Siga este enlace para ver otros tipos de publicaciones sobre el tema: TYROSINE KINASE ASSAY.
Artículos de revistas sobre el tema "TYROSINE KINASE ASSAY"
Crea una cita precisa en los estilos APA, MLA, Chicago, Harvard y otros
Consulte los 50 mejores artículos de revistas para su investigación sobre el tema "TYROSINE KINASE ASSAY".
Junto a cada fuente en la lista de referencias hay un botón "Agregar a la bibliografía". Pulsa este botón, y generaremos automáticamente la referencia bibliográfica para la obra elegida en el estilo de cita que necesites: APA, MLA, Harvard, Vancouver, Chicago, etc.
También puede descargar el texto completo de la publicación académica en formato pdf y leer en línea su resumen siempre que esté disponible en los metadatos.
Explore artículos de revistas sobre una amplia variedad de disciplinas y organice su bibliografía correctamente.
1
Tyner, Jeffrey W., Stephanie Willis, Michael W. N. Deininger y Brian J. Druker. "RNAi Functional Screening of the Tyrosine Kinome Identifies Therapeutic Targets in Acute Myeloid Leukemia Patients." Blood 110, n.º 11 (16 de noviembre de 2007): 208. http://dx.doi.org/10.1182/blood.v110.11.208.208.
Texto completoResumen
Abstract A large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of cancer, but identification of specific tyrosine kinases as cancer targets has been a slow process. Tyrosine kinases are thought to play a causative role in acute myeloid leukemia (AML), based in part on the high percentage of cases with phosphorylation of STAT5—a marker for activity of tyrosine kinase signaling. However, known abnormalities in tyrosine kinases in AML are restricted to internal tandem duplications of FLT3 or genetic aberrations in FLT3, c-KIT, and a few other genes. Thus, the specific tyrosine kinases that form the basis for targeted intervention have remained unclear in the majority of AML patients. Here, we report a novel assay by which cells from AML patients are functionally screened with RNAi to elucidate tyrosine kinase targets amenable to therapeutic intervention. Methods: To determine targets necessary for viability of malignant cells, we screened cell lines as well as primary cells from AML patients by electroporating siRNAs individually targeting each member of the tyrosine kinase family. Four days later, we determined the cell viability and tabulated sensitivity of the cells to any individual tyrosine kinase. Where possible, results were confirmed by treating samples with small-molecule inhibitors with activity against the genes identified by the assay. In addition, the mechanism of oncogenesis was investigated for each positive result. Results: We demonstrate that siRNA screening can identify tyrosine kinase targets containing activating mutations in JAK3 (A572V) in CMK cells (Figure 1) and c-KIT (V560G) in HMC1.1 cells. In addition, this assay identifies targets that do not contain mutations, such as JAK1 (Figure 1) and the focal adhesion kinases, yet are still crucial to the survival of the cells. We have also used this assay to determine sensitivity of numerous primary AML samples to inhibition of individual tyrosine kinases. Candidate targets found in primary samples include FLT1, PDGFR, JAK1/3, JAK2, CSF1R, ROR1, and EPHA5. Studies using small-molecule kinase inhibitors have confirmed sensitivity of specific samples to inhibition of target genes identified by the assay. Finally, the mechanism of oncogenesis and its relation to the gene target has been established in select samples with genetic abnormalities ranging from point mutations and insertional mutations to evidence of chromosomal rearrangements. Conclusions: We demonstrate that RNAi functional screening can determine sensitivity to individual tyrosine kinases, both in cell lines and in primary samples. For the first time, this technique offers the potential to match specific therapies for targeted intervention with individual patients based on a functional assay. Figure 1. CMK cells were transfected with an siRNA library individually targeting each member of the tyrosine kinase family, N-RAS, K-RAS, and non-specific controls. Cell viability was determined by an MTS assay 4 days later. Each bar represents an individual kinase with values shown as percent mean ± s.e.m (n = 3) (normalized to non-specific controls). Figure 1. CMK cells were transfected with an siRNA library individually targeting each member of the tyrosine kinase family, N-RAS, K-RAS, and non-specific controls. Cell viability was determined by an MTS assay 4 days later. Each bar represents an individual kinase with values shown as percent mean ± s.e.m (n = 3) (normalized to non-specific controls).
2
Pritz, Stephan, Gabriele Meder, Klaus Doering, Peter Drueckes, Julian Woelcke, Lorenz M. Mayr y Ulrich Hassiepen. "A Fluorescence Lifetime-Based Assay for Abelson Kinase". Journal of Biomolecular Screening 16, n.º 1 (8 de diciembre de 2010): 65–72. http://dx.doi.org/10.1177/1087057110385817.
Texto completoResumen
We present a novel homogeneous in vitro assay format and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. The assay employs a short peptidic substrate containing a single tyrosine and a single probe attached via a cysteine side chain. The structural flexibility of the peptide allows for the dynamic quenching of the probe by the nonphosphorylated tyrosine side chain. The probe responds with changes in its fluorescence lifetime depending on the phosphorylation state of the tyrosine. We use this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as an antibody against the phosphotyrosine. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay format suitable to a wide variety of other tyrosine kinases.
3
Lebakken, Connie S., Hee Chol Kang y Kurt W. Vogel. "A Fluorescence Lifetime–Based Binding Assay to Characterize Kinase Inhibitors". Journal of Biomolecular Screening 12, n.º 6 (21 de mayo de 2007): 828–41. http://dx.doi.org/10.1177/1087057107304480.
Texto completoResumen
The authors present a fluorescence lifetime—based kinase binding assay that identifies and characterizes compounds that bind to the adenosine triphosphate (ATP)—binding pocket of a range of tyrosine and serine/threonine kinases. The assay is based on displacement of an Alexa Fluor® 647 conjugate of staurosporine from the ATP-binding site of a kinase, which is detected by a change in the fluorescence lifetime of the probe between the free (displaced) and kinase-bound states. The authors screened 257 kinases for specific binding and displacement of the Alexa Fluor® 647-staurosporine probe and found that approximately half of the kinases tested could potentially be assayed with this method. They present inhibitor binding data against 4 selected serine/threonine kinases and 4 selected tyrosine kinases, using 6 commonly used kinase inhibitors. Two of these kinases were chosen for further studies, in which inhibitor binding data were compared to inhibition of kinase activity using 2 separate activity assay formats. Rank-order potencies of compounds were similar, but not identical, between the binding and activity assays. It was postulated that these differences could be caused by the fact that the assays are measuring distinct phenomena, namely, activity versus binding, and in a purified recombinant kinase preparation, there can exist a mixture of active and nonactivated kinases. To explore this possibility, the authors compared binding affinity for the probe using 2 kinases in their respective nonactivated and activated (phosphorylated) forms and found a kinase-dependent difference between the 2 forms. This assay format therefore represents a simple method for the identification and characterization of small-molecule kinase inhibitors that may be useful in screening a wide range of kinases and may be useful in identifying small molecules that bind to kinases in their active or nonactivated states. ( Journal of Biomolecular Screening 2007:828-841)
4
Kobayashi, Tomoko, Shun-Ichi Nakamura y Hirohei Yamamura. "Cytosolic Protein-Tyrosine Kinase Activities in Various Rat Tissues". Annals of Clinical Biochemistry: International Journal of Laboratory Medicine 26, n.º 2 (marzo de 1989): 164–68. http://dx.doi.org/10.1177/000456328902600213.
