Integrin {alpha}2{beta}1 Promotes Activation of Protein Phosphatase 2A and Dephosphorylation of Akt and Glycogen Synthase Kinase 3{beta}

Serine/threonine kinase Akt is a downstream effector proteinof phosphatidylinositol-3-kinase (PI-3K). Many integrins canfunction as positive modulators of the PI-3K/Akt pathway. Integrin ${alpha}$ 2ß1 is a collagen receptor that has been shown toinduce specific signals distinct from those activated by otherintegrins. Here, we found that, in contrast what was found forcells adherent to fibronectin, ${alpha}$ 2ß1-mediated cell adhesionto collagen leads to dephosphorylation of Akt and glycogen synthasekinase 3ß (GSK3ß) and concomitantly to theinduction of protein serine/threonine phosphatase 2A (PP2A)activity. PP2A activation can be inhibited by mutation in the ${alpha}$ 2 cytoplasmic domain and by a function-blocking anti- ${alpha}$ 2 antibody.Akt can be coprecipitated with PP2A, and coexpression of Aktwith PP2Ac (catalytic subunit) inhibits Akt kinase activity.Integrin ${alpha}$ 2ß1-related activation of PP2A is dependenton Cdc42. These results indicate that cell adhesion to collagenmodulates Akt activity via the ${alpha}$ 2ß1-induced activationof PP2A.

INTRODUCTION

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Introduction

The integrins are a large family of heterodimeric transmembranereceptors composed of ${alpha}$ and ß subunits (22). In additionto mediating cell-matrix interactions, integrins have been shownto activate intracellular signaling pathways which, in collaborationwith growth factor-induced signals, regulate cellular functions(46). Some integrin signaling cascades are activated via theß subunit cytoplasmic domain, and they are thereforetriggered by several integrin heterodimers. These signals includethe activation of protein tyrosine kinases of the Src and focaladhesion kinase (FAK) families (9, 47). More-recent studieshave revealed signaling events that are activated specificallyby an ${alpha}$ subunit (19). Integrins may associate with other membraneproteins, such as caveolin-1, and a subset of integrins canactivate extracellular signal-related kinase, one of the mitogen-activatedprotein kinases, via Fyn and Shc (53, 54). Some integrins interactwith other membrane proteins to regulate distinct signalingcascades. For example laminin receptor ${alpha}$ 3ß1 associateswith tetraspanin proteins and activates phosphatidylinositol-3-kinase(PI-3K) and PI-4K (4). We have shown that ${alpha}$ 2ß1 integrinspecifically activates the p38 pathway via a mechanism involvingthe ${alpha}$ 2 cytoplasmic tail and Cdc42 (25). The p38 signaling pathwayseems to regulate the expression of type I collagen and collagenase-3(25, 42), and it is required for cell migration on collagen(29).

The PI-3K/Akt pathway is activated by a wide range of extracellularstimuli, including the integrins (12), and it has been linkedto cell survival (13). Recently it was demonstrated that thedifferent variants of the cytoplasmic domain in the ß1subunit can equally activate Akt (14, 16) and that the bindingof ${alpha}$ 5ß1 to fibronectin activates Akt, unlike the bindingof ${alpha}$ 2ß1 to monomeric collagen (15). Thus the activationof Akt may be dependent on the integrin ${alpha}$ subunit.

Reversible phosphorylation of proteins is a major mechanismfor the control of cellular signaling pathways and maintenanceof homeostasis (21). Although numerous kinases have been implicatedin integrin signaling, the function and possible regulationof the corresponding phosphatases are largely unknown. Adhesionof cultured fibroblasts to extracellular matrix proteins hasbeen shown to induce recruitment and activation of SHP-2, anontransmembrane protein tyrosine phosphatase (39, 51). SHP-2seems to play an active role in integrin-mediated signalingevents, such as cell adhesion and migration (36, 62). Very littleis known about the role of protein serine/threonine phosphatasesin integrin signaling. Recent data have indicated a positiverole for protein serine/threonine phosphatase 2A (PP2A) in integrininside-out signaling. Inhibition of PP2A activity induces aselective loss of ß1 integrins from focal adhesionsites (38) and inhibits cell adhesion (11); in addition PP2Ahas been shown to colocalize with ß1 integrin at adhesionsites (38). However, the role of serine/threonine phosphatasesin modulating integrin outside-in signals remains to be studied.Many studies have demonstrated the importance of PP2A in regulatinga variety of cellular functions (52). Therefore it is likelythat PP2A activity is tightly controlled in vivo.

Cell adhesion to three-dimensional (3D) fibrillar collagen,unlike adhesion to monomeric two-dimensional collagen, inhibitscell proliferation in different cell types (15, 20, 30) andinduces specific integrin-mediated signals, which regulate geneexpression (25, 42, 44). Here, a novel ${alpha}$ 2ß1-mediatedsignaling mechanism is introduced. Using human primary fibroblastsand human osteosarcoma (Saos-2) cell clones expressing eitherthe wild-type ${alpha}$ 2 subunit or a signaling-deficient ${alpha}$ 2/ ${alpha}$ 1 chimera,we have analyzed the ability of ${alpha}$ 2ß1 integrin to regulatesignals that have been linked with cell proliferation and survival.We and others have shown that ${alpha}$ 2ß1 integrin is notinvolved in the regulation of the extracellular signal-relatedkinase mitogen-activated protein kinase pathway in responseto collagen (25, 42, 53). However, here we show that cell adhesionto 3D collagen attenuates Akt and glycogen synthase kinase 3ß(GSK3ß) phosphorylation by a mechanism involving ${alpha}$ 2ß1-inducedactivation of PP2A.

