Early Endosomal Antigen 1 (EEA1) Is an Obligate Scaffold forAngiotensin II-induced, PKC-�-dependent Akt Activation inEndosomes*□S

Received for publication, May 5, 2010, and in revised form, November 12, 2010 Published, JBC Papers in Press, November 20, 2010, DOI 10.1074/jbc.M110.141499

Rafal Robert Nazarewicz‡, Gloria Salazar‡, Nikolay Patrushev‡, Alejandra San Martin‡, Lula Hilenski‡, Shiqin Xiong‡,and R. Wayne Alexander‡§1

From the ‡Division of Cardiology, Department of Medicine, Emory University School of Medicine and §Emory University Hospital,Atlanta, Georgia 30322

Akt/protein kinase B (PKB) activation/phosphorylation byangiotensin II (Ang II) is a critical signaling event in hypertro-phy of vascular smooth muscle cells (VSMCs). Conventionalwisdom asserts that Akt activation occurs mainly in plasmamembrane domains. Recent evidence that Akt activation maytake place within intracellular compartments challenges thisdogma. The spatial identity and mechanistic features of theseputative signaling domains have not been defined. Using cellfractionation and fluorescence methods, we demonstrate thatthe early endosomal antigen-1 (EEA1)-positive endosomes area major site of Ang II-induced Akt activation. Akt moves toand is activated in EEA1 endosomes. The expression of EEA1is required for phosphorylation of Akt at both Thr-308 andSer-473 as well as for phosphorylation of its downstream tar-gets mTOR and S6 kinase, but not for Erk1/2 activation. BothAkt and phosphorylated Akt (p-Akt) interact with EEA1. Wealso found that PKC-� is required for organizing Ang II-in-duced, EEA1-dependent Akt phosphorylation in VSMC earlyendosomes. EEA1 expression enables PKC-� phosphorylation,which in turn regulates Akt upstream signaling kinases, PDK1and p38 MAPK. Our results indicate that PKC-� is a necessaryregulator of EEA1-dependent Akt signaling in early endo-somes. Finally, EEA1 down-regulation or expression of a dom-inant negative mutant of PKC-� blunts Ang II-induced leucineincorporation in VSMCs. Thus, EEA1 serves a novel functionas an obligate scaffold for Ang II-induced Akt activation inearly endosomes.

Angiotensin II (Ang II)2 is a pluripotent hormone inVSMCs that stimulates contraction, inflammation, and senes-cence, as well as growth responses resulting in VSMC hyper-trophy (1–3). These effects of Ang II are mediated primarilythrough the G protein-coupled Ang II type 1 receptor (AT1R)

(4). AT1R, once activated, moves into caveolin-enrichedplasma membrane lipid rafts where it facilitates EGF receptor(EGFR) transactivation (5). EGFR-dependent outputs emanat-ing from this domain activate at least two discrete signalingaxes (6). One is represented by Erk1/2 and its downstreamtargets and another involves activation of p38 MAPK, PDK1,Akt, and p70S6K (7, 8). This complexity of the AT1R signal-ing repertoire was anticipated by early findings in which weshowed that Ang II stimulation of phospholipase-mediatedgeneration of diacylglycerol is biphasic in VSMCs. Strategiesthat slowed or prevented internalization mechanisms inhib-ited the second, sustained phase but not the transient firstphase of signaling (9). We thus posited the existence of atleast two discrete Ang II signaling domains in VSMCs at thecell membrane and putatively in an intracellular compartment(10). Subsequently, it was found that many well describedmembrane receptor signaling pathways, such as those for EGFand insulin as well as the �2-adrenergic receptor, also gener-ate signals from various endosomal compartments, nowknown collectively as “signaling endosomes” (11–13). Multi-ple intracellular compartments provide platforms for signalgeneration by recruiting and co-localizing unique combina-tions of signaling molecules. These posited signaling loci in-clude the following: early endosomes; APPL1 (adaptor pro-tein, phosphotyrosine interaction, PH domain, and leucinezipper containing 1) endosomes; multivesicular bodies; andlate endosomes (14–17). Thus, endosomal signals may befunctionally, mechanistically, and temporally distinct fromthose generated at the cell surface (18, 19).Ang II signaling events at the plasma membrane are rela-

tively well understood, especially in the context of the canoni-cal model involving heterotrimeric G protein-coupled recep-tors. AT1Rs, for example, activate phospholipases thatgenerate second messengers such as calcium, phospholipids,and diacylglycerol, which (20) activate classical PKC isoforms(21). PKCs are a class of membrane-bound Ser/Thr kinasesthat influence the organization of signaling scaffold com-plexes and the spatial localization of the resulting output sig-nals (22). The function of PKC, in turn, can be influenced bythe scaffold without being an integral component of the com-plex (22). PKCs have been increasingly implicated in the spa-tial organization of signal propagation in disparate compart-ments in multiple cell types (30).