Texto completoResumen
Suitable assay conditions for the detection of cytosolic protein-tyrosine kinase activities in crude extracts of various rat tissues have been determined. Cytosolic protein-tyrosine kinases showed common characteristics including substrate specificity and divalent cation requirement. Using (Val5) angiotensin II and Mn2+ rather than a src-related synthetic peptide, E11G1, and Mg2+, we obtained higher activities of cytosolic protein-tyrosine kinases. Among various rat tissues tested, spleen, bone marrow, thymus, small intestine, appendix and lung, in decreasing order of total activity, contained high activities of cytosolic protein-tyrosine kinases. These results suggest that the enzyme activities in lymphatic organs and in organs closely related to cell proliferation are high. The assay system described allows the precise measurement of cytosolic protein-tyrosine kinase activity in various rat tissues, both normal and malignant.
5
Tyner, Jeffrey W., Denise K. Walters, Stephanie G. Willis, Mary Luttropp, Jason Oost, Marc Loriaux, Heidi Erickson et al. "RNAi screening of the tyrosine kinome identifies therapeutic targets in acute myeloid leukemia". Blood 111, n.º 4 (15 de febrero de 2008): 2238–45. http://dx.doi.org/10.1182/blood-2007-06-097253.
Texto completoResumen
Despite vast improvements in our understanding of cancer genetics, a large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of numerous types of cancer, but identification and validation of tyrosine kinase targets in cancer can be a time-consuming process. We report the establishment of an efficient, functional screening assay using RNAi technology to directly assess and compare the effect of individually targeting each member of the tyrosine kinase family. We demonstrate that siRNA screening can identify tyrosine kinase targets containing activating mutations in Janus kinase (JAK) 3 (A572V) in CMK cells and c-KIT (V560G) in HMC1.1 cells. In addition, this assay identifies targets that do not contain mutations, such as JAK1 and the focal adhesion kinases (FAK), that are crucial to the survival of the cancer cells. This technique, with additional development, might eventually offer the potential to match specific therapies with individual patients based on a functional assay.
6
Wu, Jinzi J., Donna R. Yarwood, Quynhchi Pham y Matthew A. Sills. "Identification of a High-Affinity Anti-Phosphoserine Antibody for the Development of a Homogeneous Fluorescence Polarization Assay of Protein Kinase C". Journal of Biomolecular Screening 5, n.º 1 (febrero de 2000): 23–30. http://dx.doi.org/10.1177/108705710000500106.
Texto completoResumen
In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions. Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available. Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 ± 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO4)LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO4)LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates. Phosphatidylserine, which enhances the kinase activity of PKC in a Ca2+-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay. The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.
7
Tyner, Jeffrey W., Luke Fletcher, Wayne Yang, Stephen T. Oh, Jason R. Gotlib, Michael WN Deininger, Brian J. Druker y Marc Loriaux. "Development of a Small-Molecule Inhibitor Screen to Rapidly Identify Key Signaling Pathways in Leukemogenesis." Blood 114, n.º 22 (20 de noviembre de 2009): 708. http://dx.doi.org/10.1182/blood.v114.22.708.708.
Texto completoResumen
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.
8
Nakayama, Grace R., Michael P. Nova y Zahra Parandoosh. "A Scintillating Microplate Assay for the Assessment of Protein Kinase Activity". Journal of Biomolecular Screening 3, n.º 1 (febrero de 1998): 43–48. http://dx.doi.org/10.1177/108705719800300106.
Texto completoResumen
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.
9
Binns, Kathleen L., Paul P. Taylor, Frank Sicheri, Tony Pawson y Sacha J. Holland. "Phosphorylation of Tyrosine Residues in the Kinase Domain and Juxtamembrane Region Regulates the Biological and Catalytic Activities of Eph Receptors". Molecular and Cellular Biology 20, n.º 13 (1 de julio de 2000): 4791–805. http://dx.doi.org/10.1128/mcb.20.13.4791-4805.2000.
Texto completoResumen
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.
10
Rivard, N., G. Rydzewska, J. S. Lods y J. Morisset. "Novel model of integration of signaling pathways in rat pancreatic acinar cells". American Journal of Physiology-Gastrointestinal and Liver Physiology 269, n.º 3 (1 de septiembre de 1995): G352—G362. http://dx.doi.org/10.1152/ajpgi.1995.269.3.g352.
Texto completoResumen
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.
11
Dailey, D., G. L. Schieven, M. Y. Lim, H. Marquardt, T. Gilmore, J. Thorner y G. S. Martin. "Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity". Molecular and Cellular Biology 10, n.º 12 (diciembre de 1990): 6244–56. http://dx.doi.org/10.1128/mcb.10.12.6244-6256.1990.
Texto completoResumen
Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.
12
Dailey, D., G. L. Schieven, M. Y. Lim, H. Marquardt, T. Gilmore, J. Thorner y G. S. Martin. "Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity." Molecular and Cellular Biology 10, n.º 12 (diciembre de 1990): 6244–56. http://dx.doi.org/10.1128/mcb.10.12.6244.
Texto completoResumen
Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.
13
Nevenzel, Hadas, Amit Zur y Doron Gerber. "A Microfluidic-Based Tyrosine Kinase and Phosphatase Assay". Biophysical Journal 102, n.º 3 (enero de 2012): 187a—188a. http://dx.doi.org/10.1016/j.bpj.2011.11.1023.
