Protein Kinase B/Akt Acts via Glycogen Synthase Kinase 3 To Regulate Recycling of {alpha}v{beta}3 and {alpha}5{beta}1 Integrins

Protein kinase B (PKB)/Akt is known to promote cell migration,and this may contribute to the enhanced invasiveness of malignantcells. To elucidate potential mechanisms by which PKB/Akt promotesthe migration phenotype, we have investigated its role in theendosomal transport and recycling of integrins. Whereas theinternalization of ${alpha}$ vß3 and ${alpha}$ 5ß1 integrinsand their transport to the recycling compartment were independentof PKB/Akt, the return of these integrins (but not internalizedtransferrin) to the plasma membrane was regulated by phosphatidylinositol3-kinases and PKB/Akt. The blockade of integrin recycling andcell spreading on integrin ligands effected by inhibition ofPKB/Akt was reversed by inhibition of glycogen synthase kinase3 (GSK-3). Moreover, expression of nonphosphorylatable activeGSK-3ß mutant GSK-3ß-A9 suppressed recyclingof ${alpha}$ 5ß1 and ${alpha}$ vß3 and reduced cell spreadingon ligands for these integrins, indicating that PKB/Akt promotesintegrin recycling by phosphorylating and inactivating GSK-3.We propose that the ability of PKB/Akt to act via GSK-3 to promotethe recycling of matrix receptors represents a key mechanismwhereby integrin function and cell migration can be regulatedby growth factors.

INTRODUCTION

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Introduction

Engagement of integrins with the extracellular matrix (ECM)is known to be required for a range of biological processes,including cell proliferation and migration and, importantly,the prevention of apoptosis and anoikis (37). It is now clearthat integrin function is influenced by the endosomal and receptorrecycling pathways, and some of the Rab GTPase- and dynamin-dependentsteps that are responsible for this have now been described(31, 35). Furthermore our laboratory has reported that growthfactor stimulation influences the Rab-dependent recycling ofintegrins (35), but the intracellular signaling pathways thatmediate this effect are unknown.

Growth factor-regulated, class I phosphatidylinositol 3-kinases[PI(3)Ks] regulate postendocytic trafficking and recycling ofvarious receptors and transporters (19, 42), and it is now establishedthat regulated recycling of the GLUT4 transporter requires theactivity of p85/p110 PI(3)K in adipocytes and muscle cells (43,45). Protein kinase B (PKB)/Akt is activated downstream of classI PI(3)Ks and is known to be involved in many cellular processes,including vesicular trafficking (25), and it is now apparentthat PKB/Akt plays a key role in regulating GLUT4 recycling(16). A number of reports have shown that inactivation of PKB/Aktopposes the ability of insulin to activate GLUT4 translocation,and constitutively active mutant PKB/Akt proteins increase deliveryof GLUT4 to the plasma membrane in the absence of insulin stimulation(13, 15, 46). It has been shown that GLUT4 is recycled to theplasma membrane via Rab4- and Rab11-dependent pathways (7, 8,49), similar to those found for integrins (35), suggesting thatGLUT4 and integrin recycling pathways may have similar mechanismsof regulation.

The various physiological functions of PKB/Akt are brought aboutby phosphorylation of several target molecules including Bad,caspase 9, Raf, and glycogen synthase kinase 3 (GSK-3) (25).GSK-3 is inactivated by phosphorylation of an N-terminal serineresidue, Ser²¹ in GSK-3- ${alpha}$ and Ser⁹ in GSK-3-ß. AlthoughGSK-3 was originally identified as a kinase that phosphorylatesglycogen synthase, more recently it has become apparent thatGSK-3 acts on a broader range of substrates including tau, ß-catenin,eukaryotic initiation factor 2B (6), and the microtubular motor,kinesin (30). PKB/Akt and phospho-GSK have both been localizedto the leading edge of migrating cells and have been shown toinfluence the motility of a number of cell types (5, 20). Inside-outactivation of integrins is also thought to be key to the coordinationof cell migration, and the activation of ${alpha}$ vß3, ${alpha}$ 5ß1, ${alpha}$ vß5, and ${alpha}$ 2ß1 integrin heterodimers is mediatedby PKB/Akt (5). Activation of integrins is likely to involvethe regulation of integrin recycling, and it is, therefore,possible that PKB/Akt and GSK-3 are involved in the transportof integrins in the endosomal pathway and/or the recycling pathway.Here we present evidence for the involvement of PI(3)K and PKB/Aktin integrin trafficking and cell spreading and show that theseprocesses are likely to be promoted by inactivation of GSK-3.

MATERIALS AND METHODS

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Materials and Methods

Expression plasmids. ${alpha}$ v, ß3, ${alpha}$ 5, and ß1 integrins and N121IRab4,wild-type Rab11 (wt-Rab11), N124IRab11, and S24NRab11 were expressedby pcDNA3 and have been described previously (35). Hemagglutinin(HA)-PKB- ${alpha}$ , HA-PKB-AAA (K179/A, T308/A, S473/A), HA-membranetargeted PKB- ${alpha}$ (memb-PKB- ${alpha}$ ), and HA-PKB kinase dead (K179/A) wereexpressed by the pCMV5 vector and have been described previously(50); they were a generous gift from D. Alessi (University ofDundee, Dundee, United Kingdom). memb-PKB-estrogen receptor(ER; conditionally active, membrane targeted) has been described(23, 27) and was a generous gift from M. McMahon (Universityof California). The green fluorescent protein (GFP)-early endosomalantigen 1 (EEA1) fusion (GFP-EEA1) was expressed by the pEGFP-C1vector. The myc-tagged human transferrin receptor (myc-hTfn-R)was expressed by the pCB7 vector and was described previously(29). The mU6pro vector was used for the expression of RNA duplexes(48, 52). A gene-specific sequence identical in the human andmurine genes encoding Akt1 and Akt2 was targeted (5'-GT CGCTGC CTG CAG TGG ACC AC-3') (11). EcoRI/NotI fragments of humanGSK-3ß-A9 and -F216 cDNAs were subcloned from pcDNA3(14) into EcoRI/HindIII sites in the pCMV5 expression vector(NotI and HindIII restriction sites were end filled to createblunt ends with T4 DNA polymerase). All plasmids were purifiedby CsCl banding prior to transfection.

