Phosphoinositide 3-OH Kinase Activates the β2Integrin Adhesion Pathway and Induces Membrane Recruitment of Cytohesin-1
1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês
10.1074/jbc.273.24.14853
ISSN1083-351X
AutoresWolfgang Nagel, Lutz Zeitlmann, Pierre Schilcher, Christiane Geiger, Johanna Kolanus, Waldemar Kolanus,
Tópico(s)Cellular Mechanics and Interactions
ResumoSignal transduction through phosphoinositide 3-OH kinase (PI 3-kinase) has been implicated in the regulation of lymphocyte adhesion mediated by integrin receptors. Cellular phosphorylation products of PI 3-kinases interact with a subset of pleckstrin homology (PH) domains, a module that has been shown to recruit proteins to cellular membranes. We have recently identified cytohesin-1, a cytoplasmic regulator of β2 integrin adhesion to intercellular adhesion molecule 1. We describe here that expression of a constitutively active PI 3-kinase is sufficient for the activation of Jurkat cell adhesion to intercellular adhesion molecule 1, and for enhanced membrane association of cytohesin-1. Up-regulation of cell adhesion by PI 3-kinase and membrane association of endogenous cytohesin-1 is abrogated by overexpression of the isolated cytohesin-1 PH domain, but not by a mutant of the PH domain which fails to associate with the plasma membrane. The PH domain of Bruton's tyrosine kinase (Btk), although strongly associated with the plasma membrane, had no effect on either membrane recruitment of cytohesin-1 or on induction of adhesion by PI 3-kinase. Having delineated the critical steps of the β2 integrin activation pathway by biochemical and functional analyses, we conclude that PI 3-kinase activates inside-out signaling of β2 integrins at least partially through cytohesin-1. Signal transduction through phosphoinositide 3-OH kinase (PI 3-kinase) has been implicated in the regulation of lymphocyte adhesion mediated by integrin receptors. Cellular phosphorylation products of PI 3-kinases interact with a subset of pleckstrin homology (PH) domains, a module that has been shown to recruit proteins to cellular membranes. We have recently identified cytohesin-1, a cytoplasmic regulator of β2 integrin adhesion to intercellular adhesion molecule 1. We describe here that expression of a constitutively active PI 3-kinase is sufficient for the activation of Jurkat cell adhesion to intercellular adhesion molecule 1, and for enhanced membrane association of cytohesin-1. Up-regulation of cell adhesion by PI 3-kinase and membrane association of endogenous cytohesin-1 is abrogated by overexpression of the isolated cytohesin-1 PH domain, but not by a mutant of the PH domain which fails to associate with the plasma membrane. The PH domain of Bruton's tyrosine kinase (Btk), although strongly associated with the plasma membrane, had no effect on either membrane recruitment of cytohesin-1 or on induction of adhesion by PI 3-kinase. Having delineated the critical steps of the β2 integrin activation pathway by biochemical and functional analyses, we conclude that PI 3-kinase activates inside-out signaling of β2 integrins at least partially through cytohesin-1. Integrins are a diverse family of heterodimeric transmembrane adhesion receptors that are present on most vertebrate cell types. They are known to play important roles either in development, or in somatic functions such as wound healing, and the regulation of complex cell-cell or cell-matrix interactions within the immune system (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar, 2Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6414) Google Scholar, 3Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2948) Google Scholar). The avidity of integrins for their ligands is dependent on the activation state of the cell on which they are expressed (4Diamond M.S. Springer T.A. Curr. Biol. 1994; 4: 506-517Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). 