Texto completo
14
Kashem, Mohammed A., Richard M. Nelson, Jeffrey D. Yingling, Steven S. Pullen, Anthony S. Prokopowicz, Jessi Wildeson Jones, John P. Wolak et al. "Three Mechanistically Distinct Kinase Assays Compared: Measurement of Intrinsic ATPase Activity Identified the Most Comprehensive Set of ITK Inhibitors". Journal of Biomolecular Screening 12, n.º 1 (12 de noviembre de 2006): 70–83. http://dx.doi.org/10.1177/1087057106296047.
Texto completoResumen
Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)–compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.
15
MELANDER, Fredrik, Tommy ANDERSSON y Karim DIB. "Fgr but not Syk tyrosine kinase is a target for beta2 integrin-induced c-Cbl-mediated ubiquitination in adherent human neutrophils". Biochemical Journal 370, n.º 2 (1 de marzo de 2003): 687–94. http://dx.doi.org/10.1042/bj20021201.
Texto completoResumen
An early and critical event in β2 integrin signalling during neutrophil adhesion is activation of Src tyrosine kinases and Syk. In the present study, we report Src kinase-dependent β2 integrin-induced tyrosine phosphorylation of Cbl occurring in parallel with increased Cbl-associated tyrosine kinase activity. These events concurred with activation of Fgr and, surprisingly, also with dissociation of this Src tyrosine kinase from Cbl. Moreover, the presence of the Src kinase inhibitor PP1 in an in vitro assay had only a limited effect on the Cbl-associated kinase activity. These results suggest that an additional active Src-dependent tyrosine kinase associates with Cbl. The following observations imply that Syk is such a kinase: (i) β2 integrins activated Syk in a Src-dependent manner, (ii) Syk was associated with Cbl much longer than Fgr was, and (iii) the Syk inhibitor piceatannol (3,4,3′,5′-tetrahydroxy-trans-stilbene) abolished the Cbl-associated kinase activity in an in vitro assay. Effects of the mentioned interactions between these two kinases and Cbl may be related to the finding that Cbl is a ubiquitin E3 ligase. Indeed, we detected β2 integrin-induced ubiquitination of Fgr that, similar to the phosphorylation of Cbl, was abolished in cells pretreated with PP1. However, the ubiquitination of Fgr did not cause any apparent degradation of the protein. In contrast with Fgr, Syk was not modified by the E3 ligase. Thus Cbl appears to be essential in β2 integrin signalling, first by serving as a matrix for a subsequent agonist-induced signalling interaction between Fgr and Syk, and then by mediating ubiquitination of Fgr which possibly affects its interaction with Cbl.
16
Yu, C. L., R. Jove y S. J. Burakoff. "Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase." Journal of Immunology 159, n.º 11 (1 de diciembre de 1997): 5206–10. http://dx.doi.org/10.4049/jimmunol.159.11.5206.
Texto completoResumen
Abstract The Lck protein, a Src family tyrosine kinase, plays a critical role in T cell maturation and activation. Dysregulation of Lck expression or Lck kinase activity has been implicated in T cell leukemias from mice to humans, although the mechanism underlying Lck-mediated oncogenesis is still largely unclear. We report here that both DNA binding activities and tyrosine phosphorylation of STAT3 and STAT5, but not STAT1, are constitutively enhanced in the mouse T cell lymphoma LSTRA, which is a well-characterized cell line that overexpresses Lck protein and exhibits high levels of Lck kinase activity. Furthermore, Janus kinase 1 (jak1) and Jak2 protein tyrosine kinases are constantly activated in these cells, as determined by their autophosphorylation in an in vitro kinase assay and increased levels of tyrosine phosphorylation on immunoblots. Therefore, like Src-transformed cells, Lck-overexpressing LSTRA cells also exhibit constitutive activation of distinct Jak and STAT proteins.
17
Pazdrak, K., L. Justement y R. Alam. "Mechanism of inhibition of eosinophil activation by transforming growth factor-beta. Inhibition of Lyn, MAP, Jak2 kinases and STAT1 nuclear factor." Journal of Immunology 155, n.º 9 (1 de noviembre de 1995): 4454–58. http://dx.doi.org/10.4049/jimmunol.155.9.4454.
Texto completoResumen
Abstract The activation of eosinophils by IL-5 plays a crucial role in the pathogenesis of allergic and parasitic disorders. IL-5 has recently been shown to activate Lyn and Jak2 tyrosine kinases, MAP kinases, and STAT1 nuclear factor. We have previously reported that TGF-beta blocks the IL-5-induced activation of eosinophils. In this study, we investigated the effect of TGF-beta on the IL-5-induced signaling molecules in eosinophils. Purified eosinophils from mildly allergic patients were preincubated with TGF-beta and then stimulated with IL-5. The cell lysates were then immunoprecipitated and blotted with antiphosphotyrosine Abs. The activity of the kinases was further studied in the immune-complex kinase assay. We found that TGF-beta inhibited the tyrosine phosphorylation of multiple proteins in eosinophils. The identity of some of the proteins was established by immunoprecipitation. We found that TGF-beta inhibited tyrosine phosphorylation of Lyn, Jak2, and a 44-kDa MAP kinase. In further experiments, it blocked the activation of the above kinases as determined by immune-complex kinase assay. TGF-beta also inhibited phosphorylation of the STAT1 (p91) nuclear protein in eosinophils. We believe that the inhibition of Lyn, Jak2, MAP kinase, and the STAT1 nuclear protein may underlie the inhibitory activity of TGF-beta on eosinophils.
18
Laniyonu, Adebayo, Seiji Eto, Jerry H. Wang y Morley D. Hollenberg. "Detection of sarcoma virus family tyrosine kinase activity in coronary arterial tissue". Canadian Journal of Physiology and Pharmacology 73, n.º 11 (1 de noviembre de 1995): 1552–60. http://dx.doi.org/10.1139/y95-214.
Texto completoResumen
We have observed that the tyrosine kinase inhibitors genistein and tyrphostin can selectively block angiotensin II mediated and vasopressin-mediated contractions in porcine coronary arterial strips, without affecting the action of acetylcholine. Therefore, we assessed the presence of tyrosine kinase activity in the porcine coronary artery tissue, using an assay specific for sarcoma virus (src) related tyrosine kinases. In both membrane and cytosolic fractions of porcine coronary artery, we detected src-related tyrosine kinase activity that could be inhibited by both genistein and tyrphostin. The tyrosine kinase activity in membrane extracts was separated into two peaks by sequential chromatography on hydroxylapatite and Mono-Q columns. Protein in both peaks exhibited Western blot cross-reactivity with anti-src antibodies and contained tyrosine kinase activity that was inhibited by genistein and tyrphostin. We conclude that porcine coronary artery tissue contains src-related tyrosine kinase activity. However, because of the comparatively low sensitivity of the isolated src kinase activity towards genistein and tyrphostin, compared with the much higher sensitivity of the contractile response to these inhibitors, a direct role for c-src in the regulation of contractions elicited by agonists such as angiotensin II and arginine vasopressin cannot yet be assigned.Key words: tyrosine kinase, genistein, tyrphostin, coronary artery, angiotensin II, vasopressin.