Cell culture and transfection. NIH 3T3 mouse fibroblasts were grown in Dulbecco's modifiedEagle's medium (DMEM) as described previously (35). For assaysof receptor internalization, integrin recycling, cell spreading,and receptor localization by immunofluorescence, NIH 3T3 fibroblastswere grown to 50% confluence, fed with fresh DMEM containing10% fetal calf serum, and transfected with Fugene 6 (Roche)according to the manufacturer's instructions. The ratio of Fugene6 to DNA was maintained at 3 µl of Fugene 6:1 µgof DNA.

For the ¹²⁵I-transferrin recycling assays, transfections werecarried out by using the Amaxa Nucleofector system. Briefly,cells were grown to 80% confluence, removed by trypsinization,washed in phosphate-buffered saline (PBS), and resuspended inAmaxa solution R together with 10 µg of DNA for eitherwt-PKB- ${alpha}$ , PKB-AAA, Mu6pro, or Mu6pro-akt1/2. Following electroporation(in Nucleofector; program T-20), the cells were replated ineight-well dishes. All experiments were carried out 24 h posttransfection,and cells were serum starved for 30 min prior to labeling ofcell surface receptors.

Internalization and recycling assays. Receptor internalization and integrin recycling assays wereperformed as described previously (35). Detection of biotinylatedreceptors was by capture enzyme-linked immunosorbent assay (ELISA)as previously described (35) with microtiter wells coated with5 µg of monoclonal anti-human ß3 (Pharmingen;clone VI-PL2) and ${alpha}$ 5 (Pharmingen; clone VC5) integrins or anti-humantransferrin receptor (CD71) (Pharmingen; clone M-A712)/ml. ¹²⁵I-transferrinrecycling assays were performed essentially as described previously(47) with some modifications. Briefly, serum-starved cells wereincubated with ¹²⁵I-labeled transferrin (NEN; NEX212; 0.1 µCi/well)for 1 h at 4°C in PBS with 1% (wt/vol) bovine serum albumin(BSA). The tracer was allowed to internalize for 15 min at 22°C(to label early endosomes) or 30 min at 37°C (to label therecycling compartment). Tracer remaining at the cell surfacewas removed by incubation with acid-PBS (corrected to pH 4.0by the addition of HCl) at 4°C for 6 min, and the tracerwas allowed to recycle at 37°C in serum-free DMEM supplementedwith 1% BSA and 50 µM desferoxamine (Sigma; D9533). Thequantity of ¹²⁵I recycled into the medium is expressed as apercentage of the number of counts incorporated during the internalizationperiod.

For internalization assays 100 nM wortmannin (Sigma; W1628)and 60 µM LY294002 (Calbiochem; 440202) were added duringthe last 10 min of the serum starvation period and maintainedthroughout the internalization period. For integrin recyclingassays, these reagents were added for the last 10 min of theinternalization period and maintained throughout the recyclingperiod. For experiments involving the inhibition of GSK-3, 20mM LiCl (Sigma; L-8895), 10 µM SB216763 (Affiniti; EI-312),and 30 µM SB415286 (Affiniti; EI-311) were included onlyduring the recycling period.

Immunofluorescence. For tracking the endocytic transport of receptors, NIH 3T3 fibroblastswere plated onto glass coverslips and grown to 50% confluenceand then transfected with human ${alpha}$ vß3 integrin or humantransferrin receptor in combination with either GFP-EEA1 orwt-Rab11 and either wt-PKB- ${alpha}$ or dominant negative PKB-AAA byusing Fugene 6. Under the transfection conditions employed thePKB constructs were found to be expressed in all integrin andtransferrin receptor-expressing cells. Surface receptors weretagged by incubation with 2 µg of a mouse anti-ß3monoclonal antibody (Pharmingen; clone VI-PL2; dialyzed againstPBS to remove sodium azide)/ml or 60 µg of Texas Red-conjugatedtransferrin (Molecular Probes; T-2875)/ml for 30 min at 4°Cin PBS containing 0.1% (wt/vol) BSA. The tracer was allowedto internalize for 15 min at 22°C or for 30 min at 37°C,and the cells were rapidly cooled to 4°C. Tracer remainingat the cell surface was removed by a low-pH wash at 4°Cas described above, and the cells were fixed in 4% paraformaldehydeand permeabilized in 0.2% Triton X-100. Internalized anti-ß3was visualized with Texas Red-conjugated anti-mouse antibody(Southern Biotechnology; 1030-07), and the cells were counterstainedwith rabbit anti-Rab11 (Zymed; 71-5300) followed by fluoresceinisothiocyanate (FITC)-labeled anti-rabbit antibody (SouthernBiotechnology, 4050-02) where appropriate.

