title: Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.
abstract: OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.
authors.affiliation: OSI Pharmaceuticals, Inc., 1 Bioscience Park Drive, Farmingdale, NY 11735, USA.
authors.forename: Filippo
authors.initials: F
authors.lastname: Petti
authors.forename: April
authors.initials: A
authors.lastname: Thelemann
authors.forename: Jen
authors.initials: J
authors.lastname: Kahler
authors.forename: Siobhan
authors.initials: S
authors.lastname: McCormack
authors.forename: Linda
authors.initials: L
authors.lastname: Castaldo
authors.forename: Tony
authors.initials: T
authors.lastname: Hunt
authors.forename: Lydia
authors.initials: L
authors.lastname: Nuwaysir
authors.forename: Lynn
authors.initials: L
authors.lastname: Zeiske
authors.forename: Herbert
authors.initials: H
authors.lastname: Haack
authors.forename: Laura
authors.initials: L
authors.lastname: Sullivan
authors.forename: Andrew
authors.initials: A
authors.lastname: Garton
authors.forename: John D
authors.initials: JD
authors.lastname: Haley
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D006801:Humans; D007946:Leukemia, Mast-Cell / Q000201:enzymology*; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D019009:Proto-Oncogene Proteins c-kit / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D011804:Quinolines / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D013876:Thiophenes / Q000494:pharmacology*; D046888:Tissue Array Analysis; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; C505207:OSI 930; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011804:Quinolines; D013876:Thiophenes; D019009:Proto-Oncogene Proteins c-kit