Focal Adhesion Kinase (FAK) Binds RET Kinase via Its FERM Domain, Priming a Direct and Reciprocal RET-FAK Transactivation Mechanism
Focal Adhesion Kinase (FAK) Binds RET Kinase via Its FERM Domain, Priming a Direct and Reciprocal RET-FAK Transactivation Mechanism
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DOI：
10.1074/jbc.m110.168500
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发表时间：
2011-05-13
影响因子：
4.8
通讯作者：
Isacke, Clare M.
中科院分区：
文献类型：
--
作者：
Plaza-Menacho, Ivan;Morandi, Andrea;Isacke, Clare M.
RET是否能够直接磷酸化和激活下游靶点，而不依赖于含有Src同源性2或磷酸酪氨酸结合结构域的蛋白质的结合，以及胞质激酶的反式机制是否可以调节RET功能和信号传导，这些都在很大程度上尚未探索。在这项研究中，寡肽阵列用于筛选底物直接磷酸化的纯化重组野生型和致癌RET激酶结构域的存在或不存在的小分子抑制剂。通过酶动力学、体外激酶和基于细胞的实验验证了肽阵列的结果。粘着斑激酶（FAK）作为RET激酶的直接底物的鉴定揭示了（i）由RET直接磷酸化FAK Tyr-576/577和FAK相互磷酸化RET组成的RET-FAK反式激活机制，其关键是能够拯救激酶受损的RET K758 M突变体，以及（ii）FAK通过其FERM结构域结合RET。有趣的是，这种相互作用在RET磷酸化后被消除，表明RET与FAK的FERM结构域的结合是RET-FAK反式激活的启动步骤。最后，我们的数据表明，FAK抑制剂可用作多发性内分泌瘤2型肿瘤患者的潜在治疗剂，因为FAK激酶抑制剂NVP-TAE 226治疗和通过siRNA下调FAK均降低RET磷酸化和信号传导以及肿瘤和表达致癌RET的转染细胞系的增殖和存活。
Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely unexplored. In this study, oligopeptide arrays were used to screen substrates directly phosphorylated by purified recombinant wild-type and oncogenic RET kinase domain in the presence or absence of small molecule inhibitors. The results of the peptide array were validated by enzyme kinetics, in vitro kinase, and cell-based experiments. The identification of focal adhesion kinase (FAK) as a direct substrate for RET kinase revealed (i) a RET-FAK transactivation mechanism consisting of direct phosphorylation of FAK Tyr-576/577 by RET and a reciprocal phosphorylation of RET by FAK, which crucially is able to rescue the kinase-impaired RET K758M mutant and (ii) that FAK binds RET via its FERM domain. Interestingly, this interaction is abolished upon RET phosphorylation, indicating that RET binding to the FERM domain of FAK is a priming step for RET-FAK transactivation. Finally, our data indicate that FAK inhibitors could be used as potential therapeutic agents for patients with multiple endocrine neoplasia type 2 tumors because both, treatment with the FAK kinase inhibitor NVP-TAE226 and FAK down-regulation by siRNA reduced RET phosphorylation and signaling as well as the proliferation and survival of tumor and transfected cell lines expressing oncogenic RET.