Cell spreading assays. Cell spreading assays were carried out on 24-well tissue cultureplates which were coated overnight at 4°C with fibronectin(Sigma; F-1141) or vitronectin (Sigma; V-8379) at a concentrationof 20 µg/ml and then blocked with 2% BSA. Cells were allowedto spread for 1 h and, where appropriate, 100 nM wortmannin,60 mM LY294002 (Calbiochem; 440202), 20 mM LiCl, and 30 µMSB415286 (Affiniti; EI-311) were added 5 min after cell attachmentand maintained throughout the spreading period. Attached cellswere fixed for 1 min in 0.2% glutaraldehyde containing 5 mMEGTA, and ß-galactosidase-expressing cells were visualizedby incubation with 5 mM potassium ferricyanide and 1 mg of X-Gal(5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside)overnight at 37°C. To obtain an index of cell spreading,the area of cells expressing ß-galactosidase was determinedby delineation of the cell envelope using the National Institutesof Health Image software.

RESULTS

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Results

Integrin and transferrin receptor endocytosis does not require PI(3)K or PKB/Akt. To determine whether the activity of PI(3)K is necessary forthe endocytosis of ${alpha}$ vß3 and ${alpha}$ 5ß1 integrinsand Tfn-R, receptor internalization was measured following treatmentof NIH 3T3 fibroblasts with 100 nM wortmannin. The assays wereconducted in the presence of 0.6 mM primaquine in order to negatethe effect that membrane recycling has on measurements of endocytosis.All three receptors were internalized rapidly following transferof the cells to 37°C, and the rate and extent of endocytosiswere unaffected by wortmannin (Fig. 1A to C). PKB/Akt is activateddownstream of wortmannin-sensitive PI(3)Ks, so, to oppose theactivity of this, we used a mutant PKB/Akt (PKB-AAA) in whichthe activator phosphorylation sites Thr³⁰⁸ and Ser⁴⁷³ as wellas the Lys¹⁷⁹ in the kinase domain have been replaced by alanineresidues (50). As indicated in Fig. 1D, expression of PKB-AAAopposed both the basal and growth factor-induced phosphorylationof the well-characterized PKB/Akt substrate GSK-3ß.Given the insensitivity of internalization to wortmannin treatment,it seemed unlikely that integrin internalization would be sensitiveto the expression of PKB-AAA. Indeed, the dynamics of integrinand Tfn-R internalization were the same irrespective of overexpressionof wild-type or dominant negative PKB constructs (Fig. 1E to G).These data indicate that integrin and Tfn-R endocytosisdoes not require the activity of PI(3)K or PKB/Akt.

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FIG. 1. Receptor internalization does not require PKB/Akt. NIH 3T3 fibroblasts were transfected with human ${alpha}$ vß3 (h ${alpha}$ vß3) (A and E), h ${alpha}$ 5ß1 (B and F), or hTfn-R (C and G) together with wt-PKB or PKB-AAA. Cells were serum starved for 30 min at 37°C with the inclusion of 100 nM wortmannin (WMN) or dimethyl sulfoxide (DMSO) for the last 10 min of the starvation period and then surface labeled with 0.2 mg of N-hydroxysuccinimide-S-S-biotin/ml at 4°C for 30 min. Internalization was determined by warming the cells to 37°C for the times indicated in the presence of 0.6 mM primaquine. Biotin was released from proteins remaining at the cell surface by incubation with ß-mercaptoethanesulfonic acid (MESNa), and biotinylated proteins were determined by capture ELISA with microtiter wells coated with monoclonal antibodies against hß3 (A and E) and h ${alpha}$ 5 (B and F) integrins or human CD71 (C and G). Values are means ± standard errors of the means (n > 4). For analysis of cellular phospho-GSK-3 levels, cells were transfected with wt-PKB or PKB-AAA by using the Amaxa Nucleofector. Cells were then serum starved and incubated in the presence and absence of 10 ng of PDGF-BB/ml for 10 min. Cell lysates were then probed for the presence of GSK-3ß phosphorylated at Ser⁹ and total GSK-3ß by Western blotting (D).

Trafficking to early endosomes and the recycling compartment does not require PKB/Akt. Following internalization, recycling receptors are known topass through early endosomes and then to accumulate in the Rab11-positiverecycling compartment prior to their return to the plasma membrane.To determine whether this was influenced by the activity ofPKB/Akt, cells were pulse-labeled with an anti-ß3monoclonal antibody or Tfn and incubated at 22°C for 15min or at 37°C for 30 min. Tracer remaining on the cellsurface was removed by a low-pH wash at 4°C, and internalizedtracer was visualized with respect to GFP-EEA1 (a marker forearly endosomes) or Rab11 (a marker for the perinuclear recyclingcompartment). Following the shorter internalization period,both ${alpha}$ vß3 and Tfn-R became closely colocalized withEEA1 in early endosomes (Fig. 2A to D). Longer internalizationtimes resulted in these receptors being transported out of earlyendosomes, and, after 30 min at 37°C, both ${alpha}$ vß3and Tfn-R accumulated in the perinuclear recycling compartmentand colocalized closely with Rab11 (Fig. 2E to H). Furthermore,both the transport of integrin and Tfn-R to the early endosomesand their subsequent accumulation in the recycling compartmentwere identical irrespective of the expression of wt-PKB- ${alpha}$ ordominant negative PKB-AAA (Fig. 2).

