Specific beta 1 Integrin Site Selectively Regulates Akt/Protein Kinase B Signaling via Local Activation of Protein Phosphatase 2A*

Roumen PankovDagger §, Edna CukiermanDagger , Katherine ClarkDagger , Kazue MatsumotoDagger , Cornelia HahnDagger , Benoit Poulin||, and Kenneth M. YamadaDagger

From the Dagger  Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370 and the || Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-0320

Received for publication, January 27, 2003, and in revised form, March 4, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrin transmembrane receptors generate multiple signals, but how they mediate specific signaling is not clear. Here we test the hypothesis that particular sequences along the beta 1 integrin cytoplasmic domain may exist that are intimately related to specific integrin-mediated signaling pathways. Using systematic alanine mutagenesis of amino acids conserved between different beta  integrin cytoplasmic domains, we identified the tryptophan residue at position 775 of human beta 1 integrin as specific and necessary for integrin-mediated protein kinase B/Akt survival signaling. Stable expression of a beta 1 integrin mutated at this amino acid in GD25 beta 1-null cells resulted in reduction of Akt phosphorylation at both Ser473 and Thr308 activation sites. As a consequence, the cells were substantially more sensitive to serum starvation-induced apoptosis when compared with cells expressing wild type beta 1 integrin. This inactivation of Akt resulted from increased dephosphorylation by a localized active population of protein phosphatase 2A. Both Akt and protein phosphatase 2A were present in beta 1 integrin-organized cytoplasmic complexes, but the activity of this phosphatase was 2.5 times higher in the complexes organized by the mutant integrin. The mutation of Trp775 specifically affected Akt signaling, without effects on other integrin-activated pathways including phosphoinositide 3-kinase, MAPK, JNK, and p38 nor did it influence activation of the integrin-responsive kinases focal adhesion kinase and Src. The identification of Trp775 as a specific site for integrin-mediated Akt signaling supports the concept of specificity of signaling along the integrin cytoplasmic domain.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrin transmembrane receptors sense the extracellular environment and convey bidirectional signals regulating cell behavior and fate (for recent reviews, see Refs. 1-6). Signals transmitted by integrins regulate cell migration, growth, differentiation, and decisions for survival or death. Cell survival can be regulated through integrin-mediated modulation of cell shape and adhesion in cooperation with growth factors or integrin control of the activity of tyrosine and serine/threonine kinases such as FAK,1 Src, and integrin-linked kinase (see Refs. 4, 5, 7, 8, and 9-12). A variety of integrin heterodimers and mechanisms have been implicated in integrin-mediated survival of various cell types (13-24), indicating the complexity of this signaling.

An important effector of cell survival is protein kinase B (PKB), also called Akt (25-28). Akt is a Ser/Thr protein kinase that becomes fully active after dual phosphorylation of Thr308 and Ser473 (29-31). Integrins can act as positive modulators of Akt by stimulating its activation pathway involving upstream phosphoinositide 3-kinase (PI3K) (18, 19, 32).

Recently, it was shown that an integrin can also regulate the inactivation of Akt by affecting protein phosphatase 2A (PP2A) (33). PP2A is an abundant and ubiquitous Ser/Thr phosphatase that is involved in the regulation of a wide variety of cellular activities. Its substrate specificity is tightly regulated by subunit composition, post-translational modifications, and association with different intracellular components (see Refs. 34-36). PP2A can dephosphorylate and inactivate Akt (37-40); it can also associate with the beta 1 integrin (41), suggesting the possibility of a close spatial/functional relationship between these molecules.

Although beta  integrin subunit cytoplasmic domains are relatively short (except for beta 4, which is 1000 amino acids, the rest range between 46 and 70 residues), they are saturated with binding sites for cytoplasmic proteins that participate in integrin-mediated signaling and cytoskeletal interactions (see Refs. 1, 42, and 43). We hypothesized that different sequences within the integrin tail are essential for regulation of distinct pathways of signal transduction. In fact, a site conserved between different integrin subunits might be dedicated to a unique signaling pathway, such that a specific integrin mutation would affect only one out of many integrin-mediated signaling pathways.

Previous mutational studies of beta  integrin cytoplasmic domains have focused mainly on the possible roles of phosphorylation sites along the integrin tail (44-48). They have documented the importance of these residues for basic cellular processes including adhesion, organization of the cytoskeleton and focal adhesions, and cell migration without identifying effects on specific signaling pathways.

To test our hypothesis of site-specific integrin regulation of signaling, we tried to identify an amino acid residue within the beta 1 integrin cytoplasmic domain that was relatively specifically involved in integrin-mediated survival signaling. Here we report the identification of tryptophan 775 in the beta 1 integrin cytoplasmic domain as being both necessary and specific for Akt activation and cell survival. This residue was found to be involved in interactions that regulate the local activity of PP2A within integrin-organized complexes. We also found that Akt is a constituent of these complexes and is a substrate for PP2A. The interactions mediated by Trp775 appear to be specific for Akt signaling, since we were unable to identify any other integrin-mediated signaling pathways affected by mutating this residue.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Antibodies-- Antibodies to human beta 1 integrins included rat monoclonal antibody 9EG7 (Pharmingen), mouse monoclonal antibodies 12G10 (49), TS2/16 (50), and K20 (Immunotech), and rabbit antibody Rab 4080 (51). Anti-mouse alpha 5 integrin antibody (monoclonal antibody 1928) was from Chemicon. Antibodies against actin, tubulin, and vinculin were from Sigma; anti-total Akt, anti-phospho-Akt, anti-phospho-PDK1, and anti-total PDK1 antibodies and the Akt kinase assay kit were from Cell Signaling; anti-PP2A and anti-phospho-FKHRL1 were from Upstate Biotechnology, Inc.; anti-phospho-FAK and anti-phospho-Src were from BioSource; anti-phospho- and anti-total p38 and anti-phospho- and anti-total mitogen-activated protein kinase (MAPK) were from New England Biolabs; anti-Bcl-2 and anti-phospho- and anti-total c-Jun N-terminal kinase (JNK) were from Santa Cruz; and anti-alpha -actinin was from ICN. Anti-talin rabbit polyclonal antibody was generously provided by Dr. Keizo Takenaga (NIDCR, NIH, Bethesda, MD). Cy3- and fluorescein isothiocyanate-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories.

Sheep anti-mouse magnetic microbeads (4.5 µm) were purchased from Dynal. Okadaic acid, AG1433, PD98059, and UO126 were from Calbiochem. Human plasma fibronectin was purified as previously described (52). The pHA262pur puromycin resistance plasmid was generously provided by Dr. Hein te Riele (Netherlands Cancer Institute) (53).

Generation of Mutant beta 1A cDNAs-- Human beta 1A integrin cDNA was kindly provided by Erkki Ruoslahti (Burnham Institute). Point mutations in the integrin cDNA were introduced by the QuikChangeTM site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol using the primers listed in Table I. The resulting constructs were sequenced to confirm the presence of the desired mutations and the absence of any other alterations introduced during manipulation.


