title: Early endosomal antigen 1 (EEA1) is an obligate scaffold for angiotensin II-induced, PKC-alpha-dependent Akt activation in endosomes.
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.
authors.affiliation: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Rafal Robert
authors.initials: RR
authors.lastname: Nazarewicz
authors.forename: Gloria
authors.initials: G
authors.lastname: Salazar
authors.forename: Nikolay
authors.initials: N
authors.lastname: Patrushev
authors.forename: Alejandra
authors.initials: A
authors.lastname: San Martin
authors.forename: Lula
authors.initials: L
authors.lastname: Hilenski
authors.forename: Shiqin
authors.initials: S
authors.lastname: Xiong
authors.forename: R Wayne
authors.initials: RW
authors.lastname: Alexander
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D051381:Rats; D017207:Rats, Sprague-Dawley; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology*; D033921:Vesicular Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: C000631434:Pdk1 protein, rat; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D014662:Vasoconstrictor Agents; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D000804:Angiotensin II; C546845:mTOR protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D051571:Protein Kinase C-alpha; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases