Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930
Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930
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DOI：
10.1158/1535-7163.mct-05-0114
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发表时间：
2005-08-01
影响因子：
5.7
通讯作者：
Haley, JD
中科院分区：
文献类型：
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作者：
Petti, F;Thelemann, A;Haley, JD
OSI-930是Kit、KDR和血小板衍生生长因子受体酪氨酸激酶的有效噻吩抑制剂，用于选择性抑制肥大细胞白血病细胞系HMC-1中胞膜突变型Kit下游的酪氨酸磷酸化。Kit激酶活性的抑制导致Kit的快速去磷酸化和下游信号通路的抑制。Ras-Raf-Erk（磷酸化Erk，磷酸化p38）、磷脂酰肌醇-3 '激酶（磷酸化p85，磷酸化Akt，磷酸化S6）以及转录信号传导途径的信号转导和激活因子（磷酸化STAT 3/5/6）的衰减通过亲和液相色谱串联质谱法、免疫印迹法和固定细胞团的组织微阵列进行测量。为了更全面地定义由激酶抑制暂时改变的Kit信号传导的其他组分，使用了新型多重定量同量异位肽标记方法。这种方法允许通过时间表达模式对蛋白质进行聚类。Kit激酶在激酶抑制后迅速去磷酸化，被证明可以调节Shp-1和BDP-1酪氨酸磷酸酶以及磷酸酶相互作用蛋白PSTPIP 2。通过抑制Kit激酶活性，减弱了与SH 2结构域衔接子[生长因子受体结合蛋白2（Grb 2）、Cb 1、Slp-76]和SH 3结构域衔接子（HS 1、coronin、CD 2BP 3）的相互作用。观察到Kit与非受体酪氨酸激酶Fes/Fps、Fer、Btk和Syk之间的功能串扰。Kit的抑制调节了与控制粘着斑的途径（桩蛋白、亮帕辛、p130 CAS、FAK 1、Src家族激酶林恩、Wasp、Fh 1 -3、G25 K、Ack-1、Nap 1、SH 3 P12/桥蛋白）和septin-actin复合物（NEDD 5、cdc 11、actin）的磷酸化依赖性相互作用。同量异序蛋白定量和表达聚类、免疫印迹和组织微阵列策略的组合使用允许在肥大细胞白血病模型中通过突变Kit抑制调节的时间测量信号通路。
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), Cb1, 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, Fh1-3, 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.