19
Bicocca, Vincent, Bill H. Chang, Markus Muschen, Brian J. Druker y Jeffrey W. Tyner. "ROR1 as a Therapeutic Target In E2A-PBX1-Positive Acute Lymphoblastic Leukemia". Blood 116, n.º 21 (19 de noviembre de 2010): 539. http://dx.doi.org/10.1182/blood.v116.21.539.539.
Texto completoResumen
Abstract Abstract 539 Background: Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. These lesions can result in aberrant tyrosine kinase expression and activity required for leukemogenesis. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. The role of E2A-PBX1 in the development of acute leukemia remains unclear. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the E2A-PBX1 ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against E2A-PBX1-positive ALL cells. Methods: To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients by electroporating siRNAs individually targeting each member of the tyrosine kinase gene family. Four days later, we determined the cell viability and tabulated sensitivity of the cells to any individual tyrosine kinase. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cell lines and primary ALL cells by treating samples with a library of small-molecule inhibitors consisting of 90 cell-permeable inhibitor compounds. Inhibitors are plated at four serial dilutions to allow IC50 calculations. The effect of each drug on cell viability is determined at day three by an MTS cell viability assay. Results: The RAPID assay identified a unique sensitivity to the receptor tyrosine kinase-like orphan receptor 1 (ROR1) in a subject identified with E2A-PBX1-positive B cell precursor pediatric ALL. Similar sensitivity was not observed in patients of other leukemic backgrounds or ALL patients of alternative cytogenetic subtypes. Examination of mononuclear cells from this patient by reverse-transcriptase-PCR revealed overexpression of the ROR1 transcript compared with ALL patients lacking the E2A-PBX1 fusion product. Examination of 12 additional E2A-PBX1-positive ALL patient samples revealed universal overexpression of ROR1 within the E2A-PBX1 background. Analysis of E2A-PBX1-positive cell lines and early passage xenograft cells showed both overexpression of ROR1 and sensitivity to siRNA-mediated ROR1 silencing, confirming the ROR1 dependent survival observed in primary cells with the RAPID assay. Finally, since ROR1 is defined as a tyrosine kinase, we performed a kinase inhibitor screen and identified universal sensitivity of E2A-PBX1-positive cell lines and patient samples to the FDA-approved drug dasatinib. Hence, dasatinib is suggested as a potential therapeutic for E2A-PBX1-positive ALL patients. Conclusion: The cell surface receptor ROR1 is consistently overexpressed in E2A-PBX1-positive ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Finally, the kinase inhibitor dasatinib is suggested as a novel therapeutic tool for treatment of E2A-PBX1-positive ALL based on universal sensitivity of E2A-PBX1 samples to this kinase inhibitor. Disclosures: Druker: Molecular MD: Equity Ownership.
20
Freiberg, Gail, Julie Wilkins, Caroline David, James Kofron, Yong Jia, Gavin C. Hirst, David J. Burns y Usha Warrior. "Utilization of Microarrayed Compound Screening (μARCS) to Identify Inhibitors of p56lck Tyrosine Kinase". Journal of Biomolecular Screening 9, n.º 1 (febrero de 2004): 12–21. http://dx.doi.org/10.1177/1087057103259667.
Texto completoResumen
Protein tyrosine kinases play critical roles in cell signaling and are considered attractive targets for drug discovery. The authors have applied μARCS (microarrayed compound screening) technology to develop a high-throughput screen for finding inhibitors of the p56lck tyrosine kinase. Initial assay development was performed in a homogeneous time-resolved (LANCE™) format in 96-well microplates and then converted into the gel-based μARCS format. The μARCS methodology is a well-less screening format in which 8640 compounds are arrayed on a microplate-sized piece of polystyene and subsequently assayed by placing reagents cast in agarose gels in contact with these compound sheets. A blotting paper soaked with adenosine triphosphate is applied on the gel to initiate the kinase reaction in the gel. Using this screening methodology, 300,000 compounds were screened in less than 40 h. Substantial reagent reduction was achieved by converting this tyrosine kinase assay from a 96-well plate assay to μARCS, resulting in significant cost savings. ( Journal of Biomolecular Screening 2004: 12-21)
21
Lee, Jennifer Y., Sheri Miraglia, Xiongwei Yan, Elana Swartzman, Susan Cornell-Kennon, Julia Mellentin-Michelotti, Charles Bruseo y Dennis S. France. "Oncology Drug Discovery Applications Using the FMAT™ 8100 HTS System". Journal of Biomolecular Screening 8, n.º 1 (enero de 2003): 81–88. http://dx.doi.org/10.1177/1087057102239668.
Texto completoResumen
High-throughput screening (HTS) for potential anticancer agents requires a broad portfolio of assay platforms that may include kinase enzyme assays, protein-protein binding assays, and functional cell-based apoptosis assays. The authors have explored the use of fluorometric microvolume assay technology (the FMAT™ 8100 HTS System) in three distinct homogeneous HTS assays: (1) a Src tyrosine kinase enzyme assay, (2) a Grb2-SH2 protein-peptide interaction assay, and (3) an annexin V binding apoptosis assay. Data obtained from all three assays suggest that the FMAT system should facilitate the implementation of homogeneous assays for a wide variety of molecular targeted and cell-based screens. ( Journal of Biomolecular Screening 2003:81-88)
22
Hsu, Jonathan, Jun Zhang, Chris Kitson, Seng-Lai Tan, Satwant Narula, Julie A. DeMartino y Cheng Liao. "Development of a Pharmacodynamic Assay Based on PLCγ2 Phosphorylation for Quantifying Spleen Tyrosine Kinase (SYK)–Bruton’s Tyrosine Kinase (BTK) Signaling". Journal of Biomolecular Screening 18, n.º 8 (23 de mayo de 2013): 890–98. http://dx.doi.org/10.1177/1087057113489881.