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Overexpression of cytohesin-1 or subdomain constructs in the Jurkat T cell line was shown to have pronounced in vitro effects on the binding of αLβ2 to its ligand, the intercellular adhesion molecule 1 (ICAM-1). Whereas the overexpression of full-length cytohesin-1 resulted in a constitutive adhesion of αLβ2, expression of the PH domain construct specifically inhibited the activation of LFA-1 in a dominant negative fashion. Since the PH domain was not found to be mediating the interaction with the integrin cytoplasmic domain, it has been postulated that its unidentified cellular ligand may be an upstream component of the inside-out signaling pathway of αLβ2. The finding that the overexpressed, isolated Sec7 domain acted only as a partial agonist pointed in the same direction (29Kolanus W. Nagel W. Schiller B. Zeitlmann L. Godar S. Stockinger H. Seed B. Cell. 1996; 86: 233-242Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). PH domains are structural modules present in more than 100 proteins that play known or postulated roles in signal transduction. It is a commonly found thread that PH domains may aid in membrane recruitment of proteins through their interactions with phosphorylated ligands present at the inner leaflet of cellular membranes (30Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 31Pawson T. Nature. 1995; 373: 573-579Crossref PubMed Scopus (2234) Google Scholar, 32Lemmon M.A. Falasca M. Ferguson K.M. Schlessinger J. Trends Cell Biol. 1997; 7: 237-242Abstract Full Text PDF PubMed Scopus (148) Google Scholar). Although a subgroup of PH domains is capable of interacting with tyrosine-phosphorylated proteins (30Lemmon M.A. Ferguson K.M. Schlessinger J. 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Interestingly, certain PH domains showin vitro binding preference to lipid compounds, which arein vivo phosphorylation products of PI 3-kinase. The PH domains of Akt, Btk, and of GRP-1, a close homolog of cytohesin-1, have been demonstrated to belong to the latter group because they bind either phosphatidylinositol (3,4)-bisphosphate (Akt), or phosphatidylinositol (3,4,5)-trisphosphate (PIP3) with high affinity (38Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1254) Google Scholar, 39Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C. Driscoll P.C. Waterfield M.D. Panayotou G.R.A. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (495) Google Scholar, 40Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (372) Google Scholar, 41Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.-S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). One of the important topics that remain to be addressed is the characterization of the lymphocyte inside-out signaling pathway proximal to the integrin cytoplasmic domains. In this study, we show for the first time that a constitutively active version of PI 3-kinase suffices to activate the αLβ2 adhesion pathway in a T cell line. Functional, biochemical, and cell biological evidence is provided, which suggests that cytohesin-1 is located downstream of PI 3-kinase and that it is regulated by the recruitment of its PH domain to the plasma membrane. Cytoplasmic immunoglobulin fusion proteins have been described previously (29Kolanus W. Nagel W. Schiller B. Zeitlmann L. Godar S. Stockinger H. Seed B. Cell. 1996; 86: 233-242Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). A cDNA fragment for the PH domain of murine Btk (corresponding to amino acids 8–170) has been subcloned into pcIgTkg vaccinia expression vector (29Kolanus W. Nagel W. Schiller B. Zeitlmann L. Godar S. Stockinger H. Seed B. Cell. 1996; 86: 233-242Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). The cDNAs for Myc-P110* and Myc-P110*Δ(917–950) were subcloned into the pTkg vaccinia expression vector (70Kolanus W. Romeo C. Seed B. Cell. 1993; 74: 171-183Abstract Full Text PDF PubMed Scopus (305) Google Scholar). Jurkat E6 cells were infected with recombinant vaccinia viruses as described (29Kolanus W. Nagel W. Schiller B. Zeitlmann L. Godar S. Stockinger H. Seed B. Cell. 1996; 86: 233-242Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). 