Abstract 5277: Central Role of Early Endosomal Antigen 1 (EEA1) in Organization of Angiotensin II Signaling Leading to Akt Activation in Early Endosome
Akt activation is a major output of Angiotensin II (AngII) signaling. Abundant evidence indicates that AngII signaling occurs in multiple signaling compartments. The mechanisms organizing post-membrane events are incompletely understood. Akt activation requires EGFR transacti-vation at the cell membrane and also requires the endocytotic machinery. We hypothesized that Akt activation appears within the endosomal compartment. In vascular smooth muscle cells, inhibition of endocytosis by phenylarsine oxide (10 μM), concanavalin A (0.3 mg/ml) or overexpression of dynamin K44A mutant, reduce by 90% AngII-induced Akt phosphorylation at Thr308 and Ser473. Cell fractionation shows that Akt phosphorylation occurs mainly in early endosomal fractions after 3 min of AngII (0.1 μM). We posited that the early endosomal marker and scaffolding protein, early endosome antigen 1 (EEA1), is crucial for organizing early endosomal AngII-Akt signaling. Cell fractionation, immunostaining and co-immunoprecipitation studies show that AngII recruits EEA1, AT1R, EGFR, PDK1, PAK1, PKC-alpha and Akt into early endosomes to form a signaling complex. We demonstrate that PAK1 and PKC-alpha are essential components of the EEA1-binding complex required for Akt activation. siRNA knockdown of EEA1 decreases AngII-induced ( 0.1 μM, 3 and 5 min) PDK1, PKC-alpha and Akt phosphorylation by 90%. AngII-induced (0.1 μM, 5 min) EGFR transactivation or EGFR degradation after 30 and 60 min of EGF stimulation (50 ng/ml) was unaffected. Further, downregulation of EEA1 prevents Akt activation by mislocation of critical components of the signaling complex. Taken together, we demonstrate a novel function for EEA1 itself as a central organizer of AngII-Akt signaling in early endosomes.