Добірка наукової літератури з теми "TYROSINE KINASE ASSAY"

Abstract Abstract 708 Aberrantly activated tyrosine kinases and their associated signaling pathways are critical to leukemogenesis and primary acute myeloid leukemia (AML) cell viability. While aberrant kinase activation has been confirmed in a significant percentage of AML, constitutive phosphorylation of STAT5, a marker of tyrosine kinase activation, is present in the majority of AML samples indicating that as yet unidentified tyrosine kinases can be aberrantly activated and contribute to leukemogenesis. Efforts to identify activating tyrosine kinase mutations using high-throughput sequencing have identified low frequency mutations of uncertain functional significance. Because these studies failed to detect additional high-frequency kinase mutations, the identity and mechanism of tyrosine kinase activation may be unique in many AMLs. To avoid the imitations of high-throughput sequencing, we have developed a functional assay that can rapidly and simultaneously identify therapeutic targets while providing therapeutic options. Methods: To rapidly identify activated kinase pathways in individual, primary AML samples, we have developed a small-molecule inhibitor array which includes 90 small-molecule, cell-permeable inhibitor compounds including a core of 36 tyrosine kinase inhibitors that covers the majority of the tyrosine kinome. Many of the inhibitors are available for clinical use or are in clinical development. In this assay, inhibitors were placed in 96-well plates at four serial dilutions to allow IC50 calculations. Three days after adding primary AML cells to each well, we performed an MTS cell viability assay to evaluate the effect of each inhibitor on cell viability. Because most inhibitors affect multiple kinases, we compared target specificities of compounds that decrease primary AML cell viability with those that have no effect to identify potential targets. Results: In preliminary proof-of-principal experiments, we tested leukemia cell lines with known activating tyrosine kinase mutations and Ba/F3 cell lines expressing activated tyrosine kinases. Appropriate inhibitor sensitivity profiles were obtained in CMK cells which depend on a JAK3 A572V mutation for viability, MKPL-1 cells with an activating CSF1R translocation, and in a Ba/F3 line expressing JAK2 V617F. In addition to the primary target, downstream targets were frequently identified; MKPL-1 cells also showed sensitivity to phosphoinositol 3-kinase and NFKB inhibitors. Thus, not only primary targets but the downstream signaling pathways critical to leukemic cell viability can be highlighted using this assay. To date, we have analyzed approximately 150 primary leukemia and lymphoma samples. In some cases, targets could be identified by comparison of overlapping kinase specificities for compounds that decreased leukemic cell viability and subtraction of possible kinase targets inhibited by compounds that had no effect on viability. However, many cases exhibited complex, often unique, inhibitor sensitivity profiles that complicated target identification. Comparison with sensitivity profiles for known aberrantly activated kinases was useful when available. Accordingly, additional leukemia cell lines and Ba/F3 lines that depend on a single aberrantly activated tyrosine kinase for viability are being evaluated. Automated scripts that correlate the leukemic cell inhibitor sensitivity with the inhibitor target specificity are also in preparation. Conclusions: These preliminary data demonstrate that the small-molecule inhibitor functional assays can rapidly identify disease causing genes, provide insights into their mechanism of action, and suggest therapeutic options. The distinct patterns of tyrosine kinase sensitivity in these samples support the hypothesis that tyrosine kinases and related pathways contributing to leukemogenesis in each patient may be different and that targeted therapy will be most effective when administered on an individualized basis. Disclosures: Druker: OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding.

Protein kinases, a class of enzymes that phosphorylate certain tyrosine, serine, and threonine residues, play an important role in cellular functions and are important targets in drug discovery research. Thus, it is of interest to develop a simple assay that can be used to measure protein kinase activity toward specific substrates and is suitable for the high throughput screening (HTS) of potential kinase inhibitors. The scintillation proximity concept has been successfully applied for measuring specific kinase activity using surfaces passively coated with a peptide substrate. In this study, we evaluated kinase assay performance on three ScintiStrip platforms: unmodified surface, streptavidin-coated surface, and streptavidin covalently attached to surface. The high affinity of streptavidin toward biotin-linked peptide substrates makes it a unique platform for measuring specific incorporation of radiolabeled phosphate into selected substrates of specific enzymes in the presence of others. Therefore, this assay may be used with cell extracts containing impure kinases as well as with purified enzymes. The scope of this assay was demonstrated with purified tyrosine kinases (e.g., p60c-src kinase) and A431 cell extracts. This scintillation proximity assay is universal, simple, rapid, accurate, and can be adapted for use with robotics for HTS.

ABSTRACT Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

Cholecystokinin (CCK) is the major pancreatic secretagogue and acinar cell mitogen. This study was performed to determine by which effector systems CCK regulates tyrosine kinases, phosphatidylinositol (PtdIns) 3-kinase, and phospholipase D (PLD) activities. Pancreatic acini loaded with [3H]myristic acid or [3H]inositol were used to assay PLD and PtdIns 3-kinase. G protein activation with NaF increased particulate and crude cytosolic tyrosine kinase and PLD activities. PLD activation was pertussis toxin sensitive. Inhibition of phospholipase C (PLC) slightly reduced caerulein-stimulated particulate tyrosine kinase and blocked crude cytosolic tyrosine kinase activity without affecting caerulein-induced PLD activity. Ca2+ is an important factor in caerulein stimulation of tyrosine kinase and PLD activities. Protein kinase C and tyrosine kinase inhibition abolished caerulein-activated particulate and crude cytosolic tyrosine kinase and PtdIns 3-kinase activities without any effect on PLD. Wortmannin inhibited PLD and PtdIns 3-kinase activation. Caerulein-induced amylase secretion was partially reduced by tyrosine kinase inhibition, with no effect from wortmannin. Caerulein can stimulate a pertussis toxin-insensitive G protein, leading to particulate tyrosine kinase activation and a Ca(2+)-sensitive cytosolic tyrosine kinase through PLC activation. However, PLD activation by caerulein is pertussis toxin sensitive, cytosolic Ca2+ sensitive, and independent of previous PLC and tyrosine kinase activation.