                              
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Table I
Oligonucleotide primers used to generate beta 1 integrin mutants
Mismatches with the template are underlined. FW, forward primer; RV, reverse primer.

Cell Culture, Transfection, and Establishment of Stable Cell Lines-- The GD25 beta 1-null fibroblast cell line was a generous gift from Reinhard Fässler (Lund University). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml fungizone. Plasmid DNAs encoding wild type or mutant human beta 1A integrins were co-transfected together with the puromycin selection vector pHA262pur by electroporation as described (51). Pools of mixed populations of stable transfectant cells expressing comparable levels of each of the wild type or mutant integrins were established by selection in medium containing 10 µg/ml puromycin and by four consecutive fluorescence-activated cell sortings with anti-human beta 1 integrin antibody K20 performed over a 3-month period. Cells expressing the W775A mutation were used up to passage 10 after establishing a pool of positive cells. Due to increased apoptosis of the mutant transfectant, a beta 1 integrin-negative subpopulation becomes detectable after this passage.

Integrin Activation by Clustering and Isolation of Integrin-based Complexes-- Sheep anti-mouse IgG magnetic beads (4.5 µm; Dynal) were coated with TS2/16 or K20 antibodies according to the manufacturer's instructions. GD25 cells expressing beta 1 WT or W775A integrins were trypsinized and serum-starved in suspension in DMEM with 1% bovine calf serum for 3 h. The serum-deprived cells were mixed with TS2/16-coated beads (5 beads/cell), and clustering and activation of integrins was allowed to continue for 1 h at 37 °C with rotation. The complexes of beads and bound cells were collected by a magnetic particle concentrator (Dynal) and washed once with phosphate-buffered saline, and then the cells were lysed and prepared for Western blot analysis.

For the isolation of integrin-based complexes, serum-starved cells were mixed with beads (five beads/cell) coated with K20 antibody and processed as described (51). Briefly, the complexes of beads and bound cells were isolated with a Dynal magnetic concentrator and were washed with 1 ml of cold CSK buffer (50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, protease inhibitor mixture (Roche Applied Science), 1 mM sodium vanadate, 50 mM NaF, and 1 mM phenylmethylsulfonyl fluoride in 10 mM PIPES, pH 6.8) without detergent. The pellet containing cell-bead complexes was extracted with 0.5 ml of cold CSK buffer containing 0.5% Triton X-100 and sonicated for 10 s with a 50-watt ultrasonic processor (model GE 50 set at amplitude 20; Aldrich). The bead-protein complexes were washed five times with 1 ml of cold CSK buffer containing detergent and prepared for Western blot analysis or PP2A assay. Phosphatase inhibitors were omitted from the CSK buffer when integrin-based complexes were prepared for assaying PP2A.

Co-immunoprecipitation and Immunoblotting-- GD25 cells expressing beta 1 WT or W775A integrins were plated on fibronectin-coated dishes (5 µg/ml) in DMEM with 1% bovine serum albumin. After overnight incubation, the cells were solubilized on ice in Nonidet P-40 buffer (137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 20 mM Tris-HCl, pH 8.0, 1 mM sodium vanadate, 50 mM NaF) containing protease inhibitors and 1 mM phenylmethylsulfonyl fluoride. Homogenates were clarified by centrifugation at 20,000 × g for 15 min at 4 °C. Immunoprecipitates were obtained using 4 µg of the indicated antibodies and GammaBindTM Plus SepharoseTM (Amersham Biosciences). Protein complexes bound to beads were solubilized in reducing SDS-PAGE sample buffer and resolved on 4-12% gradient gels (Novex). After electrotransfer to nitrocellulose membranes (Novex), the filters were blocked (5% nonfat dry milk in 150 mM NaCl, 50 mM Tris HCl, 0.1% Tween 20, pH 7.4) and probed with the indicated phosphospecific or general antibodies, followed by the appropriate secondary horseradish peroxidase-conjugated antibodies. Immunoblots were visualized using the ECL system and Hyperfilm x-ray film (Amersham Biosciences).

Flow Cytometry, Immunofluorescence, and Apoptosis Assay-- For flow cytometry, cells were detached with trypsin-EDTA, washed with culture medium, and incubated with the indicated antibodies for 30 min on ice in PBA buffer (phosphate-buffered saline, 0.5% bovine serum albumin, 0.02% NaN3). Stained cells were washed, incubated with fluorescein isothiocyanate-conjugated secondary antibodies in the same buffer for an additional 30 min, washed again, and analyzed using a FACSCalibur flow cytometer (BD Biosciences). For cell sorting, trypsinized cells were washed and stained in complete medium with fluorescein isothiocyanate-conjugated anti-human beta 1 integrin antibody K20 (Immunotech) for 30 min on ice, washed with complete medium without phenol red, and sorted using a FACStar plus cell sorter (BD Biosciences).

Cells for immunofluorescence analysis were plated on fibronectin (5 µg/ml) precoated glass coverslips (12 mm; Carolina Biological Supply Co.) and cultured overnight in the absence of serum. Samples were fixed with 4% paraformaldehyde in phosphate-buffered saline containing 5% sucrose for 20 min and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 3 min. Primary antibodies were used at 10 µg/ml and visualized with secondary CY3- or fluorescein isothiocyanate-conjugated antibody.

For assaying apoptosis induced by serum starvation, cells were cultured in the absence of serum for 72 h. After this period, the cells were fixed and stained without permeabilization with 2 µM Hoechst 33342 fluorochrome for 5 min. Stained samples were mounted in GEL/MOUNTTM (Biomeda Corp.) containing 1 mg/ml 1,4-phenylenediamine (Fluka) to reduce photobleaching. Immunofluorescent images were obtained with a Zeiss Axiophot microscope equipped with a Photometrics CH350 cooled CCD camera. Digital images were obtained using MetaMorph 3.5 software (Universal Imaging). The same software was used to score the number of apoptotic (bright) and nonapoptotic (dark) nuclei from each of two randomly chosen fields from samples obtained from three separate experiments.

Akt Dephosphorylation, Akt Kinase, MAPK, and PP2A Phosphatase Assays-- Cells were cultured overnight on fibronectin-coated dishes (5 µg/ml) in DMEM, 1% bovine serum albumin and then stimulated with 20 ng/ml PDGF BB for 15 min, washed, and cultured in the same medium containing 30 µM PDGF kinase inhibitor AG 1433. Samples were collected at the indicated time points after PDGF stimulation and were prepared for Western blot analysis with anti-phospho-Akt (Ser473) antibody. In some experiments, the cells were pretreated for 15 min with 1 µM okadaic acid before stimulation with PDGF, and the same concentration of this PP2A inhibitor was present throughout the entire experiment.

Akt kinase activity was evaluated using an Akt kinase assay kit from Cell Signaling according to the manufacturer's protocol. Briefly, cells cultured as described above were lysed, endogenous Akt was immunoprecipitated, and its ability to phosphorylate a recombinant GSK-3alpha /beta polypeptide containing residues surrounding Ser21 and Ser9 was assessed with phosphospecific antibodies against these serine residues.