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FIG. 2. Transport of ${alpha}$ vß3 and Tfn-R to early endosomes and the recycling compartment does not require PKB/Akt. NIH 3T3 fibroblasts were transfected with GFP-EEA1 in combination with human ${alpha}$ vß3 (h ${alpha}$ vß3; A and B) or hTfn-R (C and D) together with wt-PKB (A and C) or PKB-AAA (B and D). Alternatively, cells were transfected with Rab11 in combination with h ${alpha}$ vß3 (E and F) or hTfn-R (G and H) together with wt-PKB (E and G) or PKB-AAA (F and H). Surface receptors were tagged by incubation with a mouse anti-ß3 monoclonal antibody (A, B, E, and F) or Texas Red-conjugated Tfn (C, D, G, and H), and the tracer was allowed to internalize for 15 min at 22°C (A to D) or 30 min at 37°C (E to H). Tracer remaining at the cell surface was removed by a low-pH wash at 4°C, and the cells were fixed and permeabilized with detergent. Internalized anti-ß3 was visualized with Texas Red-conjugated anti-mouse antibody (A, B, E, and F), and the cells were counterstained with rabbit anti-Rab11, followed by detection with a FITC-conjugated anti-rabbit antibody (E to H). GFP-EEA1 and FITC fluorescence is shown in green, Texas Red fluorescence is shown in red, and yellow indicates colocalization of the two fluorophores. The boxed areas are shown enlarged by a factor of 3.9 in panels B', D', F', and H'. Scale bar, 16 µm.

Integrin, but not Tfn-R, recycling requires PI(3)K and PKB/Akt. Having established that both integrin internalization and itstransport through early endosomes to the recycling compartmentwere independent of PKB/Akt, we proceeded to determine whetherthe recycling of integrins from these compartments to the plasmamembrane was regulated by PI(3)K and PKB/Akt. Initially we concentratedon the platelet-derived growth factor (PDGF)-regulated ${alpha}$ vß3recycling that we have previously shown to be dependent on Rab4(35). Cells were surface labeled, internalization was allowedto proceed for 15 min at 22°C to allow integrin to accumulatein early endosomes, and recycling was determined in the presenceand absence of PDGF as described previously (35). Wortmannintreatment and expression of PKB-AAA both suppressed PDGF-stimulated ${alpha}$ vß3 recycling by ${approx}$ 50% (Fig. 3A), indicating that growthfactor-stimulated integrin recycling was dependent, at leastin part, on the PI(3)K/PKB signaling axis.

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FIG. 3. The requirement for PI(3)K and PKB/Akt in integrin recycling. NIH 3T3 fibroblasts were transfected with human ${alpha}$ vß3 (h ${alpha}$ vß3; A and B) or h ${alpha}$ 5ß1 (C) in combination with wt-PKB, PKB-AAA, Mu6pro, Mu6pro-akt1/2, N121IRab4 (DN-Rab4), or N124IRab11 (DN-Rab11). Serum-starved cells were surface labeled with 0.2 mg of N-hydroxysuccinimide-S-S-biotin/ml for 30 min at 4°C, and internalization was allowed to proceed for 15 min at 22°C (A) or 30 min at 37°C (B and C). Where appropriate 100 nM wortmannin (WMN) or 60 µM LY294002 was included in the last 10 min of the internalization period. Biotin remaining at the cell surface was removed by exposure to ß-mercaptoethanesulfonic acid (MESNa) at 4°C, and internalized integrin was chased back to the cell surface at 37°C for 10 min in the absence (open bars; control) and presence (solid bars) of 10 ng of PDGF-BB/ml for 10 min (A) or for 30 min at 37°C in the absence of PDGF (B and C). Wortmannin and LY294002 were included in the recycling medium where indicated. Cells were then reexposed to MESNa, and biotinylated integrin was determined by capture ELISA using microtiter wells coated with anti-human ß3 (A and B) or anti-human ${alpha}$ 5 (C) integrin monoclonal antibodies. The proportion of integrin recycled to the plasma membrane is expressed as a percentage of the pool of integrin labeled during the internalization period. Values are means ± standard errors of the means (n > 10). To test the ability of the Akt1/Akt2 hairpin RNA to suppress cellular PKB/Akt levels, cells were transfected with Mu6pro or Mu6pro-akt1/2 by using the Amaxa Nucleofector. Cells were lysed for 24 and 48 h following transfection, and the lysates were probed for the presence of PKB/Akt and vinculin (as a loading control) by Western blotting (D).

Recycling of ${alpha}$ vß3 and ${alpha}$ 5ß1 integrins occursfrom the perinuclear recycling compartment in the absence ofgrowth factor addition. We wished, therefore, to determine thedependence of this basal recycling pathway on PKB/Akt. Cellswere surface labeled, and internalization was allowed to proceedfor 30 min at 37°C to accumulate integrin in the perinuclearrecycling compartment. Recycling was then determined in theabsence of growth factor. Dominant negative PKB-AAA profoundlyinhibited the recycling of ${alpha}$ vß3 (Fig. 3B) and ${alpha}$ 5ß1(Fig. 3C) from the recycling compartment, as did wortmanninand the structurally unrelated inhibitor of PI(3)K, LY294002.

Overexpression of inactive forms of kinases can result in "sideways"inhibition of other pathways under the control of the same upstreamkinases (25). For instance, overexpression of PKB-AAA may preventphosphorylation of PKC- ${zeta}$ by 3-phosphoinositide-dependent proteinkinase 1 (2). RNA interference (RNAi) has previously been employedto reduce the cellular levels of PKB/Akt (11, 52), so we usedthis approach to corroborate results obtained with PKB-AAA.We constructed a U6 hairpin RNAi expression vector directedagainst a nucleotide sequence that is found in both the murineAkt1 and Akt2 genes (and that is also identical to the correspondinghuman sequences for these kinase genes). To test the efficacyof this vector in suppressing cellular levels of the PKB/Aktprotein, we performed Western blot analysis on lysates fromcells transfected with Mu6pro-akt1/2 and compared these lysateswith lysates from cells transfected with a control vector (Mu6pro).Expression of Mu6pro-akt1/2 reduced cellular levels of PKB/Aktby approximately fourfold, and this suppression was evident48 h following transfection (Fig. 3D). We therefore determinedthe effect of Mu6pro-akt1/2 on integrin recycling. Indeed, RNAiof PKB/Akt suppressed recycling of both ${alpha}$ vß3 (Fig.3B) and ${alpha}$ 5ß1 (Fig. 3C) integrins, indicating that endogenousPKB/Akt is required for delivery of these integrins from therecycling compartment to the plasma membrane.

