Early Endosomal Antigen 1 (EEA1) Is an Obligate Scaffold for Angiotensin II-induced, PKC-α-dependent Akt Activation in Endosomes

• Type of Material: Article
• Authors: Rafal Robert Nazarewicz, Emory University; Gloria Salazar, Emory University; Nikolay Patrushev, Emory University; Alejandra San Martin Almeyda, Emory University; Lu Hilenski, Emory University; ShiQin Xiong, Emory University; R Wayne Alexander, Emory University
• Language: English
• Date: 2011-01-28
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://www.jbc.org/content/286/4/2886
• Title of Journal or Parent Work: Journal of Biological Chemistry
• Volume: 286
• Issue: 4
• Start Page: 2886
• End Page: 2895
• Grant/Funding Information: This work was supported, in whole or in part, by National Institutes of Health Grants UO1 HL80711 and HL60728.
• Keywords: Akt PKB; EEA1; Endosomes; Smooth Muscle; Endocytosis; VSMCs; Signal Transduction; PKC Alpha; Angiotensin II; Hypertrophy
• Research Categories: Health Sciences, Medicine and Surgery; Chemistry, Biochemistry

Abstract

Akt/protein kinase B (PKB) activation/phosphorylation by angiotensin II (Ang II) is a critical signaling event in hypertrophy of vascular smooth muscle cells (VSMCs). Conventional wisdom asserts that Akt activation occurs mainly in plasma membrane domains. Recent evidence that Akt activation may take place within intracellular compartments challenges this dogma. The spatial identity and mechanistic features of these putative signaling domains have not been defined. Using cell fractionation and fluorescence methods, we demonstrate that the early endosomal antigen-1 (EEA1)-positive endosomes are a major site of Ang II-induced Akt activation. Akt moves to and is activated in EEA1 endosomes. The expression of EEA1 is required for phosphorylation of Akt at both Thr-308 and Ser-473 as well as for phosphorylation of its downstream targets mTOR and S6 kinase, but not for Erk1/2 activation. Both Akt and phosphorylated Akt (p-Akt) interact with EEA1. We also found that PKC-α is required for organizing Ang II-induced, EEA1-dependent Akt phosphorylation in VSMC early endosomes. EEA1 expression enables PKC-α phosphorylation, which in turn regulates Akt upstream signaling kinases, PDK1 and p38 MAPK. Our results indicate that PKC-α is a necessary regulator of EEA1-dependent Akt signaling in early endosomes. Finally, EEA1 down-regulation or expression of a dominant negative mutant of PKC-α blunts Ang II-induced leucine incorporation in VSMCs. Thus, EEA1 serves a novel function as an obligate scaffold for Ang II-induced Akt activation in early endosomes.