For assaying MAPK activity and Akt phosphorylation after spreading on fibronectin, cells were trypsinized, washed with trypsin inhibitor, and rotated for 30 min in DMEM, 1% bovine serum albumin at 37 °C. Equal numbers of cells were plated on dishes precoated with 5 µg/ml fibronectin and blocked with 1% heat-denatured bovine serum albumin. Homogenates of cells in suspension were analyzed by Western blotting with anti-phospho-MAPK and anti-phospho-Akt antibodies after the indicated plating times. Relative phosphorylation levels for each time point were calculated as a ratio between densitometry readings obtained using anti-phospho-MAPK and anti-total MAPK antibodies or anti-phospho-Akt and anti-actin antibodies.

PP2A activity was measured with a Ser/Thr phosphatase assay kit (Upstate Biotechnology) according to the manufacturer's protocol. In some experiments, a specific phospho-Akt1/PKB peptide (Upstate Biotechnology) was used as a substrate. Total PP2A activity was measured after immunoprecipitation of the PP2Ac catalytic subunit with anti-PP2A antibody clone 1D6. PP2A activity in integrin-based complexes was measured after their isolation with anti-beta 1 integrin antibody K20. After the phosphatase reaction, each sample was analyzed by Western blotting with anti-PP2A antibodies for determination of PP2A protein content. PP2A activity was calculated as the ratio between released free phosphate measured as absorbance at 650 nm with the phosphatase assay kit and the densitometry signal for PP2A according to Western blotting.

PI3K Assay and Measurement of Phosphoinositide Content-- Cell lysates were obtained from serum-starved cells after overnight plating on fibronectin as described above. The lysates were immunoprecipitated with agarose-conjugated 4G10 anti-phosphotyrosine antibody or with a mixture of isoform-specific anti-p110 PI3K or anti-p85 PI3K antibodies. PI 3-kinase was assayed as described (54). After thin layer chromatography on LK6D plates (Whatman), 32P-labeled phosphoinositides were detected by autoradiography.

For phosphoinositide measurement, serum-starved cells plated on fibronectin were washed with phosphate-free DMEM and labeled with 0.5 mCi/ml ortho[32P]phosphate (Amersham Biosciences) for 1 h. Phospholipids were extracted from the cells as previously described (55), and the chloroform phase was vacuum-dried and subjected to deacylation. Samples were dissolved in methylamine solution (33% methylamine in ethanol (Fluka)/water/n-butanol; 5/2/1 (v/v/v)), incubated at 53 °C for 1 h, and vacuum-dried. The dried material was dissolved in water, and the acyl moieties were removed by two chloroform extractions. The aqueous phases containing the glycerophosphoinositol phosphates were analyzed by HPLC with a Partisil SAX 10 column using a 70-min nonlinear gradient of 0-1 M NH4H2PO4 at a flow rate of 1.2 ml/min. Radioactivity in the eluate was monitored with an on-line Flow-One detector (Packard). Retention times of individual glycerophosphoinositol phosphates were determined using radiolabeled standards. Results are expressed as the percentage of total phospholipid radioactivity attributed to each deacylated phosphoinositide species.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrin-mediated Regulation of Akt Activity and Cell Survival Depends on a Single Conserved Tryptophan within the Integrin Cytoplasmic Domain-- To test our hypothesis that a particular sequence or site might be involved in selective integrin signaling, we focused on integrin-regulated Akt (PKB) signaling, which is relevant to cell survival but is poorly understood. We first identified the amino acid residues that are conserved between six human beta  integrin cytoplasmic tails and substituted an alanine residue sequentially for each of them in the human beta 1 integrin sequence (Fig. 1A). We omitted the well studied conserved NPXY motifs known to be important for a variety of basic integrin functions including integrin localization, focal adhesion organization, and cell migration (56, 57), because mutations in these NPXY regions could affect signal transduction indirectly in complex ways.


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Fig. 1.   Substitution of the conserved tryptophan 775 residue of the beta 1 cytoplasmic domain inhibits integrin-mediated Akt signaling. A, conserved amino acid residues (boldface type and underlined) in the human beta 1A integrin cytoplasmic domain were identified as residues that are found five or more times at comparable sites in human beta 1, beta 2, beta 3, beta 5, beta 6, and beta 7 sequences. They were separately mutated to Ala in order to generate a set of point mutant human beta 1 integrins. B, each mutant was stably expressed in beta 1-null mouse GD25 cells and FACS-sorted with anti-human integrin antibody K20 to obtain mixed cell populations with similar levels of integrin expression. The stable transfectant cell lines were plated overnight on fibronectin in the absence of serum and lysed, and the levels of Akt phosphorylation were determined by Western blotting with antibody against phospho-Ser473 Akt. Note that cells expressing the integrin W775A mutation show a 5-fold decrease in Akt phosphorylation. C, the expression of comparable levels of mutant integrins was verified by Western blotting with anti-integrin beta 1 cytoplasmic domain antibody (Rab 4080). Actin was used as the internal control for loading. *, p < 0.001.

Each of the point-mutated human beta 1 integrins was stably transfected into beta 1-null mouse GD25 cells (58). Mixed populations of positive transfectants with similar levels of integrin expression (Fig. 1C) were obtained by fluorescence-activated cell sorting (see "Experimental Procedures") in order to avoid potential variations associated with the use of single clones or different expression levels of the mutated integrins.

The first screen was for effects on the activity of the serine/threonine protein kinase Akt/PKB, which plays a central role in the survival of a wide range of cell types (25-28). The activity of Akt, which is regulated by phosphorylation, was probed with phosphospecific antibodies. The level of phosphorylation on Ser473 in each of the sorted cell lines was determined on whole cell lysates in comparison with Akt phosphorylation in cells expressing wild type beta 1 integrin (Fig. 1B). Only one mutation (W775A) among the nine conserved integrin sites tested by alanine mutagenesis showed a statistically significant (p < 0.001) decrease in Akt phosphorylation. This result suggested that the tryptophan residue at position 775 is important for integrin-regulated Akt activation. This mutant was used in all subsequent studies.

Full activation of Akt occurs via phosphorylation of both Thr308 in the kinase domain and of Ser473 in the carboxyl-terminal regulatory domain of the molecule (29-31). In W775A-expressing cells, both residues showed a 5-fold decrease in phosphorylation when the cells were grown in the absence of serum (Figs. 2A and 3A) and a 2-fold decrease in the presence of 10% fetal bovine serum (not shown) when compared with parallel beta 1 WT transfectants.