MATERIALS AND METHODS

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Materials and Methods

Plasmids, adenoviruses, and antibodies. The ${alpha}$ 2 integrin and the chimerical ${alpha}$ 2/ ${alpha}$ 1 integrin expression constructshave been described previously (25, 43). In ${alpha}$ 2/ ${alpha}$ 1 the intracellulardomain of ${alpha}$ 2 was replaced with one from ${alpha}$ 1 integrin. Cdc42^Asn17,Cdc42QL, Rac1^Asn17, and RhoA^Asn19 were provided by J. C. Lacal(Consejo Superior de Investigaciones Cientificas, Madrid, Spain).pCMVHA-Akt and pCMVHA (hemagglutinin [HA] vector) were providedby K. Vuori (Burnham Institute), the HA-PP2Ac expression constructwas provided by D. Brautigan (University of Virginia) (8), andpCMV was from Invitrogen. The adenovirus vector for the expressionof membrane-targeted, active Akt (myrAkt) was obtained fromK. Walsh (Tufts University School of Medicine) and was constructedas described previously (17, 18). The antibodies against Akt,the HA probe, and Cdc42 were purchased from Santa Cruz Biotechnology.Anti-PP2A was a gift from D. Brautigan. The phospho-specificantibodies were from New England Biolabs. The anti- ${alpha}$ 2 (MCA743)and anti- ${alpha}$ 1 (MCA1133) integrin antibodies were from Serotec.

Collagen gels. Collagen gels were prepared from bovine dermal collagen, whichcontains 95% type I collagen and 5% type III collagen (Cellon).Eight volumes of Cellon were mixed with 1 volume of 10-fold-concentratedDulbecco's modified Eagle medium (DMEM) and 1 volume of 0.05M NaOH in HEPES buffer (0.2 M) and kept on ice. Cells were trypsinized,resuspended in 1/10 gel volume of DMEM supplemented with inhibitorsor anti-integrin antibodies, mixed into neutralized Cellon solution,and transferred into cell culture plates. The collagen was allowedto polymerize for 1 h at 37°C, after which 1 gel volumeof DMEM was added, the gels were detached from the sides ofthe cell culture plates, and incubation was continued for varioustimes. Cells from various Saos-2 cell clones were cultured onplastic and harvested directly from the plate or kept in suspension(1 ml of DMEM) for 1 h. The release of cells from collagen gelwas performed as described previously (25).

Immunoblot analysis. Cells were released from collagen, washed once with ice-coldphosphate-buffered saline, and lysed in Laemmli sample buffer.The samples were sonicated, fractionated by sodium dodecyl sulfate-10or 12% polyacrylamide gel electrophoresis, and transferred toa Hybond ECL membrane (Amersham Corp.). Western blotting wasperformed as described previously (25) with New England Biolabsantibodies at the dilution of 1:1,000 and other antibodies atthe dilution of 1:250. Specific binding of antibodies was detectedwith peroxidase-conjugated secondary antibodies and visualizedby an enhanced chemiluminescence detection system (Amersham).

Transfections and kinase assay. For the Akt kinase assay, subconfluent cells plated on 10-cm-diameterdishes were transfected using 18 µl of Fugene 6 transfectionreagent (Boehringer Mannheim) and 6 µg of either emptyvector, HA vector, or PP2Ac expression vector together with3 µg of pCMVHA-Akt. At 36 h after transfections, the cellswere treated with collagen gel for 1.5 h, and a kinase assaywas performed as described previously (18). Briefly, equal amountsof protein in cell lysis buffer (1% NP-40; 10% glycerol; 137mM NaCl; 20 mM Tris-HCl [pH 7.4]; 20 mM NaF; 1 mM phenylmethylsulfonylfluoride[PMSF]; leupeptin, antipain, and pepstatin [2 µg/mleach]) from each sample were preincubated with protein G-Sepharosefor 30 min at 4°C. After centrifugation, an anti-HA antibodywas added together with protein G-Sepharose and 2 mg of bovineserum albumin/ml. Immunoprecipitation was performed at 4°Cfor 2 h. The immunoprecipitates were washed twice with celllysis buffer, twice with H₂O, and twice with kinase buffer (20mM HEPES [pH 7.2], 10 mM MgCl₂, 10 mM MnCl₂), and they wereincubated in 50 µl of kinase buffer containing 2 µgof myelin basic protein (Boehringer Mannheim) and [ ${gamma}$ -³²P]ATP(5 µM, 10 µCi; Amersham) at room temperature for30 min. Kinase reactions were terminated by adding sodium dodecylsulfate sample solution. The reactions were run on 15% polyacrylamidegels and subjected to autoradiography. Okadaic acid (1 nM) wasincluded at every stage to inhibit any in vitro phosphataseactivity from influencing the kinase activity after the lysisof the cells. The in vitro kinase assays for PI-3K (3, 58) wereperformed in the presence of [ ${gamma}$ -³²P]ATP and PI (Avanti PolarLipids). The formed [³²P]PI was measured using thin-layer chromatographyand autoradiography.