6 h after infection, cells were labeled with 12 μg/ml bisbenzimide H33342 fluorochrome trihydrochloride (Calbiochem) for 30 min at 37 °C, collected by centrifugation, resuspended in Hanks' buffered saline solution (HBSS), and delivered to 96-well plates (Nunc, Maxisorp) at 1.5 × 105/well. Prior to adhesion, plates were coated with goat anti-human IgG (Fcγ-specific) antibody at 0.85 μg/well for 90 min at 25 °C, blocked with 1% (w/v) bovine serum albumin in PBS, incubated with culture supernatants from COS cells expressing ICAM-1-Rg fusion protein, and subsequently used in the assay. Where indicated in the figures, cells were incubated with 100 nmwortmannin (Sigma) 0.5 h prior to the adhesion assay. Cells were then allowed to adhere for 1 h at 37 °C, and unbound cells were carefully washed off with 3 × 300 μl of HBSS. Bound cells were assayed in 100 μl of 2% (v/v) formaldehyde in PBS using a fluorescence plate reader (Cytofluor II, PerSeptive). The signal of 1.5 × 105 cells/well at 490 nmcorresponds to 100% adhesion. Each value is the mean of two determinations carried out in triplicate. Displacement assay for [3H]IP4 binding activity was carried out according to Ref. 71Yagisawa H. Hirata M. Kanematsu T. Watanabe Y. Ozaki S. Sakuma K. Tanaka H. Yabuta N. Kamata H. Hirata H. Nojima H. J. Biol. Chem. 1994; 269: 20179-20188Abstract Full Text PDF PubMed Google Scholar with modifications; 5 μm of purified, polyhistidine-tagged PH domains derived from expression inEscherichia coli BL21 (29Kolanus W. Nagel W. Schiller B. Zeitlmann L. Godar S. Stockinger H. Seed B. Cell. 1996; 86: 233-242Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar) were rebound to 10 μl of nickel-agarose at 20 nm imidazole in MBSD (50 mm Mops, pH 6.8, 100 mm NaCl, 0.1% Igepal CA-630) in a final volume of 40 μl. After incubation with 10 nm [3H]IP4 for 10 min on ice, increasing amounts of unlabeled inositol phosphate were added for 15 min. Then samples were transferred to ultraspin microfilters (M r cut-off: 105) and washed with 50 μl of MBSD by centrifugation at 1500 × g . Protein-bound radioactivity was eluted by MBSD, containing 200 mm imidazole. Samples were collected by centrifugation and counted by solid phase scintillation. Values were corrected for nonspecific binding of 10 nm[3H]IP4 to nickel-agarose. Each value represents the mean of two determinations carried out in duplicate. Purified polyhistidine-tagged full-length cytohesin-1 derived from expression in E. coli BL21 at 10 μm was mixed with 10 nm[3H]IP4 and [3H]d-Ins(1,4,5)P3, respectively, and with 20 μm of the appropriate unlabeled isomer in MBSD. The mixture was applied to an Amersham Pharmacia Biotech Fast Desalting Column HR. The elution position of the protein was monitored at 215 nm with the SMART system (Amersham Pharmacia Biotech). Scintillation counting was used to detect the elution position of the radioactively labeled inositol phosphates. Binding specificity was assessed with a mixture of 10 nm[3H]IP4 and purified GST. Cells which had been infected with recombinant vaccinia viruses or uninfected cells were collected by centrifugation and resuspended on ice in 0.5 ml of ice-cold hypotonic solution (HS: 10 mm Hepes, pH 7.5, 10 mm KCl, 10 mm MgCl2, 0.5 mm dithiothreitol) containing 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride. Then cells were sheared, the nuclei were removed, and the supernatant cytosol was collected as described (72Meller N. Liu Y.C. Collins T.L. Bonnefoyberard N. Baier G. Isakov N. Altman A.R.A. Mol. Cell. Biol. 1996; 16: 5782-5791Crossref PubMed Google Scholar). The cytosolic fraction was brought to a final concentration of 1% (v/v) Igepal CA-630 and 150 mm NaCl and used directly for immunoprecipitation. The pellet was resuspended, washed with HS, and centrifuged at 15,000 × g for 15 min. The resulting pellet was resuspended in HS containing 1% (v/v) Igepal CA-630 and 150 mm NaCl, centrifuged, and the supernatant representing the particulate fraction was subjected to immunoprecipitation at Protein A-Sepharose employing an antigen-purified antibody directed against cytohesin-1 (29Kolanus