For comparison we also investigated the requirement for PI(3)Kand PKB/Akt for the recycling of the Tfn-R. NIH 3T3 fibroblastswere transfected with the Amaxa Nucleofector system, which,in our hands, results in the expression of PKB-AAA in >95%of the cells (data not shown). Transfected cells were allowedto internalize ¹²⁵I-labeled human transferrin for either 15min at 22°C or 30 min at 37°C, and the return of thelabeled transferrin into the medium was measured during a subsequentchase period at 37°C. Consistent with a previous report(34), Tfn-R recycling was partially inhibited by expressionof dominant negative Rab11 (Fig. 4C). However, recycling of¹²⁵I-Tfn from either early endosomes (Fig. 4A) or the recyclingcompartment (Fig. 4B and C) was completely unaffected by inhibitionof PI(3)K or expression of PKB-AAA. These data indicate thatgrowth factor-regulated "short-loop" recycling and basal "long-loop"recycling of integrins are regulated by the PI(3)K/PKB signalingaxis, whereas Tfn-R recycling is independent of the activityof PKB/Akt.

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FIG. 4. ¹²⁵I-Tfn recycles independently of PI(3)K and PKB/Akt. NIH 3T3 fibroblasts were transfected with wt-PKB, PKB-AAA, or S24NRab11 (DN-Rab11) by using the Amaxa Nucleofector. Cells were then serum starved and incubated with ¹²⁵I-labeled transferrin for 1 h at 4°C. The tracer was allowed to internalize for 15 min at 22°C (A) or 30 min at 37°C (B and C), with 100 nM wortmannin (WMN) or 60 µM LY294002 being included in the incubation for the last 10 min of the internalization time. Tracer remaining at the cell surface was removed, and the internalized ¹²⁵I-transferrin was allowed to recycle in the absence and presence of wortmannin or LY294002 for the times indicated. The quantity of ¹²⁵I recycled into the medium is expressed as a percentage of the number of counts incorporated during the internalization period. Values are means ± standard errors of the means (n = 8).

Active PKB/Akt drives ${alpha}$ vß3 integrin recycling. We wished to determine the effect of constitutively active memb-PKBon ${alpha}$ vß3 integrin recycling. Initially, attempts tomeasure this directly indicated that it was not possible toload ${alpha}$ vß3 integrin into early endosomes upon expressionof memb-PKB. Indeed following expression of memb-PKB, ${alpha}$ vß3appeared to remain on the cell surface (Fig. 5A). This couldbe due to either the ability of memb-PKB to stimulate recyclingfrom the early endosome or conversely may represent a blockadeof ${alpha}$ vß3 endocytosis. To distinguish these possibilities,we determined the internalization of ${alpha}$ vß3 in memb-PKB-transfectedcells in the presence and absence of primaquine. Upon additionof primaquine, ${alpha}$ vß3 accumulated rapidly within thecell, indicating that memb-PKB does not oppose internalizationbut acts to drive the recycling of integrin such that its endosomalpool is too small to be detected easily at steady state (Fig.5B).

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FIG. 5. Active PKB/Akt drives ${alpha}$ vß3 integrin recycling. (A and B) NIH 3T3 fibroblasts were transfected with human ${alpha}$ vß3 (h ${alpha}$ vß3) integrin in combination with constitutively active PKB (memb-PKB) or PKB-AAA. Cells were serum starved and surface labeled with 0.2 mg of N-hydroxysuccinimide (NHS)-S-S-biotin/ml for 30 min at 4°C. Internalization was initiated by warming to 37°C for the times shown in the absence (open symbols) and presence (closed symbols) of 0.6 mM primaquine. Biotin was released from proteins remaining at the cell surface, and biotinylated integrin was determined by capture ELISA using microtiter wells coated with an anti-human ß3 integrin monoclonal antibody. (C) Cells were transfected with h ${alpha}$ vß3 in combination with the conditionally active fusion memb-PKB and the hormone-binding domain of the ER (memb-PKB-ER). Serum-starved cells were surface labeled with 0.2 mg of NHS-S-S-biotin/ml for 30 min at 4°C, and internalization was allowed to proceed for 15 min at 22°C. Biotin remaining at the cell surface was removed by exposure to ß-mercaptoethanesulfonic acid (MESNa) at 4°C, and internalized integrin was chased back to the cell surface at 37°C for 15 min in the absence (open bars; control) and presence (solid bars) of 300 nM 4-HT. Cells were then reexposed to MESNa, and biotinylated integrin was determined by capture ELISA and expressed as for Fig. 3. Values are means ± standard errors of the means (n = 12).