* This work was supported, in whole or in part, by National Institutes ofHealth Grants UO1 HL80711 and HL60728.

□S The on-line version of this article (available at http://www.jbc.org) con-tains supplemental Figs. 1–3.

1 To whom correspondence should be addressed: Dept. of Medicine, EmoryUniversity Hospital, 1364 Clifton Rd. NE, Atlanta, GA 30322. Tel.: 404-727-1749; Fax: 404-727-3099; E-mail: ralexan@emory.edu.

2 The abbreviations used are: Ang II, angiotensin II; VSMC, vascular smoothmuscle cells; EEA1, early endosomal antigen-1; AT1R, Ang II type 1 recep-tor; EGFR, EGF receptor; PLA, proximity ligation assay; DN-PKC, PKC-�dominant negative mutant; mTOR, mammalian target of rapamycin;mTORC, mTOR complex.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 4, pp. 2886 –2895, January 28, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Although Akt/PKB is a central node in initiating majorpathophysiological outcomes, including Ang II-induced hy-pertrophy in blood vessels, the mechanisms of its activationare not fully understood. In the conventional model, Akt acti-vation mainly occurs at the plasma membrane in lipid raftsand nonlipid raft microdomains (23, 24). We and others (15,25–28) reported that general inhibitors of endocytosis and/orintracellular membrane trafficking modulate signaling eventsinitiated at the plasma membrane. For example, a dynamindominant negative mutant, concanavalin A and methyl-�-cyclodextrin prevent Akt activation in several cell types (27,29). More specifically, methyl-�-cyclodextrin inhibits AngII-dependent activation of Akt in VSMCs (29). Recent experi-ments infer mechanistic insights. The APPL1 containing en-dosome is a transitional structure leading to the PI3K-depen-dent formation of EEA1-positive early endosomes (16).Down-regulation of APPL1 by siRNA inhibits Akt activation/phosphorylation. Akt but not p-Akt interacts with APPL1through its PTB domain (30, 31). p-Akt was not detected inAPPL1 endosomes, which raises the question of whether p-Akt is formed in the downstream EEA1-positive endosomes.Definitive understanding of the mechanisms by which AngII-induced signals activate Akt in VSMCs requires definitionof the subcellular compartment(s) in which these eventsoccur.EEA1 endosomes were recognized originally as organelles

contributing to the trafficking and degradation of plasmamembrane components and inactivation of signaling mole-cules (32). EEA1 is a membrane bound, endosome fusion-promoting protein and an effector of the small GTPase Rab5that regulates early endocytosis events (33, 34). Together,these proteins enable early endosome formation. BecauseEEA1 is not found in other endosomal compartments, it hasbecome a de factomarker of the early endosome. Recent evi-dence infers that EEA1 endosomes may organize and com-partmentalize signaling events. In neutrophils stimulated byplatelet-activating factor, EEA1 protein binds to the NADPHoxidase p40phox-p67phox subunit complex (35). Platelet-activating factor also stimulates Akt1 association with bothp40- and p67-phox. Association of Akt directly with EEA1was not demonstrated and the phosphorylation of Akt wasalluded to but not directly addressed (35). Thus, EEA1 mayplay a functional role in organizing signaling events.We posited that Ang II activation/phosphorylation of Akt

in VSMCs occurs in EEA1-containing early endosomes. Here,we show that EEA1 is required for activation of PKC-�, whichis upstream of Akt phosphorylation. Our study provides com-pelling evidence for a novel physiological role of EEA1 as asignal organizer/scaffold for PKC-�-dependent Akt activationin early endosomes.

EXPERIMENTAL PROCEDURES

Materials—Antibodies to EEA1 and dynamin were pur-chased from BD Biosciences. Rabbit polyclonal antibodies toAkt1, APPL1, EGFR, and PKC-� were from Santa Cruz Bio-technology (San Diego, CA). Antibodies to p-Akt (Ser-473,Thr-308), p-Erk1/2 (Thr-202/Tyr-204), Erk1/2 MAP kinase(9102), p-mTOR (Ser-2448), p-PDK1 (Ser-241) were from

Cell Signaling Technology, Inc. (Danvers, MA). All otherchemicals and reagents, including DMEM, were from Sigma.Cell Culture, Adenovirus Transduction, and Ang II