Texto completoResumen
Spleen tyrosine kinase (SYK) and Bruton’s tyrosine kinase (BTK) are key mediators in coupling cell surface receptors, such as the B-cell receptor (BCR), to downstream signaling events affecting diverse biological functions. There is therefore tremendous interest in the development of pharmacological inhibitors targeting the SYK-BTK axis for the treatment of inflammatory disorders and hematological malignancies. A good pharmacodynamic (PD) assay, ideally a blood-based assay that measures proximal events, is warranted for evaluation of such inhibitors. In platelets, collagen-induced activation of membrane glycoprotein GPVI is dependent on the SYK-BTK axis. Here, we report the development of a novel immunoassay that uses the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) to measure GPVI-mediated phosphorylation of phospholipase C γ2 (PLCγ2), a direct substrate of SYK and BTK, in platelets. The assay was validated using SYK or BTK inhibitors and generated IC50 correlated with those from the BCR-induced B-cell activation assay. Furthermore, this assay showed good stability and uniformity over a period of 24 h in different donors. Interestingly, compound IC50 values using blood from patients with rheumatoid arthritis were slightly higher compared with those produced using samples from healthy donors. This novel platelet PLCγ2 phosphorylation-based immunoassay should serve as a promising PD assay for preclinical and clinical development of inhibitors targeting the SYK-BTK axis.
23
Babcook, John, Julian Watts, Ruedi Aebersold y Hermann J. Ziltener. "Automated nonisotopic assay for protein-tyrosine kinase and protein-tyrosine phosphatase activities". Analytical Biochemistry 196, n.º 2 (agosto de 1991): 245–51. http://dx.doi.org/10.1016/0003-2697(91)90461-2.
Texto completo
24
Beeler, J. F., W. J. LaRochelle, M. Chedid, S. R. Tronick y S. A. Aaronson. "Prokaryotic expression cloning of a novel human tyrosine kinase". Molecular and Cellular Biology 14, n.º 2 (febrero de 1994): 982–88. http://dx.doi.org/10.1128/mcb.14.2.982-988.1994.
Texto completoResumen
Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.
25
Beeler, J. F., W. J. LaRochelle, M. Chedid, S. R. Tronick y S. A. Aaronson. "Prokaryotic expression cloning of a novel human tyrosine kinase." Molecular and Cellular Biology 14, n.º 2 (febrero de 1994): 982–88. http://dx.doi.org/10.1128/mcb.14.2.982.
Texto completoResumen
Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.
26
Gaudry, M., C. Gilbert, F. Barabe, PE Poubelle y PH Naccache. "Activation of Lyn is a common element of the stimulation of human neutrophils by soluble and particulate agonists". Blood 86, n.º 9 (1 de noviembre de 1995): 3567–74. http://dx.doi.org/10.1182/blood.v86.9.3567.bloodjournal8693567.
Texto completoResumen
The functional responsiveness of human neutrophils is known to be initiated and modulated by protein tyrosine phosphorylation. The regulation of the levels of tyrosine phosphorylation is most likely the result of the coordinated actions of tyrosine kinases and phosphatases, which have so far been only very partially characterized. In the present study, we present evidence demonstrating that the stimulation of neutrophils by a variety of agonists (soluble as well as particulate) leads to the activation of the src-related tyrosine kinase lyn. The stimulation of tyrosine kinase activity of lyn was detected using an immune kinase assay as well as an in situ labeling technique. Phosphoaminoacid analysis of lyn indicated that the autophosphorylation of the kinase was exclusively on tyrosine residues. The time course of the activation of lyn is consistent with its playing a role in the early tyrosine phosphorylation responses of neutrophils. The ability of agonists with widely varying functional end responses to stimulate the activity of lyn indicates that this event plays a key and central role in the control of the activation of human neutrophils.
27
Sills, Matthew A., Donna Weiss, Quynhchi Pham, Robert Schweitzer, Xiang Wu y Jinzi J. Wu. "Comparison of Assay Technologies for a Tyrosine Kinase Assay Generates Different Results in High Throughput Screening". Journal of Biomolecular Screening 7, n.º 3 (junio de 2002): 191–214. http://dx.doi.org/10.1177/108705710200700304.
Texto completoResumen
In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC50 values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC50 values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.
28
O'Bryan, J. P., R. A. Frye, P. C. Cogswell, A. Neubauer, B. Kitch, C. Prokop, R. Espinosa, M. M. Le Beau, H. S. Earp y E. T. Liu. "axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase". Molecular and Cellular Biology 11, n.º 10 (octubre de 1991): 5016–31. http://dx.doi.org/10.1128/mcb.11.10.5016-5031.1991.
Texto completoResumen
Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.
29
O'Bryan, J. P., R. A. Frye, P. C. Cogswell, A. Neubauer, B. Kitch, C. Prokop, R. Espinosa, M. M. Le Beau, H. S. Earp y E. T. Liu. "axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase." Molecular and Cellular Biology 11, n.º 10 (octubre de 1991): 5016–31. http://dx.doi.org/10.1128/mcb.11.10.5016.
Texto completoResumen
Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.
30
Lamaze, C. y S. L. Schmid. "Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase." Journal of Cell Biology 129, n.º 1 (1 de abril de 1995): 47–54. http://dx.doi.org/10.1083/jcb.129.1.47.
Texto completoResumen
EGF-receptor (EGF-R) tyrosine kinase is required for the down-regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of kinase-deficient receptors occurs inefficiently and at the same basal rate of endocytosis of unoccupied receptors or receptors lacking any cytoplasmic domain. Sequestration of deletion mutants of the EGF-R that lack autophosphorylation sites also requires an active tyrosine kinase. This suggests that a tyrosine kinase substrate(s) other than the EGF-R itself, is required for its efficient ligand-induced recruitment into coated pits. Addition of a soluble EGF-R tyrosine kinase fully and specifically restores the recruitment of kinase-deficient EGF-R into coated pits providing a powerful functional assay for identification of these substrate(s).
31
Konopka, J. B. y O. N. Witte. "Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products". Molecular and Cellular Biology 5, n.º 11 (noviembre de 1985): 3116–23. http://dx.doi.org/10.1128/mcb.5.11.3116-3123.1985.
Texto completoResumen
The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.
32
Konopka, J. B. y O. N. Witte. "Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products." Molecular and Cellular Biology 5, n.º 11 (noviembre de 1985): 3116–23. http://dx.doi.org/10.1128/mcb.5.11.3116.
Texto completoResumen
The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.
33
Patel, Jay, Christopher J. Stanley y Stuart M. Wilson. "Homogeneous Assay for Tyrosine Kinase: Use of Bacteriophage Antibody Conjugates in an Assay for p56lck Kinase". Clinical Chemistry 48, n.º 10 (1 de octubre de 2002): 1860–62. http://dx.doi.org/10.1093/clinchem/48.10.1860.
Texto completo
34
Tyner, Jeffrey W., Stephen Spurgeon, Luke B. Fletcher, Wayne Yang, Tibor Kovacsovics, Brian J. Druker y Marc M. Loriaux. "A Small-Molecule Inhibitor Screen Rapidly Identifies Therapeutic Targets and Individualized Therapeutic Strategies In Patients with Acute and Chronic Leukemias". Blood 116, n.º 21 (19 de noviembre de 2010): 2754. http://dx.doi.org/10.1182/blood.v116.21.2754.2754.
Texto completoResumen
Abstract Abstract 2754 The development of more effective and less toxic therapies for acute and chronic leukemias will require the identification of the molecular abnormalities contributing to leukemogenesis and the identification of drugs that specifically block the activity of these lesions. We hypothesize that aberrantly activated tyrosine kinase signaling pathways play a critical role in the pathogenesis of a substantial proportion of leukemia cases, and our preliminary data suggest that the molecular abnormalities causing aberrant kinase activation are unique in a significant number of patients. Thus, effective therapies for leukemia will need to be determined on an individual patient basis. To address this need, we have developed a function-first, small-molecule kinase inhibitor assay that can identify therapeutic targets in tyrosine kinase signaling pathways in primary leukemia samples and provide individualized therapeutic options in a clinically relevant time frame. Methods: To rapidly identify drug sensitivity profiles and activated kinase pathways in individual, primary leukemia 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 collectively target the majority of the tyrosine kinome. Many of the inhibitors are available for clinical use or are in clinical development. Inhibitors were placed in 96-well plates at four serial dilutions to allow IC50 calculations. Three days after adding primary leukemia cells to each well, we performed a tetrazolium based cell viability assay to evaluate the effect of each inhibitor. Because most inhibitors affect multiple kinases, we utilized automated scripts to compare target specificities of compounds that uniquely decreased primary leukemia cell viability to identify potential targets. Results: In preliminary proof-of-principal experiments, we tested leukemia cell lines and primary leukemia samples with known activating tyrosine kinase mutations and Ba/F3 cell lines expressing activated tyrosine kinases. As expected, all cells showed hypersensitivity to compounds with activity against the primary, mutated target. In addition, downstream targets were frequently identified. For example, MKPL-1 cells, which depend on an activating CSF1R translocation for viability, also showed sensitivity to phosphoinositol 3-kinase and NFKB inhibitors. To date, we have fully analyzed approximately 150 primary myeloid and lymphoid leukemia samples. Hierarchical clustering of IC50 data for individual patients identifies activated pathways characteristic to specific leukemia subtypes. Pathways include PI3K activation in acute lymphoblastic leukemia, SRC kinase and BTK activation in chronic lymphocytic leukemia, FLT3 and KIT activation in AML patients, and MEK kinase activation in chronic myelomonocytic leukemia. Importantly, the results show heterogeneous inhibitor sensitivity profiles and potential kinase targets for individual samples even within diagnosis groups supporting a need for individualized targeted therapies. We are currently utilizing inhibitor assay results for clinical trial development. Approximately 40% of samples show sensitivity to at least one FDA approved drug in the inhibitor panel, and we are developing phase II proof-of-concept trials to test the ability of the inhibitor assay to predict effective targeted therapies for individual patients. Conclusions: Our data demonstrate that the small-molecule inhibitor functional assay can rapidly identify genes contributing to leukemogenesis, provide insights into their mechanism of action, and suggest therapeutic options. The unique patterns of inhibitor sensitivity in many samples support the hypothesis that tyrosine kinases and related pathways contributing to leukemogenesis in each patient may be different. These findings, in turn, support the concept that targeted therapy will be most effective when administered on an individualized basis. By utilizing our pre-clinical assay to select individualized leukemia therapies, we hope to create a platform upon which we can rapidly test the effectiveness of individualized kinase therapy and apply this information to enhance development of new drugs and new drug combinations in leukemia patients. Disclosures: Kovacsovics: Celator Pharmaceuticals: Research Funding. Druker:Molecular MD: Consultancy, Equity Ownership. Loriaux:Celator Pharmaceuticals: Research Funding.
35
SIERKE, Susan L., Kunrong CHENG, Hong-Hee KIM y John G. KOLAND. "Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein". Biochemical Journal 322, n.º 3 (15 de marzo de 1997): 757–63. http://dx.doi.org/10.1042/bj3220757.
Texto completoResumen
The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidine-tagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptor-derived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-length ErbB3 proteins also yielded no evidence of catalytic activity. A fluorescence assay previously used to characterize the nucleotide-binding properties of the EGF receptor indicated that the ErbB3 protein was unable to bind nucleotide. The hisTKD-B3 protein was subsequently found to be an excellent substrate for the EGF receptor protein tyrosine kinase, which suggested that in vivophosphorylation of ErbB3 in response to EGF could be attributed to a direct cross-phosphorylation by the EGF receptor protein tyrosine kinase.
36
Angeles, Thelma S., Catherine Steffler, Becky A. Bartlett, Robert L. Hudkins, Robert M. Stephens, David R. Kaplan y Craig A. Dionne. "Enzyme-Linked Immunosorbent Assay for trkA Tyrosine Kinase Activity". Analytical Biochemistry 236, n.º 1 (abril de 1996): 49–55. http://dx.doi.org/10.1006/abio.1996.0130.
Texto completo
37
Zhang, Zheng, Hava Avraham y David M. Cohen. "Urea and NaCl differentially regulate FAK and RAFTK/PYK2 in mIMCD3 renal medullary cells". American Journal of Physiology-Renal Physiology 275, n.º 3 (1 de septiembre de 1998): F447—F451. http://dx.doi.org/10.1152/ajprenal.1998.275.3.f447.
Texto completoResumen
Two cytosolic tyrosine kinases, focal adhesion kinase (FAK) and the newly described FAK homolog, related adhesion focal tyrosine kinase (RAFTK, also called PYK2 and CAKβ), have been implicated in signaling to multiple mitogen-activated protein kinase (MAPK) pathways. Therefore, the ability of NaCl and urea to activate these kinases was investigated by in vitro kinase assay and anti-phosphotyrosine immunoblotting. RAFTK was promptly but only transiently activated by urea (within 1 min; 45%), whereas NaCl activated this kinase at 1, 5, 15, and 30 min of treatment (35–60%). In contrast, FAK exhibited only subtle regulation by the two solutes; however, the time course of induction was distinct for each solute. NaCl activated FAK at 1, 5, and 15 min (25–40%), whereas urea-inducible FAK activation (30%) was not evident until fully 15 min of treatment. At 5 min of treatment with increasing concentrations of solute, both urea and NaCl activated RAFTK in a dose-dependent and comparable fashion, culminating in an approximately twofold activation at 800 mosmol/kgH2O solute. Consistent with these data, solute treatment also enhanced tyrosine phosphorylation of RAFTK.