Phosphatase assay. Subconfluent human osteosarcoma cells and human primary fibroblastswere treated with collagen for various times in the presenceor absence of antibodies (function-blocking anti- ${alpha}$ 2, nonfunctionalanti- ${alpha}$ 1 immunoglobulin G [IgG]; 5 µg/ml) or inhibitors(bisindolylmaleimide [BIM], 10 µM; SB203580, 20 µM;Calbiochem) or were lysed directly on the plate. Alternatively,cells plated on 10-cm-diameter dishes were transfected using20 µl of Fugene 6 transfection reagent (Boehringer Mannheim)and 10 µg of expression vector pCEV either alone or withCdc42^Asn17, Cdc42QL, Rac1^Asn17, or RhoA^Asn19. Thirty-six hoursafter transfections, the cells were placed inside a collagengel for 2 h. Cells were released from collagen as describedpreviously (25) and lysed in phosphatase lysis buffer (20 mMHEPES [pH 7.4]; 10% glycerol; 0.1% NP-40; 1 mM EGTA; 30 mM ß-mercaptoethanol;1 mM PMSF; leupeptin, antipain, and pepstatin [2 µg/mleach]). The protein phosphatase assay system (Life Technologies)was used to determine phosphatase activity according to manufacturer'sinstructions. Briefly, ³²P-labeled phosphorylase (a substratefor both PP1 and PP2) was allowed to react with 2 to 20 µgof protein from cell lysate for 10 min in 30°C, after whichthe proteins were precipitated with 20% trichloroacetic acidand the radioactivity released was measured by scintillationcounting. Protein content was determined by the Bradford method,and phosphatase activity relative to protein content was calculated.

Immunoprecipitation. For immunoprecipitation, the cells were transfected with Fugene6 and 10 µg of the wt-PP2Ac-HA expression construct asdescribed above (provided by D. Brautigan, University of Virginia)(8). Eight hours after transfection the cells were infectedwith myrAkt adenovirus at a multiplicity of infection of 50and incubated for 16 h. Serum-free DMEM was added, and the incubationwas continued for 24 h. The cells were treated with 3D collagenfor 1 h under serum-free conditions, and the cells were lysedin solubilization buffer (10 mM Tris-HCl [pH 7.4], 50 mM NaCl,1% Triton X-100, 30 mM sodium pyrophosphate, 100 µM Na₃VO₄,1 mM PMSF) (1). Immunoprecipitation was performed as describedfor the Akt kinase assay, with either a specific antibody orcontrol IgG from the same species, except that the washes wereperformed six times with solubilization buffer. Western blotdetection was performed with antibodies against Akt and theHA probe and antiserum against the PP2A catalytic subunit.

RESULTS

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Results

Adhesion of ${alpha}$ 2ß1 integrin to collagen promotes dephosphorylation of Akt. A previous study suggested that integrin ${alpha}$ 5ß1-mediatedcell adhesion to fibronectin induces Akt phosphorylation, whileadhesion to monomeric collagen has no effect (32). Since monomerictwo-dimensional and fibrillar three-dimensional (3D) collagenmay have distinct properties in triggering integrin signaling(25, 42), immunoblotting was performed to determine whethercell adhesion to 3D collagen influences the Akt kinase. We examinedthe phosphorylation of Ser473 on Akt with a phospho-specificantibody, since this residue is considered to indicate activekinase (6). Serum-starved human primary fibroblasts were harvestedin the monolayer or trypsinized and replated on fibronectin-coatedplates or seeded inside collagen (for 2 h), and Akt phosphorylationwas studied by immunoblotting. Cells adhering on fibronectinshowed increased Akt phosphorylation (67% ± 11% [mean± standard error of the mean {SEM}]; four separate experiments)compared to cells cultured on plastic, while phosphorylationof Ser473 was reduced by 48% ± 3% (mean ± SEM;four separate experiments) in cells cultured inside 3D fibrillarcollagen (Fig. 1A). We also analyzed the effect of collagen-mediatedreduction of Akt phosphorylation on GSK3ß, a well-characterizedAkt substrate that is inactivated by the active Akt kinase viaphosphorylation at Ser9 (12). In only 1 h, 3D collagen induceda rapid decrease of phosphorylation of Akt, and after 3 h nearlyall Akt was in a nonphosphorylated state (Fig. 1B). Inactivationof Akt resulted in decreased phosphorylation of GSK3ß(Fig. 1B). In seven separate experiments the phosphorylationof Akt and GSK3ß in cells cultured inside collagenwas reduced by 40% ± 16% and 47% ± 17% (means± SEMs), respectively. To evaluate the role of ${alpha}$ 2ß1in collagen-induced Akt inactivation, we treated human fibroblastswith an anti- ${alpha}$ 2 integrin function-blocking monoclonal antibody(MAb). Importantly, this antibody has previously been shownto block ${alpha}$ 2ß1-initiated signals inside collagen (42).The antibody blocked collagen-induced Akt dephosphorylation(Fig. 1C). The results indicate that ${alpha}$ 2ß1 integrinfunction is involved in the observed reduction of Akt phosphorylation.