Treatments—VSMCs were isolated from male Sprague-Daw-ley rat thoracic aortas by enzymatic digestion. Cells weregrown in DMEM with 25 mM HEPES and 4.5 g/liter glucosesupplemented with 10% calf serum, 2 mM glutamine, 100units/ml penicillin, and 100 �g/ml streptomycin. For adenovi-rus-induced protein expression, cells at 40–50% confluencewere incubated with adenoviruses for 30 min in serum-freemedium. Cells were then washed and grown for 48 h in se-rum-supplemented medium. Expression of proteins wastested by Western blot using specific antibodies. For Ang IItreatments, cells were grown in serum-free DMEM overnightand incubated with 100 nM Ang II at 37 °C.Western Blot and Immunoprecipitation—VSMCs were col-

lected and lysed in lysis buffer (50 mM HEPES, pH 7.4, 50 mM

NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM PMSF, 10 �g/mlaprotinin, 10 �g/ml leupeptin, 10 mM sodium pyrophosphate,50 mM sodium fluoride, and 1mM sodium orthovanadate). Ly-sates were separated by SDS-PAGE, transferred to nitrocellulosemembranes, and incubated with specific antibodies. For immu-noprecipitation, cell lysates were clarified by centrifugation at16,000 � g for 10min, and the supernatant was incubated withspecific antibodies or IgG control. Protein-antibody complexeswere then pulled down with protein A/G PLUS-agarose beads(Santa Cruz Biotechnology). After five washes, samples wereseparated by SDS-PAGE, transferred tomembranes, and ana-lyzed byWestern blot with specific antibodies.Lipid Raft Flotation—Caveolae-enriched lipid raft fractions

were isolated by sucrose gradient flotation as described previ-ously (24). VSMCs were lysed in lysis buffer containing 1%Triton X-100 during 30 min at 4 °C, and sucrose was added toreach 1.5 M. Samples were loaded on the bottom and overlaidwith 1.2 M sucrose followed by 0.15 M sucrose prepared in thesame buffer without Triton X-100. After centrifugation at38,000 rpm (Beckman L8 ultracentrifuge) for 18 h at 4 °C,fractions were collected from top to bottom and analyzed byWestern blot.Sucrose Gradient Fractionation—VSMCs were grown on

100-mm dishes for 72 h. After overnight serum starvation,cells were stimulated with Ang II. After stimulation, all proce-dures were carried out at 4 °C. Cells were collected, washed,and resuspended in ice-cold homogenization buffer (50 mM

HEPES, pH 7.4, 0.25 M sucrose, complete mixture of proteaseinhibitors). Cell suspensions were homogenized using 30strokes with a glass dounce homogenizer. Post-nuclear frac-tion was loaded on top of a 10–50% sucrose multistep gradi-ent and sedimented at 36,000 rpm for 16 h (Beckman L8 ul-tracentrifuge). Fractions were collected from the top andanalyzed by SDS-PAGE. Distribution of specific markers wasmonitored by Western blot.Immunofluorescence—VSMCs were incubated with anti-

Akt, anti-APPL1, or anti-EEA1 for 1 h at room temperatureand then incubated in either FITC-conjugated (JacksonImmunoResearch Laboratories,West Grove, PA) or RhodamineRed X-conjugated secondary antibodies for 1 h at room tem-perature. Cells on coverslips were mounted in Vectashield

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(Vector Laboratories, Burlingame, CA) and examined usingthe 488- and 543-nm lines of the argon ion and green HeNelasers with 515/30-nm band pass and 585-nm-long pass fil-ters, respectively, in a confocal imaging system (Zeiss LSM510 META). For double-labeling experiments, FITC and Rho-damine Red X images were scanned with the multi-trackingmode on a Zeiss LSM 510 META confocal microscope. Con-trols with no primary antibody showed no fluorescence label-ing, and single-label controls were performed in double-label-ing experiments. To distinguish random color overlap fromco-localization due to either co-compartmentalization or in-teraction of proteins, Imaris Coloc software was utilized (36).Imaris software enables nonbiased co-localization analysis byproviding automatic threshold selection. All images werequantified using the automatic mode with no manual adjust-ment of the thresholds.Proximity Ligation Assay (PLA)—VSMCs were grown on

cover glasses; paraformaldehyde fixed and labeled with EEA1or APPL1, Akt, and p-Akt antibodies. Next, we followed thePLA protocol (Olink Bioscience, Uppsala, Sweden) in whichshort DNA strands are bound to antibodies of interest. In thissystem, antibodies against a protein pair are attached to shortchains of complementary DNA oligonucleotides, which hy-bridize when in close proximity. Enzymatic ligation of oligo-nucleotides generates a circularized DNA strand that servesas a template. The amplification reaction product that re-mains attached to the antibody-protein complex is visualizedthrough the hybridization of fluorescently labeled oligonu-cleotides (37, 38). A fluorescence signal occurs only when twoproteins are in close proximity (�40 nm) (38). Signals of fluo-rescent PLA probes indicating co-localization/co-compart-mentalization of two proteins (38) were acquired with a con-focal imaging system (Zeiss LSM 510 META).siRNA Transfection—siRNA for EEA1 (3�- AAGTTTCA-