38
Jiang, Chao, Ya Li, Chenghui Liu, Liying Qiu y Zhengping Li. "A general and versatile fluorescence turn-on assay for detecting the activity of protein tyrosine kinases based on phosphorylation-inhibited tyrosyl oxidation". Chemical Communications 52, n.º 85 (2016): 12570–73. http://dx.doi.org/10.1039/c6cc07035c.
Texto completo
39
Parker, Gregory J., Tong Lin Law, Francis J. Lenoch y Randall E. Bolger. "Development of High Throughput Screening Assays Using Fluorescence Polarization: Nuclear Receptor-Ligand-Binding and Kinase/Phosphatase Assays". Journal of Biomolecular Screening 5, n.º 2 (abril de 2000): 77–88. http://dx.doi.org/10.1177/108705710000500204.
Texto completoResumen
Fluorescence polarization (FP) has been used to develop high throughput screening (HTS) assays for nuclear receptor-ligand displacement and kinase inhibition. FP is a solution-based, homogeneous technique requiring no immobilization or separation of reaction components. The FP-based estrogen receptor (ER) assay is based on the competition of fluorescein-labeled estradiol and estrogen-like compounds for binding to ER. These studies determined the Kd for this interaction to be 3 nM for ERα and 2 nM for ERβ; IC50 values for 17β-estradiol, tamoxifen, 4-OH-tamoxifen, and diethylstibestrol were determined to be 5.6, 189, 26, and 3.5 nM, respectively. In a screen of 50 lead compounds from a transcriptional activation screen, 21 compounds had IC50 values below 10 μM, with one having an almost 100-fold higher affinity for ERβ over ERα. These data show that an FP-based competitive binding assay can be used to screen diverse compounds with a broad range of binding affinities for ERs. The FP-based protein-tyrosine kinase (PTK) assay uses fluorescein-labeled phosphopeptides bound to anti-phosphotyrosine antibodies. Phosphopeptides generated by a kinase compete for this binding. In c-Src kinase reactions, polarization decreased with time as reaction products displaced the fluorescein-labeled phosphopeptide from the anti-phosphotyrosine antibodies. The experimentally determined IC50 of AG 1478 was 400 pM, while Genistein did not inhibit the epidermal growth factor receptor at similar concentrations. Like the FP-based PTK assay, the protein kinase C (PKC) assay utilizes competition. PKC isoforms had different turnover rates for the peptide substrate. The IC50 for staurosporine was less than 10 nM for all PKC isoforms. Tyrosine phosphatase assays use direct binding rather than competition. Increasing concentrations of T-cell protein-tyrosine phosphatase (TC PTP) increased the rate of dephosphorylation. This change in polarization was dependent on TC PTP and was inhibited by 50,μM Na3VO4. The IC50 of Na3VO4 was 4 nM for TC PTP. These data demonstrate that a FP-based assay can detect kinase and phosphatase activity. Homogeneous, fluorescent techniques such as FP are now methods of choice for screening many types of drug targets. New HTS instrumentation and assay methods like these make FP a technology easily incorporated into HTS.
40
Atienza, Josephine M., Naichen Yu, Xiaobo Wang, Xiao Xu y Yama Abassi. "Label-Free and Real-Time Cell-Based Kinase Assay for Screening Selective and Potent Receptor Tyrosine Kinase Inhibitors Using Microelectronic Sensor Array". Journal of Biomolecular Screening 11, n.º 6 (7 de junio de 2006): 634–43. http://dx.doi.org/10.1177/1087057106289334.
Texto completoResumen
Kinases are the 2nd largest group of therapeutic targets in the human genome. In this article, a label-free and real-time cell-based receptor tyrosine kinase (RTK) assay that addresses limitation of existing kinase assays and can be used for high-throughput screening and lead optimization studies was validated and characterized. Using impedance, growth factor-induced morphological changes were quantitatively assessed in real time and used as a measure of RTK activity. COS7 cells treated with epidermal growth factor (EGF) and insulin results in a rapid increase in cell impedance. Assessment of these growth factor-induced morphological changes and levels of receptor autophosphorylation using fluorescent microscopy and enzyme-linked immunosorbent assay, respectively, demonstrates that these changes correlate with changes in impedance. This assay was used to screen, identify, and characterize a potent EGF receptor inhibitor from a compound library. This report describes an assay that is simple in that it does not require intensive optimization or special reagents such as peptides, antibodies, or probes. More important, because the assay is cell based, the studies are done in a physiologically relevant environment, allowing for concurrent assessment of a compound’s solubility, stability, membrane permeability, cytotoxicity, and off-target interaction effects. ( Journal of Biomolecular Screening 2006:634-643)
41
Smotrov, Nadya, Anjili Mathur, Ilona Kariv, Christopher M. Moxham y Nathan Bays. "Development of a Cell-Based Assay for Measurement of c-Met Phosphorylation Using AlphaScreenTM Technology and High-Content Imaging Analysis". Journal of Biomolecular Screening 14, n.º 4 (abril de 2009): 404–11. http://dx.doi.org/10.1177/1087057109331803.
Texto completoResumen
c-Met is a receptor tyrosine kinase (RTK) with a critical role in many fundamental cellular processes, including cell proliferation and differentiation. Deregulated c-Met signaling has been implicated in both the initiation and progression of human cancers and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing c-Met kinase activity. This report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen™) technology. Using AlphaScreen™, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreen™ assay were compared to data obtained from a high-content imaging (HCI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. The AlphaScreen™ assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z′ statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. The authors discuss the utility of quantifying endogenous cellular c-Met phosphorylation in lead optimization and how the modular design of the AlphaScreen™ assay allows its adaptation to measure cellular activity of other kinases. ( Journal of Biomolecular Screening 2009:404-411)
42
Pazdrak, K., D. Schreiber, P. Forsythe, L. Justement y R. Alam. "The intracellular signal transduction mechanism of interleukin 5 in eosinophils: the involvement of lyn tyrosine kinase and the Ras-Raf-1-MEK-microtubule-associated protein kinase pathway." Journal of Experimental Medicine 181, n.º 5 (1 de mayo de 1995): 1827–34. http://dx.doi.org/10.1084/jem.181.5.1827.
Texto completoResumen
Interleukin 5 (IL-5) regulates the growth and function of eosinophils. The objective of this study was to investigate the intracellular signal transduction mechanism of IL-5 in eosinophils. Purified eosinophils were stimulated with IL-5, and the involvement of various kinases was investigated by immunoblotting, immune complex kinase assay, and in situ denatured/renatured kinase assay. We found that IL-5 induced tyrosine phosphorylation and activation of a number of kinases. Two species of lyn kinases (53 and 56 kD) were present in eosinophils. Both forms were Tyr-phosphorylated and activated rapidly within 1 min. Further, lyn kinase was physically associated with the IL-5 beta receptor in eosinophils. Ras was studied by immunoprecipitation followed by thin-layer chromatography. Ras bound higher quantities of [alpha-32P]guanosine 5'triphosphate upon stimulation with IL-5. Raf-1 kinase showed increased Tyr phosphorylation on immunoblotting and increased activity in the immune complex kinase assay. Two species of MEK (MAP or Erk kinase) (41 and 45 kD) were identified in eosinophils, which underwent autophosphorylation upon stimulation. Microtubule-associated protein (MAP) kinase (p44) was Tyr-phosphorylated on immunoblotting and had increased activity in the immune-complex kinase assay. MAP kinases were also studied after metabolic radiolabeling of the cells with [32P]orthophosphates. IL-5 stimulated phosphorylation of MAP kinases in situ. Thus, we have delineated major components of an important signaling pathway in eosinophils. We believe that one of the signals generated by IL-5 receptor activation is propagated through the lyn-Ras-Raf-1-MEK-MAP kinase pathway.
43
Kinoshita-Kikuta, Emiko, Momoka Yoshimoto, Marina Yano, Eiji Kinoshita y Tohru Koike. "An assay of human tyrosine protein kinase ABL activity using an Escherichia coli protein expression system". BioTechniques 70, n.º 4 (abril de 2021): 209–17. http://dx.doi.org/10.2144/btn-2020-0154.
Texto completoResumen
ABL, a human tyrosine protein kinase, and its substrate are co-expressed in Escherichia coli. Tyrosine phosphorylation of the substrate in E. coli was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants and undetectable in T315I and M351T mutants. These comparative results demonstrated that the phosphorylation states of the mutants correlated with their activity. The bacterial co-expression system permits rapid production of tyrosine kinase variants and provides a simple approach for examining their structure–activity relationships.
44
Dong, Z., X. Qi y I. J. Fidler. "Tyrosine phosphorylation of mitogen-activated protein kinases is necessary for activation of murine macrophages by natural and synthetic bacterial products." Journal of Experimental Medicine 177, n.º 4 (1 de abril de 1993): 1071–77. http://dx.doi.org/10.1084/jem.177.4.1071.
Texto completoResumen
The purpose of these studies was to determine the intracellular signal transduction pathways of bacterial products in murine macrophages from lipopolysaccharide (LPS)-responder C3H/HeN and LPS-nonresponder C3H/HeJ mice. Both LPS and synthetic lipopeptide CGP 31362 (LPP) induced production of tumor necrosis factor alpha (TNF-alpha) in C3H/HeN macrophages. In C3H/HeJ macrophages, however, TNF-alpha was induced only by incubation with LPP. Both LPS and LPP induced tyrosine phosphorylation on proteins with apparent molecular masses of 39, 41, and 45 kD (p35, p41, and p45) in C3H/HeN macrophages, whereas in C3H/HeJ macrophages, tyrosine phosphorylation was induced only by LPP. 20-h incubation with LPS or LPP downregulated TNF-alpha production/secretion and tyrosine phosphorylation in C3H/HeN macrophages induced by additional LPS or LPP. In C3H/HeJ macrophages, however, the downregulation of TNF-alpha production and tyrosine phosphorylation were observed only with LPP. Protein kinase assays, Western blotting analyses, phenyl-Sepharose chromatography, and immunocomplex kinase assay suggested that p45 and p39 were similar or identical to mitogen-activated protein (MAP) kinase 1 and 2, respectively. Pretreatment of macrophages with LPS or LPP did not change the amount of kinase proteins but inhibited the stimulation of kinase activity by the agents. These data suggest that MAP kinases are among target proteins involved in the transduction of LPS and LPP signals that lead to activation of murine macrophages to produce/secrete TNF.
45
Park, Young-Whan, Richard T. Cummings, Lin Wu, Song Zheng, Patricia M. Cameron, Andrea Woods, Dennis M. Zaller, Alice I. Marcy y Jeffrey D. Hermes. "Homogeneous Proximity Tyrosine Kinase Assays: Scintillation Proximity Assay versus Homogeneous Time-Resolved Fluorescence". Analytical Biochemistry 269, n.º 1 (abril de 1999): 94–104. http://dx.doi.org/10.1006/abio.1999.4029.
Texto completo
46
Xue, Liang, Robert L. Geahlen y W. Andy Tao. "Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics". Molecular & Cellular Proteomics 12, n.º 10 (22 de junio de 2013): 2969–80. http://dx.doi.org/10.1074/mcp.o113.027722.
Texto completo
47
Naldini, L., E. Vigna, R. Ferracini, P. Longati, L. Gandino, M. Prat y P. M. Comoglio. "The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation". Molecular and Cellular Biology 11, n.º 4 (abril de 1991): 1793–803. http://dx.doi.org/10.1128/mcb.11.4.1793-1803.1991.
Texto completoResumen
Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.
48
Naldini, L., E. Vigna, R. Ferracini, P. Longati, L. Gandino, M. Prat y P. M. Comoglio. "The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation." Molecular and Cellular Biology 11, n.º 4 (abril de 1991): 1793–803. http://dx.doi.org/10.1128/mcb.11.4.1793.
Texto completoResumen
Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.
49
Ferrell, J. E. y G. S. Martin. "Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation". Molecular and Cellular Biology 10, n.º 6 (junio de 1990): 3020–26. http://dx.doi.org/10.1128/mcb.10.6.3020-3026.1990.
Texto completoResumen
We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.
50
Ferrell, J. E. y G. S. Martin. "Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation." Molecular and Cellular Biology 10, n.º 6 (junio de 1990): 3020–26. http://dx.doi.org/10.1128/mcb.10.6.3020.
Texto completoResumen
We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.