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FIG. 1. Differential regulation of Akt phosphorylation by extracellular matrix molecules. (A) Fibroblasts were deprived of serum for 16 h and then harvested from the cell culture dish (M, monolayer), seeded inside 3D collagen gel, or plated on fibronectin (FN) for 2 h. P-Akt, phosphorylated Akt. (B) Primary fibroblasts were deprived of serum for 16 h and then harvested from the cell culture dish (M) or seeded inside 3D collagen gel for 1 and 3 h. P-GSK3ß, phosphorylated GSK3ß. (C) Primary fibroblasts were deprived of serum for 16 h and then harvested from the cell culture dish (M) or seeded inside collagen gel (C) or treated with an anti- ${alpha}$ 2 function-blocking MAb (C + anti- ${alpha}$ 2) and placed inside collagen for 1.5 h. Western blot analyses with an antibody that recognizes Akt when it is phosphorylated at Ser473 or with an antibody that recognizes GSK3ß when it is phosphorylated are shown. Immunoblotting with an antibody that recognizes all forms of Akt was performed for reference. Densitometric quantitation of phosphorylated Akt and GSK3ß levels relative to loading is also shown (A and B).

To confirm that the effect of collagen on Akt activation ismediated by ${alpha}$ 2ß1 integrin, we used osteosarcoma cells(Saos) expressing ${alpha}$ 1ß1 and lacking endogenous ${alpha}$ 2ß1integrin. The cells were transfected to have stable expressionof wild-type ${alpha}$ 2 (Saos- ${alpha}$ 2) or ${alpha}$ 2 with the ${alpha}$ 1 cytoplasmic domain (Saos- ${alpha}$ 2/ ${alpha}$ 1)(Fig. 2A) (25). The mutant ${alpha}$ 2/ ${alpha}$ 1ß1 integrin mediatescell adhesion to collagen as efficiently as the wild-type receptorbut lacks ${alpha}$ 2 signaling functions (25). In agreement with a previousstudy (27), detachment and maintenance of the cells in suspensionresulted in complete Akt inactivation in 1 h (Fig. 2B, laneS). After 2 h inside collagen, phosphorylation of Ser473 onAkt was clearly reduced in Saos- ${alpha}$ 2 cells compared to that inSaos- ${alpha}$ 2/ ${alpha}$ 1 cells, indicating the importance of the ${alpha}$ 2 cytoplasmicdomain in Akt inactivation (Fig. 2B). This was further confirmedusing different stable cell clones. Akt phosphorylation wasmonitored over a course of some hours, and, according to thedensitometric analysis, after 6 h no phosphorylated Akt wasdetected in Saos- ${alpha}$ 2 cells, while Akt remained phosphorylatedat Ser473 in Saos- ${alpha}$ 2/ ${alpha}$ 1 cells (Fig. 2C). In correlation with thatof Akt, the phosphorylation of GSK3ß decreased insidecollagen in Saos- ${alpha}$ 2 cells while phosphorylation at Ser9 was maintainedin cells expressing the mutant ${alpha}$ 2/ ${alpha}$ 1 integrin chimera (Fig. 2D).Densitometric analysis performed in six separate experimentsshowed that the amount of phosphorylated GSK3ß in ${alpha}$ 2-expressing cells was 40% ± 22% (mean ± SEM)lower after 1 h, and 36% ± 8% (mean ± SEM) lowerafter 4 h, inside collagen than the amount in Saos- ${alpha}$ 2/ ${alpha}$ 1 cells.These results indicate that functional ${alpha}$ 2ß1 integrinis essential for the collagen-induced rapid reduction of Aktand GSK3ß phosphorylation, while integrin ${alpha}$ 1ß1does not seem to contribute to the negative regulation of Aktin the cells studied. The active role of the cytoplasmic domainof ${alpha}$ 2 in the regulation of Akt was further confirmed using Saoscells expressing an ${alpha}$ 2/ ${alpha}$ 5 integrin chimera (5). When these cellswere exposed to 3D collagen, the levels of phosphorylated Aktwere the same as those in ${alpha}$ 2/ ${alpha}$ 1 cells and higher than those inSaos- ${alpha}$ 2 cells (Fig. 2E).

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FIG. 2. Collagen-induced dephosphorylation of Akt is regulated by ${alpha}$ 2ß1 integrin in human osteosarcoma Saos cells. (A) cDNA constructs used for expression of integrin ${alpha}$ 2 and ${alpha}$ 2/ ${alpha}$ 1 (chimera) subunits in Saos cells, which lack endogenous ${alpha}$ 2ß1 integrin. TM, transmembrane domain. (B to E) Saos- ${alpha}$ 2, Saos- ${alpha}$ 2/ ${alpha}$ 1, and Saos- ${alpha}$ 2/ ${alpha}$ 5 cell clones were serum starved, detached, and cultured inside 3D collagen gel (C, collagen) for the indicated times or left in suspension (S) for 1 h. For panel E cells were cultured inside collagen for 2 h. Western blot analysis with antibodies that recognize Akt (B and E) and GSK3ß (D) when they are phosphorylated (P-Akt and P-GSK3ß, respectively) is shown. (B, D, and E) Immunoblotting with an antibody that recognizes all forms of Akt was performed for reference. (C) A separate experiment with longer incubation times inside collagen was performed with different Saos cell clones. Densitometric quantitation of phosphorylated Akt levels at different time points relative to loading is shown. If the experiment was performed with several cell clones, the clone numbers are indicated (e.g., #6).