W. Nagel W. Schiller B. Zeitlmann L. Godar S. Stockinger H. Seed B. Cell. 1996; 86: 233-242Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). Immunoprecipitates were analyzed by standard Western blot techniques. To assess that cytoplasmic contents were not trapped in the particulate fraction, lactate dehydrogenase activities were monitored as described (59Ma A.D. Brass L.F. Abrams C.S. J. Cell Biol. 1997; 136: 1071-1079Crossref PubMed Scopus (65) Google Scholar). Six hours after infection of Jurkat E6 cells with recombinant vaccinia viruses, cells were placed on poly-l-lysine-covered microscope slides for 1 h in a humidified chamber at 37 °C. Non-adherent cells were then washed off with HBSS, and adherent cells were fixed and immobilized with freshly prepared 2% (w/v) paraformaldehyde in PBS overnight at 4 °C. Subsequently, cells were permeabilized for 15 min with 0.2% (v/v) Triton X-100 in PBS, blocked with 2% (w/v) glycine in PBS, and incubated with a fluorescein isothiocyanate-labeled goat anti-human IgG (Fcγ-specific) antibody (Dianova) in PBS for 2 h at room temperature. In double labeling experiments, tetramethylrhodamine B isothiocyanate-coupled phalloidin (Sigma) at 1 μg/ml was included. After the final wash with PBS, slides were mounted on a 9:1 mixture of glycerol and 100 mm Tris/HCl, pH 9.0, containingn -propyl-gallate at 20 mg/ml as antifading reagent. Then samples were either examined on a Zeiss Axiophot microscope using a Zeiss Neofluar 40 × 1.3 oil immersion objective, or on a confocal laser scanning apparatus (Leica TCS-NT system, Leica) attached to a Leica DM IRB inverted microscope with a PLAPO 63 × 1.32 oil immersion objective. Conventional immunofluorescence images were recorded on Kodak T-MAX 400ASA film. Confocal images were collected as 512 × 512 pixel files and processed with the help of the Photoshop program (Adobe). Six hours after infection of Jurkat E6 cells with recombinant Myc-P110* and Ig control, cells were lysed in 20 mm Tris/HCl, pH 7.5, 150 mm NaCl, 10 mm KCl, 1 mm MgCl2, 0.5 mm dithiothreitol, 0.1 mm sodium orthovanadate, containing 1% (v/v) Igepal CA-630, protease inhibitors leupeptin and aprotinin at 10 μg/ml, and 1 mm phenylmethylsulfonyl fluoride. After collection of the supernatant by centrifugation at 15,000 × g , the samples were subjected to immunoprecipitation with mouse anti-c-Myc antibody (monoclonal antibody 9E10) for 12 h at 4 °C, followed by Protein A-Sepharose 6 MB (Amersham Pharmacia Biotech) for 1 h at room temperature. For detection of p110* -expression, precipitates were washed three times with lysis buffer and subjected to immunoblot analysis, using monoclonal antibody 9E10 as first antibody. The assay for immunoprecipitated PI 3-kinase activity was essentially carried out according to Ref. 73Auger K.R. Songyang Z. Lo S.H. Roberts T.M. Chen L.B. J. Biol. Chem. 1996; 271: 23452-23457Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar with modifications. Following washing of the precipitates with lysis buffer, subsequent washes were performed with 10 mm Tris/HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA three times and with kinase buffer containing 50 mm Hepes, pH 7.6, 5 mm MgCl2, 1 mm EDTA twice. Finally, the beads were resuspended in 100 μl of kinase buffer containing 20 μg of sonicated phosphatidylinositol (PI). The reaction was started by adding 1 μCi of [γ-32P]ATP and 100 μm ATP. After incubation of the sample for 20 min at 25 °C with agitation, the reaction was stopped by adding 20 μl of 6 n HCl and the radiolabeled lipid was extracted with 160 μl of chloroform:methanol (1:1) by brief mixing. Thin layer chromatography was run with the chloroform extracts on a Silica Gel 60 plate (Merck) using a solvent system of chloroform:methanol:concentrated NH3:water (60:47:2:11). Radiolabeled phosphatidylinositol was visualized by autoradiography. Large unilamellar liposomes were generated by dialysis according to (74Philippot J.R. Mutaftschiev S. Liautard J.P. Biochim. Biophys. Acta. 1983; 734: 137-143Crossref Scopus (77) Google Scholar). Liposomes