GATTCTTTACAAA) or scrambled siRNA as a control(Ambion) were transfected into VSMCs using the BasicNucleofector� kit for primary smooth muscle cells (AmaxaBiosystem, Gaithersburg, MD). The efficiency and specificityof siRNA depletion were verified by Western blot using spe-cific antibodies.[3H]Leucine Incorporation—VSMCs were grown in DMEM

containing 0.1% serum for 48 h. Next, cells were incubatedwith [3H]leucine (1 �Ci/ml) in the presence or absence of 100nM Ang II for 48 h. Before harvesting, cells were washed withphosphate-buffered saline, incubated with 5% trichloroaceticacid for 5 min on ice, and lysed with 0.4 M NaOH. Radioactiv-ity of cell-incorporated [3H]leucine was analyzed using a liq-uid scintillation counter.Statistical Analysis—All values were expressed as mean �

S.E. and analyzed using analysis of variance of the repeatedexperiments, followed by the Tukey’s post hoc test. Statisticalsignificance was accepted at p � 0.05.

RESULTS

Ang II Stimulation of Akt in VSMCs Requires Internaliza-tion and Does Not Occur in Caveolae/Lipid Raft Fractions—Ithas been reported that growth factor-mediated Akt signalingin endothelial cells is organized in caveolae/lipid rafts (24)

and, as noted earlier, Ang II-mediated signaling events inVSMCs require the transactivation of the EGFR tyrosine ki-nase in a caveolae/lipid raft-dependent manner (29). Thus, wefirst examined whether Ang II-stimulated Akt phosphoryla-tion occurs in lipid rafts in VSMCs. Ang II activates Akt witha peak between 3 and 5 min. Using flotation sucrose sedimen-tation, we isolated caveolin-enriched lipid rafts as indicated bythe presence of caveolin 1 and Rac-1 (Fig. 1A). Caveolin 1, asexpected, was also detected in noncaveolae fractions due to itslocation in multiple compartments including the Golgi andendoplasmic reticulum (39, 40). Akt was not recruited intocaveolin-enriched lipid rafts after 1 or 3 min of stimulationwith Ang II (Fig. 1A, lane 2). Phosphorylated Akt (p-Akt) lo-calized exclusively in heavy, nonlipid raft fractions, thus pro-viding evidence that Akt activation by Ang II occurs in non-lipid raft cellular compartments (Fig. 1A, lane 6).Signaling of G protein-coupled receptors (including AT1R)

is initiated at the plasma membrane; however, activation ofthe entire Ang II signaling repertoire requires internalization(9, 41). To confirm the importance of internalization for Aktactivation in VSMCs, we overexpressed the dominant nega-tive K44A dynamin mutant (DynK44A) to prevent internal-ization. Overexpression of DynK44A prevented Akt phosphor-ylation at Ser-473 and Thr-308 after 3 and 5 min of Ang IIstimulation (Fig. 1B). For control, we tested Erk1/2 activationthat was significantly but not completely inhibited underthese conditions indicating intact signaling from AT1R. Thesefindings are consistent with the notion that internalization tointracellular compartments is required for Ang II-inducedAkt phosphorylation at both of its activation sites.Akt Is Activated in EEA1 Early Endosomes—To investigate

the nature of Akt activation site(s), we performed subcellularfractionation of cells in basal and Ang II-stimulated condi-tions. Basally, Akt localized primarily in the cytosol (Fig. 2A,fractions 1 and 2). After Ang II stimulation, both Akt andpAkt co-migrated with the early endosomal markers EEA1and Rab5 (Fig. 2A, fractions 3 and 4). Migration of markers todetect cytosol (GAPDH), plasma membrane (EGFR andRac1), early endosomes (EEA1 and Rab5), late endosomes(Rab7), and caveolin-enriched fractions (Fig. 2B) were unaf-fected by 1- or 3-min incubation with Ang II (data notshown). As shown recently, Akt associates with APPL1 pro-tein in HeLa cells in response to insulin-like growth factorand in rat adipocytes in response to insulin (15, 42), raisingthe possibility that Ang II activation of Akt could occur inAPPL1 endosomes in VSMCs. The notion that this compart-ment is the precursor of EEA1 endosomes (16) and that bothcompartments contain Rab5 led us to investigate whether Aktis phosphorylated in an early endosomal compartment con-taining EEA1, APPL1, or both. To that end, we assessed theco-localization of Akt with both markers by immunofluores-cence. After 1 and 3 min of stimulation, Ang II robustly in-creased Akt co-localization with EEA1 (Fig. 3, A and C).There was a modest basal interaction between APPL1 andAkt, which was not enhanced by Ang II treatment (Fig. 3, Band C). Furthermore, we used Imaris software to visualizethe extent of co-localization of EEA1 and Akt, based onthree-dimensional images of z-stack sections. As shown in

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supplemental Fig. 1, EEA1 signals (presented on images withsurface generated by Imaris software) associate in endosomal-like structures that do not co-localize under basal conditionswith the Akt signal. Ang II stimulation changes the Akt-stain-ing pattern and brings Akt into EEA1-positive structures.Moreover, immunoprecipitation experiments showed that