Integrin ${alpha}$ 2ß1 regulates Akt via PP2A. Akt activity can be decreased by either inhibition of upstreamactivators or dephosphorylation of Thr308 and Ser473 (37). Todetermine which mechanism is involved, activation of PI-3K wastested by a direct kinase assay performed with Saos- ${alpha}$ 2 and Saos- ${alpha}$ 2/ ${alpha}$ 1cells cultured inside collagen. No constant differences betweenSaos- ${alpha}$ 2 and Saos- ${alpha}$ 2/ ${alpha}$ 1 cell clones were detected (mean of two Saos- ${alpha}$ 2and two Saos- ${alpha}$ 2/ ${alpha}$ 1 clones: 80 and 73 arbitrary units, respectively).Furthermore, inhibition of PI-3K with LY-294002 had no furthereffect on the low levels of phosphorylated Akt measured in Saos- ${alpha}$ 2cells after 1.5 h inside collagen (not shown). We thereforeconcluded that ${alpha}$ 2ß1 integrin regulates Akt phosphorylationdownstream of PI-3K. Integrin ${alpha}$ 2ß1-mediated reductionof Akt and GSK3ß phosphorylation could, indeed, beprevented by protein serine/threonine phosphatase inhibitorcalyculin A. This was observed in both the human primary fibroblastsand Saos- ${alpha}$ 2 cells cultured inside collagen (Fig. 3). The aboveexperiments strongly indicated that ${alpha}$ 2ß1 regulatesAkt dephosphorylation rather than its activation via PI-3K andphosphoinositide-dependent kinase 1 but did not resolve theexact mechanism of Akt deactivation.

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FIG. 3. Regulation of Akt phosphorylation involves serine/threonine phosphatase activity. (A) Western blot analysis with an antibody that recognizes Akt or GSK3ß when they are phosphorylated (P-Akt and P-GSK3ß, respectively). Fibroblasts were deprived of serum for 16 h and seeded inside 3D collagen gel in the presence or absence of serine/threonine phosphatase inhibitor calyculin A (10 nM). (B) Western blot analysis with an antibody that recognizes phosphorylated Akt. Saos- ${alpha}$ 2 cells were deprived of serum for 16 h and then harvested from the cell culture dish (M, monolayer) and seeded inside 3D collagen (C) gel for 2 h. Cells were cultured in the presence or absence of serine/threonine phosphatase inhibitor calyculin A (1 and 10 nM). Immunoblotting with an antibody that recognizes all forms of Akt was performed for reference (A and B).

Using a specific assay for serine/threonine phosphatases, weasked whether cell adhesion to 3D collagen could induce serine/threoninephosphatase activity and, if so, whether the differences wouldbe large enough to be detected against the background of allcellular phosphatase activity. We observed that human primaryfibroblasts seeded inside collagen have two- to fivefold (rangein three separate experiments, each with three parallel measurements)-higherphosphatase activity than cells cultured in a monolayer (Fig.4A). Interestingly, the collagen-induced phosphatase activitycould be almost completely blocked with a function-blockingantibody against anti- ${alpha}$ 2 integrin (Fig. 4A), while a controlantibody (nonfunctional anti- ${alpha}$ 1 antibody) had no effect (notshown). The phosphatase assay measures PP2A activity but isnot specific for it. However, PP2A can be distinguished fromPP1 by its over-100-fold-higher susceptibility to okadaic acid(10). The integrin-induced phosphatase activity was inhibitedby 52% with 0.1 nM okadaic acid (a very low inhibitor concentration,which does not influence PP1 [50% inhibitory concentration,15 nM]), reducing the phosphatase activity to that measuredin cells treated with collagen in the presence of an anti-integrinantibody (Fig. 4B). The data indicate that ${alpha}$ 2ß1 integrininduces PP2A-type phosphatase activity in response to collagen.

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FIG. 4. Serine/threonine phosphatase PP2A is activated inside 3D collagen gel. Fibroblasts were serum starved and then harvested from the cell culture dish (monolayer) or seeded inside collagen gel for 1.5 h. Serum-starved fibroblasts were detached and placed inside collagen in the presence or absence of either an anti- ${alpha}$ 2 function-blocking MAb (collagen + anti- ${alpha}$ 2; 2.5 µg/ml) or a nonfunctional anti- ${alpha}$ 1 MAb (2.5 µg/ml; not shown). Thereafter, cells were lysed and equal amounts of protein were assayed for PP1-PP2A activity using ³²P-labeled glycogen phosphorylase as a substrate. Phosphatase activity of cells seeded inside collagen versus that of cells cultured in a monolayer and the effect of anti- ${alpha}$ 2 MAb are shown (A). To distinguish between PP1 and PP2A type activity, the effect of okadaic acid (causes half-maximal inhibition of PP2A at 0.1 nM and has no effect on PP1 below 5 nM) was tested by an in vitro phosphatase reaction (B). Phosphatase activity relative to cell lysate protein content (mean ± SEM; n = 3) is shown.