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Fig. 2.   W775A mutation specifically affects Akt phosphorylation. A, GD25 cells expressing wild type (beta 1 wt) or tryptophan mutant (W775A) integrins were cultured overnight on fibronectin in the absence of serum in medium supplemented with 0.1 mg/ml GRGDS peptide. Phosphorylation levels of the indicated kinases were analyzed by Western blotting with phosphospecific antibodies. Tubulin levels or the total quantity of each kinase regardless of its phosphorylation state (total-MAPK; total-JNK; total-p38) were used as internal controls for loading. Boxed results were obtained from the same nitrocellulose membrane. B, cells cultured on fibronectin were serum-starved overnight, trypsinized, and maintained in suspension in medium without serum for an additional 30 min. The cells were then allowed to attach to fibronectin (5 µg/ml)-coated dishes for the indicated times. Lysates from samples taken before plating (0 time) and at the end of each time point (15-60 min) were analyzed by Western blotting with antibodies against phosphorylated and total MAPK. Densitometry obtained with anti-phospho-MAPK antibody was normalized against anti-total MAPK, and -fold changes in MAPK phosphorylation after attachment compared with suspension were calculated.


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Fig. 3.   GD25 cells expressing W775A beta 1 integrin have suppressed Akt signaling and are more sensitive to apoptosis after serum starvation. A, GD25 cells expressing wild type (beta 1 wt) or tryptophan mutant (W775A) integrins were cultured overnight on fibronectin in the absence of serum, and the levels of Akt phosphorylation of Ser (p-Akt(Ser473)) and Thr (p-Akt(Thr308)) residues were analyzed by Western blotting with phosphospecific antibodies. The total amount of Akt (total-Akt) was used as an internal control for loading. B, lysates from cells cultured as described for A were used to test Akt kinase activity in vitro. Endogenous Akt was immunoprecipitated from 50 µl (lane 1), 100 µl (lane 2), and 200 µl (lane 3) of lysates from cells expressing wild type (beta 1 wt) or tryptophan mutant (W775A) integrins. The Akt-induced phosphorylation of a recombinant GSK-3 fusion protein was measured by Western blotting with antibodies against phospho-GSK-3alpha serine 21 and phospho-GSK-3beta serine 9 (p-GSK-3alpha /beta (Ser21/9)). The amounts of immunoprecipitated Akt were verified by probing the same membrane with anti-Akt antibodies (total-Akt). C, the same cell lysates described for A were probed with phosphospecific antibodies against the Akt substrate FKHRL1 residues threonine 32 (p-FKHRL1(Thr32)) and serine 253 (p-FKHRL1(Ser253)). Tubulin was used as an internal control for the amount of protein loaded from each sample. D, cells were plated on fibronectin, serum-starved for 78 h, fixed, and then stained with Hoechst 33342 without permeabilization. Apoptotic (arrows in the insets) and nonapoptotic (arrowheads in the insets) nuclei were scored, and the percentage of apoptotic cells in each sample was determined. Bar, 100 µm.

Tryptophan Mutation Specifically Targets the Akt Signaling Pathway-- Integrins participate in a number of signaling pathways that are often complex and interdependent. To test whether mutation of the tryptophan residue of the beta 1 integrin cytoplasmic domain selectively affects only the Akt survival-related pathway or in addition affects other signaling pathways, we assayed for effects on important cellular kinases that respond to integrin activation. Activation levels of these kinases were estimated by their level of site-specific phosphorylation. Activities were measured under steady-state conditions (overnight incubation) in the presence of 0.1 mg/ml RGD peptide. The latter treatment blocks alpha vbeta 3 integrin-mediated adhesion of GD25 cells, forcing the GD25 beta 1 WT and GD25 W775A cells to depend on beta 1 integrins for attachment to fibronectin (47). The major autophosphorylation site of focal adhesion kinase (Tyr397) as well as the Src activation (Tyr418) and inhibitory (Tyr529) sites were phosphorylated to similar levels in the two transfectant cell lines (Fig. 2A, p-FAK (Tyr397), p-Src(Tyr418), and p-Src (Tyr529)), suggesting that both kinases are equally active. Comparable phosphorylation levels were also found for MAPK, JNK, and p38 MAPK (Fig. 2A, p-MAPK (Tyr202/Tyr204), p-JNK (Thr183/Tyr185), and p-p38 (Thr180/Tyr182)). Moreover, the dynamic response of MAPK following integrin engagement after plating on fibronectin was very similar, with maximal activation after 15 min and a nearly complete decline by 1 h after plating (Fig. 2B), implying that the mutant and wild type integrins had equal abilities for MAPK activation. Similar phosphorylation levels were also observed for the adapter protein Shc and Crk-associated substrate p130cas when probed with general anti-phosphotyrosine antibodies after immunoprecipitation (not shown). In summary, the phosphorylation levels of all of the integrin-responding signaling molecules tested were parallel in both beta 1 WT- and W775A-expressing cell lines, with the sole exception of Akt (Fig. 2A, p-Akt (Ser473)). These results demonstrate that the tryptophan mutation specifically affects Akt signaling, leaving other integrin-related pathways fully operational.

Downstream Effects of the W775A Mutation on the Akt Signaling Pathway-- The decrease of Akt phosphorylation in W775A integrin-expressing cells was reflected in a similar 5-fold decrease in Akt kinase enzymatic activity when compared with beta 1 WT integrin-expressing cells in an in vitro kinase assay (Fig. 3B). This deficiency in Akt activation was associated with decreased phosphorylation of FKHRL1, which is a member of the Forkhead family of transcription factors and is one of the downstream effectors of Akt. Both residues Thr32 and Ser253 of FKHRL1, which have been shown to be direct targets of Akt kinase (59), were considerably less phosphorylated in W775A-expressing cells when compared with FKHRL1 in cells expressing wild type beta 1 integrin (Fig. 3C).

The strong suppression of the Akt pathway in cells expressing the integrin tryptophan point mutant suggested that these cells would be more vulnerable to apoptosis. Indeed, when GD cell lines were subjected to serum starvation for 3 days, more than 35% of the W775A cells showed apoptotic nuclei, whereas only 5% of the cells expressing the wild type beta 1 integrin were apoptotic (Fig. 3D).

Mutation of the Tryptophan Does Not Interfere with Major Cytoplasmic Interactions and with Targeting of the Mutant Integrin to Focal Adhesions-- Introduction of amino acid substitutions might lead to conformational changes in the integrin cytoplasmic domain that could affect protein-protein interactions even distal to the mutation. Although the mutated tryptophan is not immediately adjacent to the membrane-proximal sequence KLLXXXXD, which is important for integrin heterodimer formation (60, 61), we confirmed the continued ability of the W775A integrin to dimerize with the alpha 5 subunit. Co-immunoprecipitation experiments showed that the endogenous mouse alpha 5 integrin subunit binds similar amounts of the transfected human wild type and mutated beta 1 integrins (Fig. 4A). Moreover, the amounts of alpha 5 subunit detected by FACS analysis on the surface of beta 1 WT- and W775A-expressing cells were similar, whereas the beta 1-null GD25 cells were negative, suggesting that the mutant integrin is also equally effective in stabilizing endogenous alpha  subunit cell surface expression.