EEA1 and Akt interacted in response to Ang II treatment (Fig.3D). To confirm this interaction further, we used PLA, a tech-nology that allows the detection of in vivo protein-proteininteractions (38). As shown in Fig. 3F, incubation with Aktand EEA1 antibodies generates a vesicle-like staining in basalconditions that increased after Ang II incubations, indicatingthat EEA1 and Akt interact. These results support the hypoth-esis that phosphorylation of Akt occurs in EEA1 rather thanAPPL1 endosomes and that EEA1 may facilitate Akt activa-tion. Using PLA, we found that stimulation of VSMCs withAng II led to a robust EEA1 and p-Akt interaction, as re-flected by the increase in fluorescence signal with a vesicular-like distribution (Fig. 4A). There was a basal backgroundAPPL1/p-Akt interaction that was not up-regulated with AngII stimulation. (Fig. 4B). These results are consistent with theresults from the immunofluorescence (Fig. 3A) and subcellu-lar fractionation experiments (Fig. 2), showing direct evidencethat Akt is activated in EEA1 early endosomes.EEA1 Down-regulation Prevents Akt Activation—The acti-

vation of Akt in EEA1 early endosomes and its interactionwith EEA1 protein support the hypothesis that EEA1 orga-nizes signaling events required for Akt activation. To investi-gate this possibility further, we knocked down EEA1 expres-sion with siRNA and assessed Akt activation. The data in Fig.5A demonstrate that silencing of EEA1 abolished Ang II-in-duced Akt phosphorylation at both Thr-308 and Ser-473 siteswithout affecting the expression level of Akt. To gain insightsregarding the specificity of EEA1 knockdown in Akt signaling,we examined activation of Erk1/2, which functions in a dis-tinctly different signaling pathway (43, 44). In contrast to theinhibition of Akt activation, Erk1/2 phosphorylation was un-affected (Fig. 5A), indicating that EEA1 has a specific role in

FIGURE 1. Akt activation by Ang II requires internalization. A, VSMCs were stimulated with 100 nM Ang II (AngII) for 1 or 3 min. Caveolin (cav)-enrichedlipid raft fractions (lanes 2 and 3) containing caveolin 1 (Cav-1) and Rac-1 were separated from non-caveolae (non-cav) fractions (lanes 5 and 6) using sucroseflotation gradients. Total and phosphorylated Akt are detectable only in noncaveolae fractions, indicating Akt is not activated in caveolin-enriched lipidrafts. B, dynamin (Dyn) K44A dominant negative mutant (lanes 4 – 6), expressed in VSMCs known to prevent endocytosis, inhibits activation of Akt, suggest-ing that Akt activation may take place within internal compartments. Overexpression of dynamin was confirmed by probing with dynamin-specific anti-body. Bar graphs represent averaged data (mean � S.E.) expressed as fold of change over control LacZ adenovirus infected cells (Ctr) (n � 3). *, p � 0.05significantly different from control conditions.

FIGURE 2. Akt is recruited to early endosomes by Ang II. Postnuclear su-pernatants of VSMCs stimulated with Ang II (100 nM for 3 min) or nonstimu-lated were separated in a 10 –50% sucrose gradient. Endosomal localizationof total and phosphorylated Akt was tested using Western blot with specificantibodies. Major fraction of phosphorylated Akt co-migrates with EEA1indicating Akt is activated within early endosomes (A). To characterize thegradient, Western blots were performed with antibodies against EEA1 andRab5 as markers of early endosomes (Early endos.; lanes 3 and 4), GAPDH forcytoplasmic fractions (Cyto; lanes 1 and 2), Rab7 for late endosomes (lane 8),and EGFR and Rac-1 for plasma membrane fractions (PM; lanes 6 – 8). Repre-sentative of three independent experiments. Bar graph (B) represents aver-aged data of A (mean � S.E.) expressed as fold of change over basal. *, p �0.05 significantly different from basal conditions. Cav-1, caveolin 1.

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the Akt signaling axis. Furthermore, transactivation of EGFR,an early event in Ang II signaling that occurs in the plasmamembrane, was also unaffected in EEA1-deficient cells com-

pared with control scrambled siRNA transfected cells (datanot shown). We also examined the effects of EEA1 down-reg-ulation on Akt downstream signaling. Silencing of EEA1 ex-pression prevented phosphorylation of mTOR at Ser-2448and S6 kinase at Ser-389 without changes in expression ofthese downstream proteins (Fig. 5B). As a control, we testedwhether other functions of early endosomes independent ofAng II signaling or internalization events were affected byEEA1 down-regulation. In particular, we examined EGF-in-duced EGFR degradation that depends on internalization andtrafficking through early endosomes (45). There were no ma-jor differences in EGF-induced EGFR degradation betweencontrol and EEA1-deficient cells (supplemental Fig. S2).These findings indicate that Ang II-induced Akt phosphoryla-tion in early endosomes and activation of its downstream tar-gets requires the presence of EEA1. Moreover, these effectsexhibit specificity for the Ang II-initiated activation of Aktand thus are not due to a broad inhibition of endocytosis-de-pendent events.EEA1 Is Required for Activation of Akt UpstreamKinases—