This was further supported by the fact that serine/threoninephosphatase activity was significantly higher (3.3- to 4.5-fold)in Saos- ${alpha}$ 2 clones than in Saos- ${alpha}$ 2/ ${alpha}$ 1 clones (Fig. 5A), furtherconfirming the role of ${alpha}$ 2ß1 in PP2A regulation andthe generality of this observation in different cell types.Introduction of ${alpha}$ 2 or ${alpha}$ 2/ ${alpha}$ 1 cDNAs into Saos cells did not aloneinfluence serine/threonine phosphatases, since both ${alpha}$ 2- and ${alpha}$ 2/ ${alpha}$ 1-expressingcells showed equal phosphatase activities when cultured on fibronectinand since the phosphatase activities in Saos- ${alpha}$ 2/ ${alpha}$ 1 and Saos vectorcontrol cells were the same (Fig. 5C). In accordance with theresults of the measurement done in fibroblasts, in Saos cellsthe ${alpha}$ 2ß1-induced phosphatase activity was predominantlyPP2A, since 0.2 nM okadaic acid inhibited the activity by 60%(Fig. 5B). The activation of PP2A by ${alpha}$ 2ß1 integrinrepresents a novel integrin-mediated signaling mechanism, wherean integrin ${alpha}$ subunit specifically activates a serine/threoninephosphatase.

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FIG. 5. An integrin ${alpha}$ 2 cytoplasmic tail is needed for activation of a serine/threonine phosphatase PP2A. Saos- ${alpha}$ 2 and Saos- ${alpha}$ 2/ ${alpha}$ 1 cells were serum starved, detached, and cultured inside collagen for 1.5 h (A and B) or cultured on fibronectin (C). Measurement of PP1-PP2A phosphatase activity was done from cell lysates using ³²P-labeled glycogen phosphorylase as a substrate. Two different Saos- ${alpha}$ 2 and Saos- ${alpha}$ 2/ ${alpha}$ 1 single-cell clones were tested. The effect of 0.1 nM okadaic acid (OA) (inhibits half maximally PP2A but has no effect on PP1) was tested in the in vitro phosphatase reaction (B). Phosphatase activity relative to cell lysate protein content is shown. If the experiment was performed with several cell clones, the clone numbers are indicated (e.g., #2).

Akt forms a complex with PP2A. Recently, PP2A has been shown to form a complex with a numberof kinases (1, 42, 57, 58). In most cases, PP2A inactivatesthe kinases. Here, we examined whether Akt associates physicallywith PP2A. Human primary fibroblasts were transfected with theendogenous catalytic subunit of PP2A (PP2Ac) containing a HAtag or with an empty HA tag vector only. The anti-HA immunoprecipitateswere Western blotted with an anti-Akt antibody, and the Aktprotein was found in HA-PP2Ac-transfected samples (Fig. 6A). Thecontrol samples had some Akt but much less than the samplesfrom HA-PP2Ac-transfected cells (Fig. 6A). The matrix (fibronectinor collagen) had no effect on the coprecipitation (Fig. 6A).In another set of experiments human primary fibroblasts wereinfected with adeno-myrAkt to increase the level of active kinasein the cells. PP2Ac was detected in Akt immunoprecipitates.Neither Akt nor PP2A could be detected in precipitates preparedwith control IgG (not shown). We also verified the interactionbetween Akt and PP2Ac in fibroblasts transfected with a vectorencoding HA-tagged PP2Ac and infected with adeno-myrAkt (notshown). To confirm the specificity of the antibodies used, Westernblotting was performed on whole-cell extracts from collagen-stimulated,nontransfected human primary fibroblasts. Anti-PP2A and anti-Aktantibodies detected a single specific band, and anti-HA showedno unspecific binding to cellular proteins (Fig. 6B).

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FIG. 6. PP2A interacts with Akt in transfected fibroblasts in vivo. (A) Human primary fibroblasts were transfected with Fugene 6 reagent and 6 µg of plasmid encoding the HA-tagged PP2A catalytic subunit (HA-PP2Ac) or with empty plasmid (HA vector). After 1 h inside collagen or on fibronectin, the cells were harvested, lysed, and immunoprecipitated (i.p.) with anti-HA antibody. The immunoprecipitates were analyzed by Western blotting with antibodies against Akt or the PP2A catalytic subunit. (B) Immunoblots from whole-cell lysates (nontransfected) using the same antibodies, indicating that the antibodies against Akt and PP2A recognize the corresponding wild-type proteins and that the anti-HA antibody shows no unspecific binding to cellular proteins.

