Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRgamma (Fc receptor gamma) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRgamma chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP-76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cgamma2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (approximately 10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRgamma chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti-(alpha2 integrin) antibodies Ha1/29 and HMalpha2, but not by blockade of alphaIIbbeta3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI-FcRgamma chain complex, and is facilitated by binding of collagen to integrin alpha2beta1.
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PMID:Glycoprotein VI/Fc receptor gamma chain-independent tyrosine phosphorylation and activation of murine platelets by collagen. 1528 2

Small molecule inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were developed as antagonists of protein tyrosine kinases implicated in cancer progression. Derivatives from this compound class are effective against most of the imatinib mesylate-resistant BCR-ABL mutants isolated from advanced chronic myeloid leukemia patients. Here, we established an efficient proteomics method employing an immobilized pyrido[2,3-d]pyrimidine ligand as an affinity probe and identified more than 30 human protein kinases affected by this class of compounds. Remarkably, in vitro kinase assays revealed that the serine/threonine kinases Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) and p38alpha were among the most potently inhibited kinase targets. Thus, pyrido[2,3-d]pyrimidines did not discriminate between tyrosine and serine/threonine kinases. Instead, we found that these inhibitors are quite selective for protein kinases possessing a conserved small amino acid residue such as threonine at a critical site of the ATP binding pocket. We further demonstrated inhibition of both p38 and RICK kinase activities in intact cells upon pyrido[2,3-d]pyrimidine inhibitor treatment. Moreover, the established functions of these two kinases as signal transducers of inflammatory responses could be correlated with a potent in vivo inhibition of cytokine production by a pyrido[2,3-d]pyrimidine compound. Thus, our data demonstrate the utility of proteomic methods employing immobilized kinase inhibitors for identifying new targets linked to previously unrecognized therapeutic applications.
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PMID:Chemical proteomic analysis reveals alternative modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors. 1547 68

To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein-ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein-ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affinity ranking of the mixture components. The technique requires no radiolabel incorporation or direct biochemical assay, no modification or immobilization of the compounds or target protein, and all reaction components, including any buffers or cofactors required for protein stability, are free in solution. We demonstrate the method for several compounds of wide structural variety against representatives of the most important protein classes in contemporary drug discovery, including novel ATP-competitive and allosteric inhibitors of the Akt-1 (PKB) and Zap-70 kinases, and previously undisclosed antagonists of the M(2) muscarinic acetylcholine receptor, a G-protein coupled receptor (GPCR). The theoretical basis of the technique is analyzed mathematically, allowing quantitative estimation of binding affinities and, in the case of allosteric interaction, absolute determination of binding cooperativity. The method is readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.
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PMID:A general technique to rank protein-ligand binding affinities and determine allosteric versus direct binding site competition in compound mixtures. 1556 78

Peroxynitrite, the product of the reaction between *NO and O2*-, is a strong oxidant and nitrating molecule, and it has been recently consideredas a component of some important signaling pathways. Herein, we report the effect of peroxynitrite on glucose uptake in 3T3-L1 adipocytes. Peroxynitrite stimulated glucose uptake and this effect was inhibited by citochalasin B, indicating the participation of facilitated GLUT transporters. Peroxynitrite-induced glucose uptake was not related to intracellular ATP, nor to external or internal calcium, but it was inhibited by the phosphatidylinositol 3-kinase (PI3-K) inhibitor, wortmannin. Additionally, we also found that peroxynitrite did not activate the insulin receptor nor the PI3-K downstream signaling protein kinase B (PKB/Akt). The dose-dependent inhibitory action of wortmannin suggests that peroxynitrite activates glucose transport without affecting GLUT transporters translocation.
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PMID:Peroxynitrite activates glucose uptake in 3T3-L1 adipocytes through a PI3-K-dependent mechanism. 1557 46

During the past year, crystal structures of the PDK-1, ITK, Aurora-A, c-KIT and FLT-3 protein kinases in complex with several ATP-competitive inhibitors have been determined. Some structures have crystallized in catalytically active conformations, whereas others appear to be in inactive or native conformations. The differences between these two classes of structures provide further understanding of how kinase activity may be self-regulated in the cellular environment and how phosphorylation can modulate signalling at a molecular level. All of these structures provide a basis for designing selective protein kinase inhibitors of use in the treatment of cancer and autoimmune disease.
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PMID:Novel protein kinases and molecular mechanisms of autoinhibition. 1558 94