To investigate the mechanism by which EEA1 regulates Aktactivation, we explored whether EEA1 modulates Akt up-stream kinases. In many cells, mTOR complex 2 (mTORC2)has been implicated in Akt activation; however, such a role inVSMCs has not been established. Prolonged treatment withrapamycin inhibits, phosphorylation at Ser-2448 andSer-2481, markers of activation of mTORC1 and mTORC2,respectively (46, 47). As shown in supplemental Fig. S3 pro-longed rapamycin treatment of VSMCs inhibited Ang II-in-duced activation of both complexes as indicated by lack ofphosphorylation of the specific activation sites. Akt phosphor-ylation, however, was not affected. These data indicate that incontrast to other model systems (24, 46, 47), mTORC2 maynot directly participate in Akt activation in VSMCs, at least inresponse to Ang II.Next, we examined the effects of EEA1 silencing upon the

following: activation of PDK1, a well established kinase forAkt Thr-308 phosphorylation (48–50); p38 MAPK, whichwas reported as a kinase upstream of Akt Ser-473 (51); andPKC-�, which was recently established as an Akt regulator(24, 52). We first examined whether these kinases are re-cruited to EEA1 endosomes in response to Ang II. Similar toAkt, PKC-�, PDK1, and p38 MAPK move from the cytosol toEEA1 containing fractions after 3 min of Ang II stimulation(Fig. 6A). Consistent with observations in other cell types, weobserved a transient translocation of PKC-� and PDK1 toplasma membranes after 1 min of Ang II stimulation. Next,we tested the role of EEA1 in Akt upstream signaling kinaseactivation. We found that down-regulation of EEA1 inhibited

FIGURE 3. Akt co-localizes with EEA1. A and B, VSMCs incubated with or without (100 nM Ang II for 1 or 3 min) were fixed with 4% paraformaldehyde andprocessed for immunofluorescence as described under “Experimental Procedures.” Confocal images were acquired in samples co-stained with antibodiesagainst EEA1 (A, red) or APPL1 (B, red) and Akt (green). Nuclei were localized by DAPI (blue). The co-localization channel (Colo) generated by overlappingpixels of EEA1 or APPL1 and Akt immunofluorescent signals using Imaris software shows that Akt co-localize with EEA1 in response to Ang II, but no signifi-cant changes were observed for APPL1 endosomes. C, bar graph represents averaged data of A and B (mean � S.E.) expressed as fold of change over basal.*, p � 0.05 significantly different from basal conditions. NC represents negative control in which the primary anti EEA1 antibody was omitted. D, EEA1 wasimmunoprecipitated (IP) from VSMCs. Ang II (100 nM) stimulates association of Akt and EEA1. E, bar graph represents quantified data of D expressed as foldof change over basal. F, interaction of EEA1 and Akt after Ang II (100 nM) visualized as a red fluorescent signal detected by PLA (as described under “Experi-mental Procedures”). Blue signal of DAPI indicates nuclei.

FIGURE 4. Akt is activated in EEA1 early endosomes. Phosphorylated Aktlocalize in close proximity (�40 nm) to EEA1 (A), but no increase in signalwas detected for APPL1 (B) as indicated by the red fluorescent signal gener-ated by PLA in VSMCs stimulated with Ang II (100 nM for 1 or 3 min). Theblue signal indicates nuclei stained with DAPI.

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Ang II-induced PDK1, p38 MAPK, and PKC-� phosphoryla-tion (Fig. 6B). To test whether PKC-� is upstream of Akt ki-nases in VSMCs, we overexpressed a PKC-� dominant nega-tive mutant (DN-PKC). We observed that the DN-PKCreduced Akt, p38 MAPK, and PDK1 phosphorylation in re-sponse to Ang II (Fig. 6C). Thus, PKC-� acts as an importantregulator of Ang II-Akt signaling pathway by modulating theactivation of both Akt Ser-473 and Thr-308 upstream kinasesin early endosomes.EEA1 and PKC-� Are Required for Ang II-induced

[3H]Leucine Incorporation in VSMCs—Akt has an establishedrole in Ang II-induced [3H]leucine incorporation implicatedin VSMC hypertrophy (51). Thus, we tested whether down-regulation of EEA1 expression or overexpression of DN-PKC-� prevents Ang II-induced [3H]leucine incorporation.As shown in Fig. 7, A and B, both interventions significantlyinhibited Ang II-induced [3H]leucine incorporation, confirm-ing the physiological relevance of EEA1 and PKC-� for AngII-induced Akt activation in early endosomes.