By transient cotransfections using PP2Ac together with taggedAkt, we assessed whether PP2A is directly responsible for thedephosphorylation of Akt. Cells were lysed, and Akt was immunoprecipitatedin the presence of okadaic acid (1 nM) to prevent PP2A actionduring and after cell lysis. Overexpression of PP2Ac reducedAkt activity in an in vitro kinase assay of serum-starved Saos- ${alpha}$ 2/ ${alpha}$ 1cells that were stimulated with collagen for 1 h (Fig. 7). Inthree separate experiments the reduction in Akt activity was36% ± 23%. Thus, overexpression of PP2A was able to reduceAkt phosphorylation in cells where active endogenous Akt couldbe observed due to the inability of the mutant integrin to activatePP2A. The data suggest a tight interrelationship between PP2Aactivity and Akt dephosphorylation.

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FIG. 7. Overexpression of PP2A inactivates Akt. The effect of the HA-PP2A catalytic subunit or HA vector on the kinase activity of cotransfected Akt was tested. Akt was immunoprecipitated from transfected (36 h posttransfection) and serum-starved Saos- ${alpha}$ 2/ ${alpha}$ 1 cells after 1.5 h of treatment inside collagen. Akt activity was determined by the ability to phosphorylate myelin basic protein (MBP). A control blot shows that the overexpression of the PP2A catalytic subunit has no effect on the amount of Akt protein in cells.

PP2A activation by ${alpha}$ 2ß1 integrin is independent of p38 and dependent on Cdc42. The mechanism of PP2A activation by ${alpha}$ 2ß1 integrin wasstudied. We have previously shown that integrin ${alpha}$ 2ß1stimulates p38 kinase via small GTPase Cdc42 (25). This ledus to study the possible role of Cdc42 in ${alpha}$ 2ß1-mediatedactivation of PP2A. Saos- ${alpha}$ 2 cells were transiently transfectedwith a dominant-negative Cdc42 mutant (Cdc42DN). Transfectionefficiency was determined and seemed to vary between 25 and50% (not shown). Cdc42DN effectively inhibited integrin ${alpha}$ 2ß1-inducedactivation of PP2A. In five separate experiments PP2A activitywas 53% ± 13% lower in Cdc42DN-transfected cells thanin vector control cells. The inhibitory effect of Cdc42DN wasfound to be statistically significant (Student t test; P <0.016). Figure 8A shows a representative experiment. We alsoperformed control assays to show that the transfections hadno effect on the amount of PP2A in cells (Fig. 8A). Dominant-negativeRhoA and Rac1 mutants had no effect on PP2A activity in collagen-stimulatedSaos- ${alpha}$ 2 cells (Fig. 8A). In contrast, the active Cdc42 mutantincreased PP2A activity to some extent (not shown).

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FIG. 8. Activation of PP2A by ${alpha}$ 2ß1 integrin is dependent on Cdc42. (A) The effect of the transfected dominant-negative mutant forms of Cdc42 (Cdc42DN), Rac1 (Rac1DN), and RhoA (RhoADN) on the activation of PP2A in Saos- ${alpha}$ 2 cells. Phosphatase activity relative to cell lysate protein content is shown. Immunoblotting with antibodies against PP2A and Cdc42 was performed to show that Cdc42 was overexpressed in transfected cells and that it had no effect on the amount of PP2A protein in cells. (B) Serum-starved fibroblasts were detached and left untreated or treated with inhibitor BIM I (10 µM) or SB203580 (20 µM) and placed inside collagen for 1 h. Phosphatase activity relative to cell lysate protein content is shown (mean ± SEM). BIM I, four separate experiments; SB203580, two separate experiments (n = 3 for each).

The roles of other previously identified ${alpha}$ 2ß1 downstreameffectors, p38 kinase (25) and protein kinase C- ${zeta}$ (PKC- ${zeta}$ ) (61),were also studied. Inhibition of p38 kinase with SB203580 didnot influence PP2A activity, while PKC inhibitor BIM I, capableof inhibiting the PKC- ${zeta}$ isoform at the concentration used, showedsome effect (Fig. 8B). However, this inhibition was not foundto be statistically significant, and the high BIM I concentrationmay also inhibit other kinases. The inability of p38 inhibitionto influence PP2A activity indicates that ${alpha}$ 2ß1 mustregulate at least two distinct signaling functions, namely,the activation of PP2A and p38.

DISCUSSION

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Discussion

PP2A comprises a family of protein serine/threonine phosphatases.They can interact with a substantial number of proteins andcontribute to the regulation of numerous signaling pathways(26). Active PP2A can inhibit the cell cycle, predominantlyat the G₂/M checkpoint (33), induce apoptosis (13, 48), andact as a tumor suppressor (45). In this study we demonstratethat ${alpha}$ 2ß1 integrin modulates Akt and GSK3ßphosphorylation in response to cell adhesion to 3D collagenby activating PP2A. In general integrin-mediated cell adhesionis known to trigger initial tyrosine phosphorylation events,such as the activation of FAK and Src family kinases, that resultin the activation of downstream signaling pathways, a numberof which involve the activity of various serine/threonine kinases(50). However, relatively little is known about how specificprotein tyrosine and serine/threonine phosphatases functionto regulate integrin-mediated signals. The suggestion that theactivation of PP2A in response to collagen is specifically mediatedby ${alpha}$ 2ß1 integrin is based on the following facts: (i)PP2A activation could be inhibited by a function-blocking antibodyto ${alpha}$ 2 integrin and (ii) human osteosarcoma cells lacking ${alpha}$ 2ß1integrin or expressing a signaling deficient, chimeric ${alpha}$ 2/ ${alpha}$ 1 subunitwere shown to be unable to activate PP2A in response to collageneven though these cells adhere to collagen similarly to the ${alpha}$ 2ß1-expressing cells (25). Thus, our results demonstratethe association of integrin function with a protein serine/threoninephosphatase. In the previous reports, a link between integrinsand protein tyrosine phosphatase SHP-2 has been recently establishedand the phosphatase function has been shown to be a positiveregulator of integrin function (23, 39). Another example ofthe interrelationship between phosphatases and integrins comesfrom studies demonstrating that the expression of dual-specificityphosphatase PTEN is essential in maintaining the anchorage dependenceof growth via negative regulation of FAK and downstream kinases(49).