The present study examines the role of Pyk2 in acid regulation of sodium/hydrogen exchanger 3 (NHE3) activity in OKP cells, a kidney proximal tubule epithelial cell line. Incubation of OKP cells in acid media caused a transient increase in Pyk2 phosphorylation that peaked at 30 seconds and increased Pyk2/c-Src binding at 90 seconds. Pyk2 isolated by immunoprecipitation and studied in a cell-free system was activated and phosphorylated at acidic pH. Acid activation of Pyk2 (a) was specific for Pyk2 in that acid did not activate focal adhesion kinase, (b) required calcium, and (c) was associated with increased affinity for ATP. Transfection of OKP cells with dominant-negative pyk2(K457A) or small interfering pyk2 duplex RNA blocked acid activation of NHE3, while neither had an effect on glucocorticoid activation of NHE3. In addition, pyk2(K457A) blocked acid activation of c-Src kinase, which is also required for acid regulation of NHE3. The present results demonstrate that Pyk2 is directly activated by acidic pH and that Pyk2 activation is required for acid activation of c-Src kinase and NHE3. Given that partially purified Pyk2 can be activated by acid in a cell-free system, Pyk2 may serve as the pH sensor that initiates the acid-regulated signaling cascade involved in NHE3 regulation.
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PMID:Pyk2 activation is integral to acid stimulation of sodium/hydrogen exchanger 3. 1559 93

Imatinib is a tyrosine kinase inhibitor that has been reported to specifically inhibit the growth of bcr-abl expressing chronic myeloid leukaemia progenitors. This drug functions by blocking the ATP-binding site of the kinase domain of bcr-abl, and has also been found to inhibit the c-abl, platelet-derived growth factor receptor, ARG and stem cell factor receptor tyrosine kinases. Reports have recently emerged demonstrating that imatinib also inhibits the growth of non-malignant haemopoietic cells. Here, we demonstrate that concentrations of imatinib within the therapeutic dose range inhibit the function of cultured monocytes (CM) from normal donors. A decrease in the response of CM to LPS was observed morphologically and functionally, with CM grown in the presence of imatinib showing decreased pseudopodia formation and inhibition of IL-6 and TNF-alpha production following LPS stimulation. Imatinib also reduced the ability of M-CSF and GM-CSF stimulated CM to phagocytose zymosan particles, with uptake of non-opsonized zymosan by M-CSF stimulated CM (M-CM) being most affected. M-CM that had been cultured in the presence of imatinib were also impaired in their ability to stimulate responder cells in a mixed lymphocyte reaction. These results demonstrate that human monocytes cultured in the presence of imatinib are functionally impaired, and suggest that imatinib displays inhibitory activity against other kinase(s) that play a role in monocyte/macrophage development.
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PMID:Imatinib inhibits the functional capacity of cultured human monocytes. 1566 Oct 41

The ability of a cell to detect an external chemical signal and initiate a program of directed migration along a gradient comprises the fundamental process called chemotaxis. Investigations in Dictyostelium discoideum and neutrophils have established that pleckstrin homology (PH) domain-containing proteins that bind to the PI3K products PI(3,4)P2 and PI(3,4,5)P3, such as CRAC (cytosolic regulator of adenylyl cyclase) and Akt/PKB, translocate specifically to the leading edge of chemotaxing cells. CRAC is essential for the chemoattractant-mediated activation of the adenylyl cyclase ACA, which converts ATP into cAMP, the primary chemoattractant for D. discoideum. The mechanisms by which CRAC activates ACA remain to be determined. We now show that in addition to its essential role in the activation of ACA, CRAC is involved in regulating chemotaxis. Through mutagenesis, we show that these two functions are independently regulated downstream of PI3K. A CRAC mutant that has lost the capacity to bind PI3K products does not support chemotaxis and shows minimal ACA activation. Finally, overexpression of CRAC and various CRAC mutants show strong effects on ACA activation with little effect on chemotaxis. These findings establish that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and adenylyl cyclase activation.
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PMID:The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis. 1566 69

Imatinib, which is an inhibitor of the BCR-ABL tyrosine kinase, has been a remarkable success for the treatment of Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemias (CMLs). However, a significant proportion of patients chronically treated with imatinib develop resistance because of the acquisition of mutations in the kinase domain of BCR-ABL. Mutations occur at residues directly implicated in imatinib binding or, more commonly, at residues important for the ability of the kinase to adopt the specific closed (inactive) conformation to which imatinib binds. In our quest to develop new BCR-ABL inhibitors, we chose to target regions outside the ATP-binding site of this enzyme because these compounds offer the potential to be unaffected by mutations that make CML cells resistant to imatinib. Here we describe the activity of one compound, ON012380, that can specifically inhibit BCR-ABL and induce cell death of Ph+ CML cells at a concentration of <10 nM. Kinetic studies demonstrate that this compound is not ATP-competitive but is substrate-competitive and works synergistically with imatinib in wild-type BCR-ABL inhibition. More importantly, ON012380 was found to induce apoptosis of all of the known imatinib-resistant mutants at concentrations of <10 nM concentration in vitro and cause regression of leukemias induced by i.v. injection of 32Dcl3 cells expressing the imatinib-resistant BCR-ABL isoform T315I. Daily i.v. dosing for up to 3 weeks with a >100 mg/kg concentration of this agent is well tolerated in rodents, without any hematotoxicity.
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PMID:A non-ATP-competitive inhibitor of BCR-ABL overrides imatinib resistance. 1567 19

Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.
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PMID:A small molecule-kinase interaction map for clinical kinase inhibitors. 1627 56


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