DISCUSSION

Previously, the primary functions associated with endo-somes were trafficking, sorting, and recycling between plasmamembrane and internal compartments. Reports in varioussystems support the concept that the internalization and en-docytotic machineries are involved directly in signal transduc-tion (14, 17, 18). Our previous work showed that a majorcomponent of Ang II signaling, including Akt activation inVSMCs, depends on internalization (9). Thus, endosomesmay serve as platforms for generating discrete activation ofdownstream pathways that refine the original agonist-medi-

ated membrane signal into one with enhanced target specific-ity. Akt is centrally involved in Ang II-induced pathologicalhypertrophy of VSMCs, but despite its importance, the mech-anisms regulating its activation by Ang II are incompletelyunderstood. In this study, we report that Ang II recruits Aktinto early endosomes in VSMCs, where it is activated and ini-tiates downstream signaling. Moreover, we provide evidencethat EEA1, a structural component of early endosomes, facili-tates activation of Akt upstream kinases and Akt itself. Wealso identify PKC-� as a key regulator of the upstream Aktkinases p38 MAPK and PDK1 and show that EEA1 andPKC-� are required for Ang II-induced [3H]leucine incorpo-ration, a process implicated in hypertrophic growth ofVSMCs. Our data are consistent with a model (Fig. 8) inwhich Ang II induces the recruitment of PKC-�, Akt, and itsupstream kinases to early endosomal compartments whereAkt becomes activated. EEA1 thus functions as a scaffold ena-bling Akt phosphorylation in early endosomes in VSMCs.Traditional models of Akt activation assigned the plasma

membrane as the primary signaling locale. More specifically,lipid rafts have been implicated as a domain where PKC-�-dependent phosphorylation of Akt occurs (24). Other investi-gators, however, reported evidence indicating that in somesignaling pathways, endocytosis is required for Akt activation(15, 26, 53). For example, the APPL1 protein, a marker ofAPPL endosomes (precursors of EEA1 early endosomes (16)),is required for Akt phosphorylation in zebrafish (15). Al-though these investigators showed that Akt co-localized withAPPL1 in response to insulin in cervical cancer cells (15), thesite of Akt phosphorylation was not defined. Also, the endo-

FIGURE 5. Down-regulation of EEA1 prevents Akt activation. A, in VSMCs, down-regulation of EEA1 by siRNA (lanes 4 – 6) compared with scramble siRNA(lanes 1–3) inhibits Ang II induced Akt phosphorylation at Ser-473 and Thr-308 but did not prevent Erk1/2 phosphorylation. B, down-regulation of EEA1(lanes 4 – 6) blunts phosphorylation of Akt downstream signaling molecules, S6 kinase, and mTOR compared with scramble siRNA (lanes 1–3). Quantifica-tions of three independent experiments are depicted in the right panel. *, p � 0.05.

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cytotic machinery is crucial for �1-adrenergic receptor-in-duced Akt phosphorylation in neonatal rat cardiac myocytes(27). Recently, it was shown that in 3T3-L1 adipocytes, Aktinteracts with endosomes containing the phosphatidylinositol3-phosphate binding, FYVE (Fab-1, YGL023, Vps27, andEEA1) domain-containing protein WDFY2 in an isoform-specific manner. Depletion of WDFY2 leads to impairedphosphorylation of Akt2 and its substrates, but not Akt1, in-

dicating that these endosomal compartments may facilitategrowth hormone signal specification generally (54). Further-more, Rab5, a GTPase regulator of endocytosis and an EEA1binding partner, is required for EGF-induced Akt phosphoryl-ation in cervical cancer cells (55). Early and recycling endo-somes also modulate lysophosphatidic acid-induced PI3K/Aktsignaling in human embryonic kidney cells (26). Although itseems clear that Akt can be activated within internal com-partments, the mechanisms and location are incompletelydefined.We found that Ang II did not recruit to or activate Akt in

caveolin-enriched lipid rafts or APPL1 endosomes. Instead,we show that Ang II-induced Akt activation is dependentupon endocytosis and that Ang II stimulation recruits a majorfraction of Akt into early endosomes, peaking at 3 min (Fig.2A), where it co-migrates with EEA1 (Fig. 2). Strikingly, phos-phorylated Akt correlates with total Akt localization and isdetected predominantly within the early endosomal fraction,further inferring that phosphorylation occurs in internalizedvesicles.p-Akt and EEA1 interaction was evaluated by PLA that in-

forms the propinquity of two proteins but does not distin-guish whether they are interacting directly or are merely co-localized in the same compartment (38). A positive signal inresponse to Ang II was observed for EEA1, but insignificant

