A critical aspect of understanding the regulation of signal transduction is not only to identify the protein-protein interactions that govern assembly of signaling pathways, but also to understand how those pathways are regulated in time and space. In this report, we have applied both gain-of-function and loss-of-function analyses to assess the role of the non-receptor protein tyrosine kinase FER in activation of the HGF Receptor protein tyrosine kinase MET. Overexpression of FER led to direct phosphorylation of several signaling sites in MET, including Tyr1349, but not the activation loop residues Tyr1234/5; in contrast, suppression of FER by RNAi revealed that phosphorylation of both a C-terminal signaling site (Tyr1349) and the activation loop (Tyr1234/5) were influenced by the function of this kinase. Adaptin β, a component of the adaptor protein complex 2 (AP-2) that links clathrin to receptors in coated vesicles, was recruited to MET following FER-mediated phosphorylation. Furthermore, we provide evidence to support a role of FER in maintaining plasma membrane distribution of MET and thereby delaying protein-tyrosine phosphatase PTP1B-mediated inactivation of the receptor. Simultaneous up-regulation of FER and down-regulation of PTP1B observed in ovarian carcinoma-derived cell lines would be expected to contribute to persistent activation of HGF-MET signaling, suggesting that targeting of both FER and MET may be an effective strategy for therapeutic intervention in ovarian cancer.

理解信号转导调控的一个关键方面，不仅是要确定控制信号通路装配的蛋白质-蛋白质相互作用，而且还要了解如何在时间和空间上调节这些通路。在本报告中，我们应用了功能获得和功能丧失分析来评估非受体蛋白酪氨酸激酶FER在激活HGF受体蛋白酪氨酸激酶MET中的作用。FER的过表达导致MET中包括Tyr1349在内的几个信号位点直接磷酸化，但没有激活环残基Tyr1234 / 5；相反，RNAi对FER的抑制作用表明C末端信号传导位点（Tyr1349）和激活环（Tyr1234 / 5）的磷酸化均受该激酶功能的影响。衔接蛋白β，FER介导的磷酸化后，将网格蛋白与包被的囊泡中的受体连接的衔接蛋白复合物2（AP-2）的一个组成部分被募集到MET。此外，我们提供证据支持FER在维持MET的质膜分布并从而延迟蛋白酪氨酸磷酸酶PTP1B介导的受体失活中的作用。在卵巢癌衍生的细胞系中同时观察到FER的上调和PTP1B的下调将有助于HGF-MET信号的持续激活，这表明靶向靶向FER和MET可能是治疗干预的有效策略在卵巢癌中。我们提供证据支持FER在维持MET的质膜分布从而延迟蛋白酪氨酸磷酸酶PTP1B介导的受体失活中的作用。在卵巢癌衍生的细胞系中同时观察到FER的上调和PTP1B的下调将有助于HGF-MET信号的持续激活，这表明靶向靶向FER和MET可能是治疗干预的有效策略在卵巢癌中。我们提供证据支持FER在维持MET的质膜分布从而延迟蛋白酪氨酸磷酸酶PTP1B介导的受体失活中的作用。在卵巢癌衍生的细胞系中同时观察到FER的上调和PTP1B的下调将有助于HGF-MET信号的持续激活，这表明靶向靶向FER和MET可能是治疗干预的有效策略在卵巢癌中。