Jozic Ivan, Blanco Gustavo, Barbieri M Alejandro
Department of Biological Sciences, Florida International University, Miami, FL 33199.
Department Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160.
Curr Cell Biochem. 2011 Dec 28;1(1):20-32.
Activation of receptor tyrosine kinases is a key feature in receptor signaling and membrane trafficking processes. In this study, we found that the insulin receptor tyrosine kinase activity is required for fusion between early endosomes. AG1024, a receptor tyrosine kinase inhibitor, blocked the endosome fusion in a concentration-dependent manner. We observed that Rab5: wild type partially rescued the fusion reaction, whereas Rab5: Q79L mutant fully rescued it. We also observed that treatment of cells with insulin receptor kinase inhibitor HNMPA-(AM) blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of insulin in intact cells. HNMPA-(AM) inhibitor also affected the endosomal co-localization of Rab5 and insulin receptor. However, the formation of Rab5: Q79L mutant-positive endosomes were not affected by the HNMPA-(AM) inhibitor. In addition, HNMPA-(AM) inhibitor affected the association of Rin1 to membrane upon insulin stimulation. Furthermore, Rin1 did not fully support endosome fusion in the presence of the AG1024 inhibitor. These results constitute the first evidence that, at least in part, the enzymatic activity of insulin receptor is required for the fusion events via the activation of Rab5.
受体酪氨酸激酶的激活是受体信号传导和膜运输过程中的一个关键特征。在本研究中，我们发现胰岛素受体酪氨酸激酶活性是早期内体之间融合所必需的。受体酪氨酸激酶抑制剂AG1024以浓度依赖的方式阻断内体融合。我们观察到，Rab5野生型部分挽救了融合反应，而Rab5 Q79L突变体则完全挽救了融合反应。我们还观察到，用胰岛素受体激酶抑制剂HNMPA-(AM)处理细胞会阻断Rab5阳性内体的形成以及完整细胞中添加胰岛素后Rab5的激活。HNMPA-(AM)抑制剂也影响Rab5与胰岛素受体的内体共定位。然而，Rab5 Q79L突变体阳性内体的形成不受HNMPA-(AM)抑制剂的影响。此外，HNMPA-(AM)抑制剂在胰岛素刺激后影响Rin1与膜的结合。此外，在AG1024抑制剂存在的情况下，Rin1不能完全支持内体融合。这些结果首次证明，至少部分胰岛素受体的酶活性是通过激活Rab5来实现融合事件所必需的。