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Fig. 4.   The W775A beta 1 mutation does not hinder pairing with the alpha 5 subunit, binding to talin, alpha -actinin, or vinculin, and focal adhesion localization. A, integrin alpha 5 subunit immunoprecipitates were prepared from parental GD25 cells (beta 1-/-) or cells expressing wild type (beta 1 wt) or tryptophan mutant (W775A) integrins after overnight incubation on fibronectin in the absence of serum and were analyzed by Western blotting for co-immunoprecipitated beta 1 integrin. Amounts of alpha 5 integrin subunit were verified with an anti-alpha 5 antibody. B, integrin-based complexes were isolated from wild type (beta 1 wt) or tryptophan mutant (W775A) integrin-expressing cells that were serum-starved in suspension for 3 h. The complexes were purified with magnetic beads coated with anti-beta 1 integrin 12G10 as described under "Experimental Procedures," and the presence of talin, alpha -actinin, and vinculin as well as beta 1 integrins was analyzed by Western blotting. C, cells were cultured overnight on fibronectin in the absence of serum, fixed, and double-stained with anti-integrin (beta 1 integrin) and anti-vinculin (vinculin) antibodies. Bar, 10 µm.

Several cytoplasmic proteins have been shown to bind directly or indirectly to the beta 1 integrin cytoplasmic domain (see Ref. 42). Among them are talin (62-64) and alpha -actinin (65, 66), which have binding sites that include Trp775. To test the effect of the tryptophan substitution on these interactions, we isolated integrin-based complexes using beads coated with anti-integrin antibodies (see "Experimental Procedures" and Ref. 51). The amounts of talin and vinculin, which may bind to beta 1 integrin via talin (67, 68), were similar, and alpha -actinin was even increased in the complexes formed by the mutant W775A integrin (Fig. 4B). Analogous results were obtained in co-immunoprecipitation experiments (not shown), suggesting that the W775A mutation does not reduce beta 1 interactions with talin, alpha -actinin, and vinculin.

Functional beta 1 integrins are capable of localizing to focal adhesions. We therefore analyzed cellular localization of the tryptophan mutant by immunofluorescence with two antibodies that recognize cation- and ligand-induced epitopes on beta 1 integrin: 12G10 (48) and 9EG7 (69, 70). Both wild type and mutant integrins reacted with 12G10 (Fig. 4C, beta 1 integrin) and 9EG7 (not shown) antibodies and co-localized similarly with the focal adhesion marker vinculin (Fig. 4C, vinculin) when plated on fibronectin. These results indicate that tryptophan substitution induces little functional change in the beta 1 integrin cytoplasmic domain, with the sole exception of Akt signaling.

Tryptophan Mutation Partially Reduces the Ligand-binding/Activation State of beta 1, yet Restoring Integrin Activation Does Not Compensate for Akt Inhibition-- Some changes in the cytoplasmic domain of integrins can affect the conformation of the extracellular portion of the molecule. This mechanism operates normally in living cells and is known as inside-out signaling (71, 72). It activates ligand binding of integrin molecules, accompanied by exposure of specific epitopes. To investigate the ligand binding/activation state of the W775A mutant, we used the 12G10 and 9EG7 monoclonal antibodies, which recognize cation- and ligand-induced epitopes on the human beta 1 integrin. FACS analysis revealed that cells expressing the tryptophan mutant bind 40% fewer 12G10 or 9EG7 antibodies when compared with cells expressing the wild type beta 1 integrin (Fig. 5A, 12G10 and 9EG7). This difference was not the result of different levels of integrin expression, because binding of the K20 monoclonal antibody, which recognizes total human beta 1 integrin regardless of its activation, was similar for the two cell lines (Fig. 5A, K20). Full stimulation of W775A to a level similar to that of the wild type integrin was obtained with the activating TS2/16 antibody (50) (Fig. 5A, TS2/16). These results suggest that the W775A substitution partially decreases the activation state of the beta 1 integrin, but this effect is reversible by integrin activators.


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Fig. 5.   W775A integrin is in a lower activation state, but its reactivation is not sufficient to restore Akt phosphorylation. A, integrin-null cells (beta 1-/-; ) or cells expressing wild type (beta 1 wt; black-square) or mutant (W775A; ) integrins were stained with anti-beta 1 integrin antibodies and analyzed by FACS. The anti-beta 1 integrin antibodies used were against ligand- and cation-induced binding sites (12G10 and 9EG7), activating (TS2/16), or capable of recognizing total beta 1 integrins regardless of activation state (K20). B, cells expressing wild type (beta 1 wt) or mutant (W775A) integrins were serum-starved in suspension and then mixed with magnetic beads coated with activating TS2/16 antibody for 30 min. Lysates from treated, or control, nontreated cells were subjected to immunoblotting analysis with phosphospecific antibodies against Akt (p-Akt (Ser473)) or focal adhesion kinase (p-FAK(Tyr473)) or with antibodies recognizing total Akt (total-Akt) or Bcl-2 (Bcl-2) proteins present in the lysates. C, cells were serum-starved overnight, trypsinized, and maintained in suspension in medium without serum for an additional 30 min. The cells were then plated on dishes precoated with fibronectin (5 µg/ml) and cultured for the indicated times. Lysates from samples taken before plating (0 h) and at the end of each time point (0.5-10 h) were analyzed by Western blotting with antibodies against phosphorylated Akt (Ser473) and actin. Phosphorylated Akt (p-Akt) concentrations in arbitrary units were calculated as the ratio between densitometry obtained with anti-phospho-Akt antibody and anti-actin antibody.

Integrin ligation and clustering have been linked to Akt activation (17, 20, 73). Our findings raised the possibility that the moderately lower activation state of the W775A integrin mutant might contribute to the observed Akt inhibition and suggested that reactivation with antibodies might compensate for this deficiency. To test this possibility directly, wild type or mutant integrins were clustered with beads coated with activating antibody TS2/16 (Fig. 5B) or activated by plating the cells on fibronectin-coated surfaces (Fig. 5C). Although the clustering and activation of beta 1 integrins induced 2-fold (after plating) to 3-fold (with beads) increases in phospho-Akt over the control in each cell line, the differences in Akt phosphorylation between beta 1 WT and W775A cells, whether treated or untreated, remained the same (Fig. 5, B, compare lines beta 1 WT (+) with W775A (+), and C, compare beta 1 WT with W775A at 0-2 h). A stable increase in the phosphorylation of Akt was observed in beta 1 WT cells 2-10 h after plating, whereas mutant integrin-expressing cells were unable to activate Akt (Fig. 5C, compare beta 1 WT with W775A, 2-10 h). 10 h after plating, W775A-expressing cells showed similar 5-fold reduction in Akt phosphorylation that was observed under steady-state conditions (Fig. 3A). The inability of clustering by activating antibodies to compensate fully for the Akt inhibition in W775A-expressing cells was also evaluated with respect to their effectiveness in the converse process of "outside-in" signaling (74). Bead-bound antibody effectively stimulated FAK autophosphorylation (Fig. 5B, p-FAK (Tyr397)) and increased Bcl-2 levels (Fig. 5B, Bcl-2) similarly in both cell lines; these events have been associated with integrin outside-in stimulation of survival signaling pathways (13, 14, 17). These results indicate that restoration of the active conformation of the extracellular domain of beta 1 integrin is not able to restore Akt activity in cells expressing the mutant W775A integrin.