To overcome the difficulty of labeling an endosomal pool ofintegrin following expression of memb-PKB, we explored the possibilityof using a membrane-targeted, conditionally active form of PKB/Akt,memb-PKB-ER. This fusion protein, in which a membrane-targetedPKB kinase domain is fused to the hormone binding domain ofthe ER, is inactive in the absence of hormone ligand. Howevermemb-PKB-ER becomes rapidly activated following the additionof 4-hydroxytamoxifen (4-HT) and elicits cellular responsesknown to be a function of the endogenous kinase (23, 27). Followingexpression of memb-PKB-ER, we were able to monitor the internalizationof ${alpha}$ vß3 into early endosomes (not shown) and foundthat the integrin did not recycle appreciably from this compartmentduring the following 15-min chase period (Fig. 5C). Howeveractivation of memb-PKB-ER at the start of the chase period byaddition of 300 nM 4-HT enhanced recycling of ${alpha}$ vß3integrin by approximately threefold (Fig. 5C). Taken togetherthese data show that, following expression of active memb-PKB, ${alpha}$ vß3 is returned to the plasma membrane from the earlyendosome without the need for growth factor stimulation, indicatingthat activation of PKB is necessary and sufficient to drivethe recycling of ${alpha}$ vß3 integrin.

PKB/Akt is necessary for cell spreading. We have previously shown that inhibition of ${alpha}$ vß3 recyclingby expression of dominant negative Rab4 results in reductionof cell spreading on vitronectin (a good ligand for ${alpha}$ vß3),whereas dominant negative Rab4 did not affect the recyclingof ${alpha}$ 5ß1 or the spreading of cells onto fibronectin(a good ligand for ${alpha}$ 5ß1) (35). We hypothesized that,as expression of PKB-AAA and RNAi of endogenous PKB/Akt affectthe recycling of both ${alpha}$ vß3 and ${alpha}$ 5ß1, PKB activitywould be necessary for cells to spread on vitronectin and fibronectin.Cells were transfected with wild-type, dominant negative, andconstitutively active PKBs or the RNAi vector for PKB/Akt (Mu6pro-akt1/2),and allowed to adhere to coverslips coated with either vitronectinor fibronectin. Cells transfected with wt-PKB, memb-PKB, andthe control Mu6pro vector spread normally on both matrices (Fig.6). However, inhibition of PKB/Akt with PKB-AAA, treatment withwortmannin, or suppression of cellular PKB/Akt with Mu6pro-akt1/2resulted in inefficient cell spreading on both matrix proteins(Fig. 6). The spread area of the cells was reduced by approximately50% following inhibition of PKB/Akt signaling (Fig. 6C and F),and the cells spread abnormally without extending a properlyorganized lamella (Fig. 6A and D). These results are consistentwith the idea that integrin recycling is indeed necessary forcell spreading on the ECM.

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FIG. 6. PKB/Akt is necessary for cell spreading on vitronectin and fibronectin. NIH 3T3 fibroblasts were transfected with wt-PKB, memb-PKB, PKB-AAA, Mu6pro, or Mu6pro-akt1/2 in combination with a ß-galactosidase transfection marker. The cells were then briefly trypsinized and allowed to adhere to either vitronectin (A to C) or fibronectin (D to F) in the presence of 10 ng of PDGF-BB/ml and in the presence and absence of wortmannin (WMN; 100 nM). Attached cells were fixed and stained for ß-galactosidase expression and then photographed with a digital camera. In panels C and F, the area of the transfected cells was then determined by delineation of the cell envelope using the National Institutes of Health Image software. Values are means ± standard errors of the means (n > 200 cells). CON, control. Cells expressing HA-tagged PKBs were visualized with a FITC-conjugated anti-HA antibody (green) and counterstained with Texas Red-conjugated phalloidin (red) (A and D). Scale bars, 16 (D) and 80 µm (E).

Active GSK-3ß suppresses integrin recycling and cell spreading. Inactivation of the serine/threonine kinase GSK-3 is now knownto be required for polarized-cell migration (12), proliferation(32), and the suppression of apoptosis (10), all processes inwhich both ${alpha}$ vß3 and ${alpha}$ 5ß1 integrins play akey role. It is well established that the PI(3)K signaling axisinactivates GSK-3 via PKB/Akt-dependent phosphorylation of Ser⁹in GSK3-ß and Ser²¹ in GSK3- ${alpha}$ (6). Therefore, it ispossible that the reduction in integrin recycling and cell spreadingthat we observe following addition of wortmannin or LY294002or expression of PKB-AAA is due to increased cellular levelsof desphosphorylated, active GSK-3. If so, pharmacological blockadeof GSK3 would relieve the inhibition of integrin recycling effectedby suppression of PKB/Akt signaling. To test this, we investigatedthe effect of LiCl and two other well-characterized inhibitorsof GSK-3 (SB415286 and SB216763 [10]) in the recycling assay.Recycling of ${alpha}$ vß3 from early endosomes was significantlyincreased by addition of 20 mM LiCl, indicating that inactivationof GSK-3 is indeed sufficient in itself to drive recycling fromthis compartment (Fig. 7A). Furthermore, addition of LiCl wasable partially to reverse the blockade of ${alpha}$ vß3 recyclingeffected by wortmannin, and LiCl, SB415286, and SB216763 restoredintegrin recycling from the Rab11 compartment in cells expressingPKB-AAA (Fig. 7B). We therefore tested the ability of GSK-3inhibitors to restore cell spreading following suppression ofPI(3)K and PKB/Akt. Interestingly, LiCl, SB415286, and SB216763completely restored spreading in cells expressing PKB-AAA, and,even following treatment with wortmannin, the addition of LiClpartially rescued cell spreading (Fig. 7C).

