蛋白质酪氨酸磷酸化在抗失巢凋亡的癌细胞中的失调变化

失巢凋亡是细胞与细胞外基质脱离发生的一种特定的凋亡方式 . 癌细胞抗失巢凋亡或失巢生存能力可以使之在转移过程中生存 . 业已发现癌细胞失巢生存与 PI3K-PKB/Akt 、 MAPK 这两条重要信号途径有关，但是 PI3K-PKB/Akt 、 MAPK 通路的上游酪氨酸激酶途径还不甚清楚 . 为此设计了一种基于 SH2-pTyr 特异性结合特性的功能性筛选方法，以期发现癌细胞失巢生存相关的酪氨酸磷酸化蛋白质，为最终明确酪氨酸激酶途径提供有力的实验依据 . 实验发现， MDCK 细胞悬浮培养后失巢凋亡，但癌细胞可以失巢生存 . 与这一现象相一致的是，悬浮培养后， MDCK 细胞中一系列 SH2 结合的酪氨酸磷酸化蛋白质水平急剧下降，而癌细胞中蛋白质酪氨酸磷酸化水平并不呈锚着依赖性 . 细胞悬浮培养后，随着培养时间的延长， MDCK 细胞中 Abl S SH2 结合的靶蛋白酪氨酸磷酸化水平逐渐降低，在 H460 肺癌细胞中经过短暂下降后升高， H1792 肺癌细胞随着培养时间的延长， Abl SH2 结合的靶蛋白酪氨酸磷酸化水平逐渐增加 . Fyn SH2 和 Crk SH2 结合的蛋白质分别为 FAK 和 p130Cas ，后者是重要的失巢生存信号 . 这些结果提示，酪氨酸磷酸化蛋白质可能赋予肺癌细胞失巢生存能力 . 结果也表明，功能性 SH2 筛查方法可以有效地发现肿瘤细胞中失巢生存相关的酪氨酸磷酸化蛋白质 .

Altered Regulation of Protein Tyrosine Phosphorylation in Anikis-resistant Tumor Cells

Anoikis is a type of apoptosis that results from cell detachment from the extracellular matrix. Resistance to anoikis may allow survival of cancer cells during metastasis. It has been found that PI3K-PKB/Akt and MAPK pathways confer anoikis resistance to cancer cells. However, the tyrosine kinase pathways upstream of PI3K-PKB/Akt and MAPK have not been adequately explored. In an attempt to identify specific phosphotyrosine-containing proteins and potential tyrosine kinase pathways involved in anoikis resistance, a functional screening method based on the specific interaction between src homologue 2 (SH2) domains and phosphotyrosine (p-Tyr) containing proteins was designed. Cell detachment rendered normal MDCK cells to undergo anoikis. However, the survival and proliferation of tumor cells was anchorage-independent. Consistent with this phenomenon, cell detachment induced rapid decrease in SH2s-binding tyrosine phosphorylated proteins in MDCK cells, while tyrosine phosphorylation of SH2-binding proteins in tumor cells was anchorage-independent. It was also found that tyrosine phosphorylation levels of Abl SH2-associated proteins decreased in detached MDCK cells. However, the tyrosine phosphorylation levels of Abl SH2-associated proteins in H460 lung tumor cells increased after a transient decrease, and the levels increased in H1792 lung tumor cells upon detachment. Using this functional screening method, some of the Fyn SH2 and Crk SH2-binding proteins were identified as FAK and p130Cas respectively, which are critical in mediating cell-matrix interactions. The present data suggest that multiple phosphotyrosine-containing proteins and potential tyrosine kinase pathways may act to support the anoikis resistance of tumor cells. The SH2-domain screening method may be an efficient way to explore anoikis-resistance-related phosphotyrosine-containing proteins and potential tyrosine kinase pathways in tumor cells.