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.