by comparing results with clinical outcome and the mutation status of the IgV(H). Using stored samples, we show significant correlation between ZAP-70 expression and clinical outcome as well as IgV(H) mutation at a cut-off point of 15%. While positive samples (>15% positivity) remain positive when kept in the laboratory environment for 48 h after initial testing, results obtained from samples from CLL patients tested after shipping at room temperature for routine testing showed no correlation with IgV(H) mutation status when 15% cut-off was used. In these samples, cut-point of 10% correlated with the IgV(H) mutation (P = 0.0001). This data suggests that although ZAP-70 positivity correlates with IgV(H) mutation status and survival, variations in sample handling and preparation may influence results. We show that IgV(H) mutation results, unlike ZAP-70 remain correlated with CD38 expression and beta-2 microglobulin in shipped samples, and ZAP-70 testing should not be used as the sole criterion for stratifying patients for therapy.
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PMID:Variations in the detection of ZAP-70 in chronic lymphocytic leukemia: Comparison with IgV(H) mutation analysis. 1690 85

Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.
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PMID:Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death. 1733 28

ITK (IL-2-inducible T cell kinase), a Tec family protein tyrosine kinase (PTK), is one of three PTKs required for T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1). Like Src and Abl family PTKs, ITK adopts an inactive, "closed" conformation, and its conversion to the active conformation is not well understood, nor have its direct substrates been identified. In a side-by-side comparison of ITK and ZAP-70 (zeta chain-associated protein kinase of 70 kDa), ITK efficiently phosphorylated Y(783) and Y(775) of PLC-gamma1, two phosphorylation sites that are critical for its activation, whereas ZAP-70 did not. SLP-76 (SH2-domain-containing leukocyte protein of 76 kDa), an adaptor required for TCR-induced activation of PLC-gamma1, was required for the phosphorylation of both PLC-gamma1 sites in intact cells. Furthermore, this event depended on the N-terminal tyrosines of SLP-76. Likewise, SLP-76, particularly its N-terminal tyrosines, was required for TCR-induced tyrosine phosphorylation and activation of ITK but was not required for the phosphorylation or activation of ZAP-70. Both ZAP-70 and ITK phosphorylated SLP-76 in vitro; thus, both PTKs are potential regulators of SLP-76, but only ITK is regulated by SLP-76. Upon TCR stimulation, a small fraction of ITK bound to SLP-76. This fraction, however, encompassed most of the catalytically active ITK. Catalytic activity was lost upon mild elution of ITK from the SLP-76-nucleated complex but was restored upon reconstitution of the complex. We propose that SLP-76 is required for ITK activation; furthermore, an ongoing physical interaction between SLP-76 and ITK is required to maintain ITK in an active conformation.
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PMID:SLP-76 mediates and maintains activation of the Tec family kinase ITK via the T cell antigen receptor-induced association between SLP-76 and ITK. 1742 Apr 79

The protein tyrosine kinase zeta-chain associated protein kinase (ZAP70), normally expressed in T cells and a subset of B cells, is solely expressed in poor prognosis chronic lymphocytic leukaemia and implicated in enhanced B cell receptor signalling. As a result, the expression of this protein provides an ideal prognostic marker for the disease. A previous study has shown differential CpG methylation of a 5' region of ZAP70 in leukaemic lymphoid cells, although no further epigenetic studies have been reported. Further investigation into the expression of ZAP70 may therefore provide targets for therapies.
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PMID:ZAP70 in chronic lymphocytic leukaemia. 1762 48


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