Tryptophan Mutation Does Not Affect Signaling Pathways Responsible for Akt Activation-- Current models for Akt regulation postulate that Akt phosphorylation depends on the activity of the upstream enzyme PI3K, and inhibitors of this kinase such as wortmannin prevent Akt activation (29, 75, 76). We tested the activity of PI3K in beta 1 WT and W775A integrin-expressing cells by comparing the amounts of phosphatidylinositol 3-phosphate produced in vitro by activated PI3K (Fig. 6, PI3K assay: PI(3)P) or total PI3K (not shown). Activated PI3K was isolated in an immune complex with anti-phosphotyrosine antibody 4G10, and total PI3K was isolated using a mixture of isoform-specific anti-p110 PI3K or anti-p85 PI3K antibodies. In both wild type and W775A integrin cell lines, the activities of PI 3-kinase were similar; both were suppressed by wortmannin (Fig. 6A). This finding suggests that the tryptophan mutation does not interfere with normal function of this kinase, although the downstream Akt from the same lysates used for PI 3-kinase assays showed marked differences in phosphorylation (Fig. 6A, WB: p-Akt (Ser473)). Wortmannin completely suppressed Akt phosphorylation in both cell lines, confirming the role of PI3K as the central upstream regulator of Akt.


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Fig. 6.   The W775A mutation does not interfere with PI 3-kinase-mediated Akt activation or PDK1 activity. A, GD25 cells expressing either wild type (beta 1 wt) or tryptophan mutant (W775A) integrins were cultured overnight on fibronectin in complete medium. The cells were treated with (+) or without (-) 100 nM wortmannin (Wortm.) for 30 min prior to lysis. The lysates were immunoprecipitated with anti-phosphotyrosine antibody 4G10, and PI3K activity in the immune complexes was assayed. Autoradiograph of 32P-labeled phosphatidylinositide 3-phosphate (PI3K assay: PI(3)P) and Western blot analysis of the same lysates with anti-phosphospecific (WB: p-Akt (Ser473)) or total (WB: total-Akt) Akt antibodies is shown. B, GD25 cells expressing either wild type (beta 1 wt) or tryptophan mutant (W775A) integrins were cultured overnight on fibronectin in the absence of serum, and the levels of PDK1 phosphorylation of Ser (p-PDK1(Ser241)) residue were analyzed by Western blotting with phosphospecific antibodies. The total amount of PDK1 (total-PDK1) was used as an internal control for loading.

The protein kinase that phosphorylates Akt on Thr308 is 3-phosphoinositide-dependent protein kinase 1 (PDK1) (30, 77). Although PDK1 is considered constitutively active, recent studies have demonstrated that autophosphorylation at Ser421 is essential for the activity of PDK1 (78) and that its plasma membrane localization is important for activation of Akt (79). PDK1 in both beta 1 WT and W775A integrin-expressing cell lines showed comparable levels of Ser421 phosphorylation (Fig. 6B) and membrane localization (not shown), suggesting that the W775A integrin mutation did not affect PDK1 function.

The activation of Akt is mediated by the PI3K products phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate. Akt binds via its pleckstrin homology domain to one or both of these phosphoinositides at the plasma membrane, which alters Akt conformation and renders it accessible to phosphorylation (75, 77). The availability of these phosphoinositides depends not only on PI3K activity but also on their rate of degradation by lipid phosphatases like phosphatase and tensin homologue deleted on chromosome 10 (PTEN) (80). To evaluate the balance between the production and degradation of these lipid mediators, we quantified the phosphoinositides of 32P-radiolabeled GD25 beta 1 WT and GD25 W775A cells. HPLC analyses of the deacylated lipid products revealed that both cell lines contained similar amounts of phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and phosphatidylinositol trisphosphate (Table II), suggesting that the primary Akt-activating pathway is not affected by this integrin mutation and thus that the low phosphorylation level of Akt in W775A integrin-expressing cells is related to a mechanism other than activation.


                              
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Table II
Phosphoinositide content of cells expressing wild-type (beta 1 WT) or mutant (W775A) integrins
Results are expressed as percentages of the total radioactivity of the phospholipid fraction attributed to each phosphoinositide species. PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol trisphosphate.

The Tryptophan Mutation Causes Local Activation of PP2A in Integrin Complexes-- The level of Akt phosphorylation at steady state is the result of an equilibrium between specific kinase and phosphatase activities that act on the Thr308 and Ser473 residues of Akt (38). To examine Akt-specific phosphatase activity, cells were stimulated with PDGF to phosphorylate and activate Akt, and samples were taken every 15 min to assess the subsequent decreases in phosphorylation of Akt. After the initial PDGF stimulation, cells were maintained in the presence of AG1433, a PDGF receptor kinase inhibitor, in order to eliminate residual receptor kinase activity and to be able to identify just the rate of dephosphorylation. Immediately after stimulation, both cell lines showed strong stimulation of Akt phosphorylation, confirming that the inhibition in W775A cells is also fully reversible by a growth factor (Fig. 7A, PDGF). Nevertheless, whereas cells expressing the wild type beta 1 integrin maintained high levels of Akt Ser473 phosphorylation, W775A cells showed a rapid decline of the signal, and by 75 min after the stimulation, it was barely detectable (Fig. 7A, W775A, 75', No treatment). Similar results were obtained even without the inhibition of residual PDGF activity with AG1433 (data not shown). These dynamics suggested that the expression of the mutant integrin could be associated with increased PP2A activity, which has been characterized as a key phosphatase that can dephosphorylate both Thr308 and Ser407 and thereby inactivate Akt (33, 37, 39, 40). In fact, treatment with okadaic acid, a specific PP2A inhibitor, completely blocked the dephosphorylation of Akt Ser473 (Fig. 7A, OA) and Akt Thr308 (not shown) in W775A cells, supporting the role of this phosphatase in the observed effects on Akt.