FIGURE 6. EEA1 and PKC-� regulation of Akt upstream signaling. A, Ang II (100 nM) induces the recruitment of Akt as well as PKC-�, PDK1, and p38 MAPKinto EEA1-containing endosomes (EE, fractions 3 and 4). Fractions were characterized as in Fig. 2A. VSMCs were transfected with scrambled (Scr, lanes 1–3) orEEA1 (lanes 4 – 6) siRNA. B, Activation of Akt upstream kinases PDK1, p38 MAPK, and PKC-� by Ang II was inhibited in EEA1 down-regulated cells. C, Ang II-induced activation of Akt and its upstream signaling kinases, p38 MAPK and PDK1, was inhibited in VSMCs overexpressing dominant negative mutant ofPKC-� (lanes 4 – 6) compared with Ad_LacZ control cells (Ctr, lanes 1–3). Quantifications of three independent experiments are depicted on the right ofWestern blot images, *, p � 0.05. PM, plasma membrane fraction; Cyto, cytoplasmic fraction.

FIGURE 7. Ang II-induced [3H]leucine incorporation requires EEA1 andPKC-�. Ang II-induced (100 nM for 48 h) VSMCs hypertrophy measured by[3H]leucine incorporation was significantly inhibited by down-regulation ofEEA1 using siRNA, compared with siScrambled (Ctr) (A) or by overexpressionof dominant negative mutant PKC-� (DN-PKC-�), compared with mocktransfected cells (Ctr) (B). *, p � 0.05 in two independent experiments per-formed in triplicate.

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changes were found for APPL1 (Fig. 4, A and B). This resultraised the possibility that EEA1 itself may play a role in Aktactivation. Moreover, EEA1 co-immunoprecipitates with Aktafter Ang II, providing additional evidence that EEA1 servesas an organizer of Akt signal generation. EEA1 siRNA notonly inhibited Akt activation but also blocked activation ofcritical Akt upstream kinases, PKC-�, p38 MAPK, and PDK1(Fig. 6B). This enabling function of EEA1 appears to be spe-cific for the Akt signaling axis in VSMCs, because EGFRtransactivation, which is localized in lipid rafts (56), andErk1/2 activation were not affected. These observations indi-cate that in VSMCs Ang II-initiated Akt phosphorylation oc-curs in early endosomes and is EEA1-dependent.PKC-� has been implicated in Akt activation in other cell

types (24, 57). For example, PKC-� is a critical organizer ofinsulin-mediated activation of Akt within lipid rafts of endo-thelial cells, as noted (24). Loss of PKC-� activity led to mislo-calization of signaling complex components and inhibition ofAkt phosphorylation. Similarly, our results show that PKC-�also coordinates the activation of Akt in Ang II-stimulatedVSMCs, at least in part, by regulating its upstream signalingkinases, p38 MAPK, and PDK1 (50, 51). These results revealPKC-� as a key regulator of the Ang II signaling leading toAkt activation in VSMCs and, together with previous studies,indicate that PKC-� is a common regulator of Akt signalingfrom both receptor tyrosine kinases (24) and G protein-cou-pled receptors (AT1R), regardless of the cellular compart-ment in which Akt phosphorylation takes place.Our previous studies established a key role of the Ang II-

Akt signaling axis in hypertrophy of VSMCs, a hallmark of

hypertensive cardiovascular disease (51). Consistent with thisobservation, we found that inhibition of the Akt pathway byeither EEA1 siRNA knockdown or expression of DN-PKC-�significantly prevents Ang II-induced [3H]leucine incorpora-tion that has been associated with hypertrophic growth inVSMCs. Our study infers physiological importance of theearly endosomal compartment in Ang II-induced Akt activa-tion and may inform more generally a novel role of EEA1 inintracellular signal organization.Thus, we show that EEA1 per se is critical for Ang II-in-

duced Akt activation/phosphorylation and provide compel-ling evidence that it acts as a scaffold for assembling proteinsignaling complexes in early endosomes. Our data reveal apreviously unrecognized role of EEA1 and early endosomes inAng II signaling.

Acknowledgments—We thank Dr. Kathy K. Griendling for manyhelpful comments and discussions. We also thank Dr. MasukoUshio-Fukai for insights and comments in the initial phase of thisproject.

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FIGURE 8. EEA1 is a scaffold for Akt activation in early endosomes. Ang IIinduces the recruitment of Akt, PDK1, and p38 MAPK and the Akt regulatorPKC-� to EEA1 endosomes to facilitate Akt activation in this compartment.Ang II-induced signaling events mediated by EEA1 and PKC-� are requiredfor the activation of S6 kinase and mTOR and subsequent hypertrophy ofVSMCs.

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Lula Hilenski, Shiqin Xiong and R. Wayne AlexanderRafal Robert Nazarewicz, Gloria Salazar, Nikolay Patrushev, Alejandra San Martin,

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

doi: 10.1074/jbc.M110.141499 originally published online November 20, 20102011, 286:2886-2895.J. Biol. Chem. 

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