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FIG. 7. Integrin recycling and cell spreading are suppressed by active GSK-3ß. (A and B) NIH 3T3 fibroblasts were transfected with human ${alpha}$ vß3 (h ${alpha}$ vß3) in combination with wt-PKB, PKB-AAA, Mu6pro, Mu6pro-akt1/2, GSK-3ß-F216, or GSK-3ß-A9. Serum-starved cells were surface labeled with 0.2 mg of N-hydroxysuccinimide-S-S-biotin/ml for 30 min at 4°C, and internalization was allowed to proceed for 15 min at 22°C (A) or for 30 min at 37°C (B). Recycling was determined as for Fig. 3; recycling conditions were 10 min at 37°C in the absence (open bars; control) and presence (solid bars) of PDGF-BB (10 ng/ml) (A) or for 30 min at 37°C in the absence of PDGF (B). One hundred nanomolar wortmannin (WMN), 20 mM LiCl, 10 µM SB216763, and 30 µM SB415286 were included during the recycling period where indicated. Values are means ± standard errors of the means (SEM; n > 10). CON, control. (C and D) NIH 3T3 fibroblasts were transfected with wt-PKB, PKB-AAA, Mu6pro, Mu6pro-akt1/2, GSK-3ß-F216, or GSK-3ß-A9 in combination with a ß-galactosidase transfection marker, and cell spreading on vitronectin was determined as for Fig. 6. Cells were spread in the presence of 100 nM wortmannin, 20 mM LiCl, and 30 µM SB415286 as indicated. Values are means ± SEM (n > 80 cells).

To extend these studies on the involvement of GSK-3 in the PKB/Aktregulation of integrin recycling, we employed two mutant versionsof GSK-3ß: GSK-3ß-F216, which is less activedue to lack of a key activation loop phosphorylation site, andGSK-3ß-A9, which cannot be phosphorylated and inactivatedby PKB/Akt. Following expression of GSK-3ß-F216 theintegrin recycling and cell spreading responses were normal,consistent with the notion that a relatively high level of inactivephospho-GSK-3 is present in cells to maintain integrin recycling.Conversely, expression of GSK-3ß-A9, which would beexpected to be active even in cells that express high levelsof activated PKB/Akt, inhibited integrin recycling and cellspreading, and, furthermore, these responses were restored bypharmacological inhibition of GSK-3 (Fig. 7). These data indicatethat PKB/Akt activates integrin recycling and cell spreadingby suppressing the activity of GSK-3.

DISCUSSION

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Discussion

Here we show that PI(3)K and its downstream target PKB/Akt areregulators of integrin recycling events. Wortmannin treatmentand blockade of PKB/Akt using dominant negative proteins andRNAi did not affect the internalization of integrins or theTfn-R, nor was the activity of PKB/Akt required for the subsequenttransport of these receptors to early endosomes or the perinuclearrecycling compartment. We found no requirement for PKB/Akt inTfn-R recycling but show that both ${alpha}$ vß3 and ${alpha}$ 5ß1integrins require active PKB/Akt to return to the plasma membrane.Experiments with mutant GSK-3ßs indicate that PKB/Aktis likely to promote integrin recycling by inactivating GSK-3,and we present the finding that, if integrin recycling and cellspreading are suppressed by expression of dominant negativePKB-AAA or RNAi of PKB/Akt, this inhibition can be relievedby inhibition of GSK-3.

PKB/Akt and membrane recycling pathways. A number of workers have reported that internalization of manyreceptors proceeds independently of PI(3)K or PKB/Akt activity(9, 39, 41), and in agreement with this we find that the internalizationof both integrin and Tfn-R is unaffected by wortmannin treatmentor blockade of PKB/Akt. In addition, our data indicate thatPKB/Akt has no role to play in the transport of ${alpha}$ 5ß1, ${alpha}$ vß3, and Tfn-R to early endosomes and their subsequentdelivery to the Rab11-positive perinuclear recycling compartment.This indicates that the inhibitory effects of PKB-AAA on integrinrecycling are not due to reduced "inward" transport of receptorsor to overt disturbance of the structure and function of theearly and recycling endosomes.

So how does PKB/Akt regulate integrin recycling? There is evidenceto suggest that PI(3)K and PKB/Akt are involved in activatingRab proteins. The possibility that Rabs may be activated downstreamof PI(3)K was first suggested by the observation that insulincan stimulate GTP exchange onto Rab4 in a fashion that was opposedby wortmannin (40). Additionally, other Rabs have been shownto be activated downstream of the PI(3)K/PKB/Akt axis. For instance,in Dictyostelium, following the sequential activation of PI(3)Kand PKB/Akt, Rab7 is recruited to macropinosomes (36), and Rab5-mediatedinternalization events also require PKB/Akt activity (3). However,it is well established that Tfn-R recycling is a Rab4/Rab11-dependentevent, and our data show that there is no requirement for PKB/Aktor for a wortmannin- or LY294002-sensitive PI(3)K in the returnof ¹²⁵I-Tfn to the plasma membrane in NIH 3T3 fibroblasts. Thisindicates that PI(3)K and PKB/Akt do not influence Rab functionper se and are, therefore, more likely to regulate the transportof integrin recycling vesicles themselves, and it will be interestingto determine whether these are distinct from the organellesthat recycle Tfn.