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Fig. 7.   Akt dephosphorylation in cells expressing W775A integrin is related to increased integrin-associated PP2A activity. A, GD25 cells expressing either wild type (beta 1 wt) or tryptophan mutant (W775A) integrins were cultured overnight on fibronectin in the absence of serum. Cells were then stimulated with 20 ng/ml PDGF-BB for 15 min, the growth factor was washed away, and the cells were maintained in medium without serum supplemented with 30 µM PDGF kinase inhibitor AG1433. Samples were taken immediately after stimulation (PDGF, down-arrow ) and at the indicated time points. The experiment was performed in the absence (No treatment) or presence of 1 µM okadaic acid (OA). Lysates from the samples were analyzed by Western blotting with anti-phospho-Ser473 Akt (WB: p-Akt (Ser473)) or anti-actin (WB: actin) antibodies. Actin was used as an internal control for loading. B, cells were cultured overnight on fibronectin in the absence of serum and lysed, and then PP2A was immunoprecipitated with monoclonal antibody clone 1D6. The immunoprecipitates were assayed for phosphatase activity in the absence (No treatment, black-square) or presence of 5 nM okadaic acid (OA, ) against a phospho-Ser473 Akt peptide (residues 467-477) using a malachite green Ser/Thr phosphatase assay kit. After the phosphatase assay, the actual amount of the PP2A in each immunoprecipitate was determined by Western blotting with antibodies against PP2A. PP2A activity is presented in arbitrary units (a.u.) calculated as the ratio between released free phosphate (absorbance at 650 nm)/min and the densitometry signal for PP2A from the Western blotting.

Activation of total cellular PP2A by the alpha 2beta 1 integrin after plating of primary fibroblasts into collagen gels has been described recently (33). However, unlike in that study, we observed no general increase in serine/threonine phosphatase activity in cellular lysates (not shown) nor differences in the activation of the total PP2A. To assay total PP2A activity, we immunoprecipitated PP2A with mouse monoclonal antibody 1D6, which does not interfere with its phosphatase activity. The immunoprecipitates were tested for activity against a general threonine phosphopeptide (KRpTIRR, where pT represents phosphothreonine) (not shown) and against a specific serine phosphopeptide derived from the sequence of Akt (KHFPQFpS473YSAS, where pS represents phosphoserine) (Fig. 7B). In both cases, the PP2A activity in beta 1 WT and W775A integrin-expressing cells was similar and was inhibited by more than 90% after treatment with 5 nM okadaic acid. These results indicate that the observed differences in putative PP2A-associated Akt dephosphorylation in W775A cells cannot be explained by changes in total PP2A activity. They instead suggest that a particular localized subpopulation of this enzyme might be responsible for the Akt inactivation.

We hypothesized that a specific subpopulation of PP2A associated with beta 1 integrins was responsible for the Akt inhibition. We first tested whether this phosphatase is present in the protein complexes organized by the integrin cytoplasmic tail. PP2A was readily detected in both wild type and mutant integrin complexes isolated with antibody-coated beads (Fig. 8A, WB: PP2A) or by immunoprecipitation (not shown). Importantly, we also found that Akt is present in the same complexes (Fig. 8A, WB: total-Akt), suggesting that Akt dephosphorylation may take place within the integrin complexes. The amount of PP2A in the complexes formed by the mutant integrin was slightly increased, but this apparent increase was difficult to reproduce consistently, and the minor increase in quantities alone could not explain the large differences in Akt phosphorylation, unless there was also altered activity of the phosphatase recruited into the complex.


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Fig. 8.   The phosphatase activity of PP2A associated with W775A integrin mutant is increased. A, cells expressing wild type (beta 1 wt) or mutant (W775A) integrins were serum-starved in suspension and incubated with magnetic beads coated with anti-beta 1 integrin antibody K20 for 30 min. The isolated integrin complexes (see "Experimental Procedures") were tested by Western blotting with the indicated antibodies. B, the integrin complexes in A were tested for phosphatase activity against the phospho-Ser473 Akt peptide (residues 467-477) using a malachite green Ser/Thr phosphatase assay kit either without inhibitors (No treatment, black-square) or in the presence of 0.5 µM inhibitor-2 (I-2, ) or 5 nM okadaic acid (OA, ). PP2A activity is presented in arbitrary units (a.u.) calculated as in Fig. 6B. Note the increase in PP2A activity present in W775A-integrin complexes when compared with the beta 1 WT complexes (p < 0.001) in the presence or absence of inhibitor-2.

To test the activity of the integrin-associated PP2A fraction, integrin complexes were isolated from beta 1 WT and W775A cells and tested in vitro for phosphatase activity against the Akt-derived phosphopeptide that includes Ser473 (Fig. 8B). The phosphatase activity in the mutant integrin complexes was 2.6 times higher than the activity in the complexes organized by the wild type beta 1 integrin (Fig. 8B, black bars, p < 0.001). This phosphatase activity was most probably due to PP2A, because the addition of 5 nM okadaic acid, a specific PP2A inhibitor at this concentration, inhibited the activity by more than 65% (66.5% for beta 1 WT and 79% for W775A complexes) (Fig. 8B, open bars). Another phosphatase that can be inhibited by okadaic acid, but only at a 100-fold higher concentration than PP2A (IC50 = 0.1 nM), is protein phosphatase 1 (IC50 = 10 nM). Because of the composite nature of the integrin complexes, we tested for the presence of contaminating protein phosphatase 1 activity. When the reactions were carried out in the presence 0.5 µM inhibitor-2, a specific inhibitor of protein phosphatase 1 with an IC50 of 2 nM, the phosphatase activity was reduced by only 5% for beta 1 WT and 8% for W775A complexes, and a similar 2.5-fold higher activity was still measured in the mutant integrin complexes (Fig. 8B, gray bars, p < 0.001). Taken together, our results suggest that the protein complexes organized by beta 1 integrin contain both a subpopulation of PP2A and its substrate Akt and that the tryptophan mutation in the integrin tail leads to a localized increase in activity of PP2A that inactivates Akt.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we tested the hypothesis that signaling selectivity exists at specific sites along integrin cytoplasmic tails. If such a site-specific function were to exist, interference with a particular site should selectively inhibit only a single signal transduction pathway. We focused on the serine/threonine kinase Akt, since it is a primary regulator of cell survival, and the mechanisms by which integrins regulate its activity are not clearly established. In support of our hypothesis, we have identified a key tryptophan residue at position 775 of the beta 1 integrin cytoplasmic domain as a specific site regulating Akt activation. Our results identify a novel mechanism employed by integrins that controls the level of Akt phosphorylation via local activity of the phosphatase PP2A.

To search for a specific signaling site, amino acid residues that are conserved between the different human beta  integrin cytoplasmic domains were identified, and their potential roles in signaling were tested by single-residue substitutions using alanine-scanning mutagenesis. Each full-length human beta 1 integrin mutant was expressed in beta 1-null GD25 cells (58). Among these beta 1 integrin mutants, only the W775A mutant demonstrated defective Akt signaling. To examine the validity of our "signaling selectivity" hypothesis, we searched for effects of this mutation on other integrin-mediated signaling pathways. Previously, the effect of mutating this residue was studied in the context of a double mutation within an interleukin-2 receptor beta 1 tail chimera in a beta 1 WT background (81). Since no specific signaling function for this residue has been identified to date, we explored the mechanism by which its mutation suppresses Akt activation.