The in vitro activity of PP2A can be regulated by phosphorylation(7) and methylation (31, 59). Cells also have specific PP2Ainhibitor proteins (34, 35). The cellular pathways regulatingPP2A activity are incompletely known, and accordingly the exactmechanism of PP2A activation by ${alpha}$ 2ß1 integrin remainsto be shown. Active Cdc42 seems indispensable in the stimulationof PP2A by ${alpha}$ 2ß1 and collagen, since expression of dominant-negativeCdc42 blocked ${alpha}$ 2ß1-mediated PP2A activation. Cdc42may regulate the formation of a PP2A-containing adhesion andsignaling complex. Indeed, a previous report indicates thatPP2A can accumulate at focal contact sites due to its interactionwith paxillin (24). Furthermore, our results suggest that thecytoplasmic domain of ${alpha}$ 2 integrin may also participate in theactivation of PP2A.

Cdc42 may be one of the prime effectors of ${alpha}$ 2ß1 signaling,since it is also required for ${alpha}$ 2ß1-mediated activationof the p38 ${alpha}$ isoform (25). p38 can activate PP2A (55), but herethe inhibition of the p38 pathway had no effect on PP2A activationby ${alpha}$ 2ß1 integrin. Additionally, a PKC inhibitor didnot affect PP2A activation. PKC- ${zeta}$ has been described as one ofthe signaling proteins activated by ${alpha}$ 2ß1-mediated bindingto collagen (60). Ceramides can concomitantly activate PKC- ${zeta}$ and PP2A, suggesting that these pathways might be connectedto each other (40).

Akt activity can, in general, be inhibited by either inhibitionof upstream activators or by direct dephosphorylation (37).The fact that here ${alpha}$ 2ß1 integrin inhibited Akt withoutconcomitant attenuation of PI-3K led us to consider a possiblelink between the observed PP2A induction and Akt inactivation.PP2A has been shown to inactivate Akt in vitro, while otherserine/threonine phosphatases, such as PP1, are unable to usephosphorylated Akt as a substrate (2). Calyculin A is able toprevent PP2A-dependent dephosphorylation of Akt (37), and herewe show that it prevented ${alpha}$ 2ß1-related Akt inactivation.Furthermore, the cotransfection experiments using the PP2A catalyticsubunit with Akt demonstrated a direct relationship betweenPP2A and Akt kinase activity.

Recent studies indicated that PP2A associates with kinases andthereby negatively regulates their function (1, 41, 56, 57).Here we suggest that activated PP2A is the mechanism behindthe modulation of Akt activity by ${alpha}$ 2ß1 integrin. Furthermore,the dephosphorylation of GSK3ß, one of the downstreamtargets of Akt, correlated with Akt deactivation. Dephosphorylationof GSK3ß leads to its activation (28). Besides theAkt/GSK-3ß pathway, PP2A has the potency to affectseveral other signaling pathways, and therefore the activationof PP2A may participate in many ${alpha}$ 2ß1-mediated cellularevents, including inhibition of the cell cycle (30).

To conclude, we have introduced a novel ${alpha}$ 2ß1-relatedsignaling pathway, namely, activation of PP2A. Protein phosphataseshave wider substrate specificity than protein kinases, and thereforethey may affect several signaling pathways. In general, theregulation of cellular phosphatase activity is less studiedthan that of kinases, and there are only a few reports indicatingtheir regulation by a specific integrin. Integrin ${alpha}$ 2ß1-mediatedPP2A activation provides a versatile mechanism to regulate numeroussignaling mechanisms and cellular functions.

ACKNOWLEDGMENTS

We thank K. Walsh, K. Vuori, J. C. Lacal, G. W. G. Wilkinson,and D. Brautigan for the vector constructs and adenovirusesused in this study. Technical assistance by M. Potila, M. Tuominen,and T. Heikkilä is gratefully acknowledged.

This study was financially supported by grants from the Academyof Finland, the Sigrid Jusélius Foundation, the FinnishCancer Association, Farmos Research Foundation, the Turku UniversityCentral Hospital, and the Technology Development Center in Finland.

FOOTNOTES

* Corresponding author. Mailing address: MediCity Research Laboratory, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. Phone: 358-2-333 7005. Fax: 358-2-333 7000. E-mail: jyrki.heino{at}utu.fi.

REFERENCES

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