It is apparent from our data that the length of time that anintegrin spends within the cell before returning to the plasmamembrane depends on the cellular activity of PKB/Akt. For instance,if cells are expressing active PKB/Akt or if PDGF is added, ${alpha}$ vß3 integrin returns to the plasma membrane from earlyendosomes. If cells are serum starved for 30 min, the integrinis transported further into the cell (i.e., to the perinuclearrecycling compartment) before being recycled. Furthermore, ifPKB/Akt is strongly suppressed by wortmannin treatment, RNAi,or expression of PKB-AAA, or indeed if cells are serum starvedfor longer times (not shown), ${alpha}$ 5ß1 and ${alpha}$ vß3integrins are not recycled efficiently from either early endosomesor the recycling compartment and accumulate within the cell.These differences are likely to be due to changes in the balancebetween the inward and outward transport of integrins in theendocytic pathway. Interactions of endocytic cargo with microtubulesand microtubular motors are necessary for movement of endocyticvesicles, and the nature of these is likely to define the cargo'sdirection of travel (38). ${alpha}$ 5ß1 has recently reportedto be associated with the kinesin-associated adaptor, kinectin(44), and kinesins mediate the transport of vesicles towardthe plus ends of microtubules at the cell periphery and, therefore,favor recycling. Experiments with GSK-3 inhibitors and mutantGSK-3ßs indicate that PI(3)K and PKB/Akt achieve theireffects on integrin transport via the inactivation of GSK-3,and it is now clear that GSK-3 phosphorylates the kinesin lightchain (30). This phosphorylation inhibits the ability of kinesinto transport vesicular cargo toward the plus ends of microtubules.Inactivation of GSK-3 by PKB/Akt may, therefore, favor the transportof integrin vesicles toward the cell periphery. Furthermore,it is now clear that GSK-3 plays a key role in the regulationof cell migration and the development of cell polarity by inducingthe interaction of adenomatous polyposis coli (APC) proteinwith the plus ends of microtubules (12). A physical link betweenAPC and kinesin has recently been reported (18), and it is possiblethat this is responsible for targeting integrins to particularcellular regions.

It is interesting that PDGF-dependent ${alpha}$ vß3 recyclingwas only partially inhibited by wortmannin treatment or expressionof PKB-AAA (Fig. 3A). Studies under way in our laboratory indicatethat the remaining component of growth factor-induced recyclingis under the control of other PDGF-activated signaling pathways,and this will be the subject of further investigation.

The endocytic pathway and integrin function. PI(3)K has long been known to be involved in inside-out regulationof integrin function (17), and a more recent report highlightsthe role that PKB/Akt may play in this process (5). PI(3)K activitymay activate integrins via the recruitment of PH domain-containingproteins to the membrane, which then influence integrin cytoplasmictails and cause subsequent heterodimer activation, as has beendemonstrated for the activation of ß2 integrins bycytohesin-1 (24). PKB/Akt may do this, although this is unlikely,as memb-PKB and memb-PKB-ER clearly promote ${alpha}$ vß3 recyclingand these are targeted to the membrane not by a PH domain butby N-terminal myristoylation (27). Another possible route fordirect regulation of integrin function by PKB/Akt is via directphosphorylation of Thr⁷⁵³ of the ß3 integrin cytodomain(22). However, in [³²P]orthophosphate labeling experiments wehave been unable to detect phosphorylation of either ${alpha}$ vß3or ${alpha}$ 5ß1 in NIH 3T3 fibroblasts (not shown), even followingexpression of constitutively active PKB/Akt.

Many of the questions surrounding integrin inside-out activationremain unsolved, and it is possible that the endosomal pathwaycontributes to this phenomenon. Indeed, a considerable proportionof active, high-affinity ${alpha}$ vß3 integrin has been observedwithin cytoplasmic vesicles in oligodendrocytes (4) and endothelialcells (21). The luminal pH of endosomes is lower than that ofthe extracellular medium, and it is well established that thisdissociates lysosome-destined ligands from receptors that arerecycled (1, 51). Therefore, if ${alpha}$ vß3 and ${alpha}$ 5ß1were internalized with their ligand-binding pockets occludedwith fragments of ECM proteins, it is likely that integrin andligand would separate in endosomes, and the integrin could thenbe returned to the plasma membrane competent to reengage theECM. A number of signaling pathways result in increased endosomeacidification (53), and recently a regulatory subunit of theV-ATPase E has been shown to bind to Sos1 (28). This associationmay form part of the mechanism whereby endosome acidificationcould be driven by receptor tyrosine kinase signaling. Miuraet al. (28) have reported that overexpression of V-ATPase inCOS cells enhanced the exchange of GTP onto Rac1. Given thatwe have found that the endosomal pathway modulates integrinfunction and that engagement of integrins is known to activateRac1 (33), it would be interesting to determine whether integrinrecycling mediates the influence of endosomal pH on Rac1 function.

Cell survival is correlated with the degree of spreading onfibronectin and vitronectin, and it is now well establishedthat integrins can prevent anoikis by enhancing binding to theECM (26). GSK-3 is also emerging as a key cell survival factor,and inhibitors of GSK-3 are known to protect against apoptosis(10). Our results are consistent with a mechanism whereby PKB/Akt-mediatedinactivation of GSK-3 promotes cell survival by maintaininga plasma membrane pool of functional antiapoptotic integrins,and in this regard it would be interesting to investigate therequirement for endosomal recycling in the maintenance of cellsurvival.

ACKNOWLEDGMENTS

This work was generously supported by grants from the WellcomeTrust.

We gratefully acknowledge Dave Critchley for critical readingand helpful comments on the manuscript. We thank Kul Sikandand Sam Wattam their invaluable assistance with the fluorescencemicroscopy. We thank Dario Alessi and Martin McMahon for thegenerous gifts of PKB constructs and Karl Matter for the RNAivector Mu6pro.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry, University of Leicester, University Rd., Leicester LE1 7RH, United Kingdom. Phone: 0116-252-5250. Fax: 0116-252-3369. E-mail: jcn2{at}le.ac.uk.

Present address: MRC Laboratory for Molecular Cell Biology,Cell Biology Unit, University College London, WC1E 6BT London,United Kingdom.

REFERENCES

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