The activation of Akt is achieved by phosphorylation at two sites, Thr308 (by PDK1) and Ser473 (by an unresolved kinase), and it is dependent on the availability of phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by PI 3-kinase (31, 75, 79). Integrins can function as upstream regulators of PI3K, thus activating Akt and cell survival (18, 19, 32). Since the W775A integrin mutant inhibited Akt phosphorylation at both residues and increased apoptosis 7-fold, we tested for the activity of PI3K and PDK1 and the levels of Akt-activating phosphoinositides present in cells with this mutant. Unexpectedly, neither the kinase activities nor relevant phosphoinositide levels differed when cells expressing wild type and mutant integrins were compared, implying that the PI3K-dependent Akt-activating pathway was not affected by the integrin mutation.

Focal adhesion kinase has been identified as an upstream intermediate in integrin survival signaling. FAK forms a complex with p130cas that activates the JNK survival pathway (13) and is capable of suppressing the p53-regulated cell death pathway (14). FAK- and Shc-dependent increases in Bcl-2 protein expression in response to integrin engagement that protect Chinese hamster ovary cells from apoptosis have also been described (17). However, the effects of the beta 1 integrin Trp775 mutation that we describe on cell survival involve a different mechanism. Analysis of FAK activation showed similar levels of phosphorylation of Tyr397 in cells expressing wild type or tryptophan mutant integrin. This major autophosphorylation is induced by integrin ligation, and this site binds Src (82). In turn, Src further activates FAK by phosphorylating it at additional sites, including Tyr861 (83), which was similarly induced in beta 1 WT and W775A integrin-expressing cells (not shown). The latter result suggested that Src is equally active in both cell lines. Indeed, comparable levels were found for phosphorylation of the activation site within the catalytic loop (Tyr418) (84) and the inhibitory site (Tyr529) (85) in the carboxyl-terminal portion of the kinase. We were also unable to find changes in JNK, p38, or Shc phosphorylation or in Bcl-2 protein levels, indicating that major kinases activated by integrins and at least several of their downstream effectors operate similarly in both cell lines and are not affected by the tryptophan mutation.

The ability of MAPK (extracellular signal-regulated kinase) to protect against cell death has also been documented (15, 16). Integrins can activate the MAPK pathway through multiple mechanisms (see Refs. 11, 12, 86), and interference with this activation may influence cell survival. Regardless of whether the tryptophan was mutated, integrins were able to elicit similar MAPK responses immediately after plating of the cells or after prolonged incubation on fibronectin. Moreover, blocking MAPK activity with the mitogen-activated extracellular signal-regulated kinase-activating kinase 1/2 inhibitors PD 98059 and U0126 did not inhibit Akt phosphorylation in GD beta 1 WT cells (not shown), suggesting that MAPK is not a mediator of Akt-regulated survival in this cell type. In summary, consistent with our hypothesis, the W775A mutation specifically affects Akt-mediated cell survival and does not interfere with other integrin-mediated signaling pathways.

Our results underscore the importance of inhibition rather than defective activation of Akt. Indeed, we found that local activation of protein phosphatase 2A accounted for the W775A integrin effect on Akt. GD25 cells stably expressing the tryptophan integrin mutation dephosphorylated Akt much faster than cells expressing wild type integrin, and this process was blocked by the specific PP2A inhibitor okadaic acid. alpha 2beta 1 integrin-induced activation of PP2A that leads to dephosphorylation of Akt and is dependent on the alpha 2 cytoplasmic domain was recently reported (33). In contrast to that study, our results point to the importance of local activation of PP2A confined within complexes organized by the mutant integrin, whereas total cellular PP2A activity was not affected by the W775A mutation. This finding, together with the fact that GD25 cells do not express alpha 2beta 1 after reconstitution of beta 1 integrin (87), suggests that the Trp775 mutation reveals a new relationship between integrins and PP2A. This phosphatase was present in both beta 1 WT and W775A integrin complexes together with its substrate Akt, but the activity of PP2A associated with the mutant integrin was 2.5 times higher. An attractive mechanism can be envisaged in which the subpopulation of PP2A associated with integrin cytoplasmic domains controls Akt phosphorylation locally in integrin molecular complexes. It is tempting to speculate that "correct" extracellular matrix interactions retain organization of normal beta 1 integrin cytoplasmic complexes in which PP2A activity remains repressed, resulting in continued Akt activation. Inappropriate matrix conditions or mutation of the tryptophan at position 775 would interfere with this process by releasing the inhibition of PP2A in integrin complexes, leading directly to altered cell survival decisions.

Trp775 is a well conserved residue present in five beta  integrin subunits (beta 1, beta 2, beta 3, beta 6, and beta 7), and to our knowledge it has not been the subject of single-residue mutational studies. It remains to be determined whether the effect on Akt activation that we describe is valid for each of the other beta  integrins or is restricted to the beta 1 integrin. The Trp775 residue resides in an integrin sequence that is implicated in alpha -actinin (66) and talin binding (88-90). Interactions of the beta  subunit with the PTB-like domain of talin have been shown to be important for integrin activation (63, 64). Alanine substitution of the Trp775 residue did not interfere with talin binding and focal adhesion localization, which has been shown to depend primarily on the integrity of the NPXY motif (56, 57, 63, 64), but it reduced the activation state of the W775A integrin by 40%, suggesting that it may affect the mechanism of inside-out integrin activation. Moreover, we found that although activation of the mutant integrin with activating antibodies promoted FAK autophosphorylation and Bcl-2 expression (similar to the wild type), it did not compensate for the inhibition of Akt. These findings imply again that local interactions involving Trp775 are more important for this specific signaling than restoration of the active conformation of the entire cytoplasmic domain. Double mutation of the tyrosines in the cytoplasmic domain of the integrin beta 1 subunit (Tyr783 and Tyr795) to phenylalanines has also been shown to affect FAK autophosphorylation but not its focal contact localization or p130cas phosphorylation (46). Although effects on other signaling pathways have not been explored, it is possible that this double mutation could represent another site of specific signaling on the beta  integrin cytoplasmic domain. These results, together with our finding that Trp775 is intimately and selectively involved in the control of Akt activation, support the concept of specificity of signaling along the integrin cytoplasmic domain. They open the exciting possibility of identifying more such distinct sites.

    ACKNOWLEDGEMENTS

We thank Dr. Susan E. LaFlamme for reagents and discussions in the initial stages of this study, Dr. Reinhard Fässler for providing GD25 beta 1 integrin-null cells, Dr. Sue Goo Rhee for valuable discussions, and Dr. Cristina Murga for assistance with the PI3K assay.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: CDBRB, NIDCR, NIH, Bldg. 30, Rm. 421, 30 Convent Dr. MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-4041; Fax: 301-402-0897; E-mail: roumen. pankov{at}nih.gov.

Present address: Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA 19111.

Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M300879200

    ABBREVIATIONS

The abbreviations used are: FAK, focal adhesion kinase; PKB, protein kinase B; PDK1, 3-phosphoinositide-dependent protein kinase 1; PP2A, protein phosphatase 2A; PDGF, platelet-derived growth factor; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; PI3K, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; PIPES, 1,4-piperazinediethanesulfonic acid; FACS, fluorescence-activated cell sorting; HPLC, high pressure liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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