contained 60% (w/w) β-palmitoyl-γ-oleoyl-l-α-phosphatidylcholine, 30% (w/w) dioleoyl-l-α-phosphatidyl-dl-glycerol, obtained from Sigma and 10% (w/w) PI(3,4,5)P3 (Mantreya, Inc.). Lipids were dried and resuspended in 10 mm Hepes, pH 7.4, 80 mm KCl, 15 mm NaCl, 0.7 mmNaH2PO4, 1 mm EGTA, containing 50 mm n -octyl-β-d-glucopyranoside. After removal of detergent by dialysis, liposomes were deposited onto an IAsys surface (FCS-0301, not derivatized) at 0.1 mg of lipid/ml of PBS. Subsequently, remaining unspecific binding sites were blocked by GST and binding of either GST-cytohesin-1 or its mutant GST-cytohesin-1 (R281C) was analyzed at 2.5 μg/ml. Cuvette was regenerated by subsequent washing with 1 n NaOH/1% (w/v) SDS, water, 1 n HCl, water, and methanol. Previous reports implicated a role of PI 3-kinase in the regulation of integrin-mediated adhesiveness (see Introduction and references therein). We therefore investigated whether a constitutively active PI 3-kinase can up-regulate lymphocyte adhesion directly. Overexpression of the catalytic subunit of mammalian PI 3-kinase is usually not sufficient for a constitutively active phenotype, but three groups have recently generated chimeric, mutant, or membrane-associated versions of PI 3-kinase, which were shown to result in an activation of cellular signaling pathways of fibroblast-like cells (42Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (517) Google Scholar, 43Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (418) Google Scholar, 44Rodriguez V.P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (501) Google Scholar, 45Reif K. Nobes C.D. Thomas G. Hall A. Cantrell D.A. Curr. Biol. 1996; 6: 1445-1455Abstract Full Text Full Text PDF PubMed Google Scholar). A chimera (p110*) that comprises the catalytic subunit (p110) of murine PI 3-kinase, fused at the amino terminus to a regulatory domain derived from the p85 subunit (42Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (517) Google Scholar), was used in this study (Fig.1 A ). A kinase deficient variant of p110*, bearing a short in-frame deletion (33 amino acids; Δ917–950) within the kinase domain, served as control (Fig.1 A ). Using recombinant vaccinia viruses, p110* or the kinase defective derivative thereof were expressed in Jurkat cells (Fig. 1 E ), and detected with a monoclonal antibody directed against the carboxyl-terminal Myc tag. Lipid kinase activity of the chimeric protein was assayed by its ability to specifically phosphorylate phosphatidylinositol in vitro (Fig. 1 E ). Subsequently the effect of p110* expression on αLβ2-mediated adhesion of Jurkat cells was analyzed. A 5-fold increase of adhesion to ICAM-1 was observed for cells that had been infected with vaccinia viruses expressing p110*, as compared with cells infected with control viruses (Fig. 1 B ). Stimulation of these cells with an anti-T cell receptor antibody, or overexpression of cytohesin-1 by recombinant vaccinia viruses, which had previously been shown to be sufficient for the activation of the αLβ2 integrin in Jurkat cells (29Kolanus W. Nagel W. Schiller B. Zeitlmann L. Godar S. Stockinger H. Seed B. Cell. 1996; 86: 233-242Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar), were used as positive controls in this assay (Fig. 1 B ). Inhibitors of PI 3-kinase activity were then used to investigate the role of PI 3-kinase in Jurkat cell adhesion to ICAM-1 in greater detail (Fig. 1 C ). We found that preincubation of Jurkat E6 cells for 0.5 h with known inhibitors of PI 3-kinase (46Shimizu Y. Hunt S.W. Immunol. Today. 1996; 17: 565-573Abstract Full Text PDF PubMed Scopus (106) Google Scholar), either 50 nm wortmannin or 50 μm LY294002, inhibited OKT3-induced adhesion completely, whereas the responses to PMA or Mn2+ were not affected, the latter result being consistent with a previous report (23Driessens M.H. van Hulten H.P. van Rijthoren E.A. Soede R.D. Roos E. Exp. Cell. Res. 1997; 231: 242-250Crossref PubMed Scopus (14) Google Scholar). These data suggest that PI 3-kinase acts downstream of the T cell antigen receptor in the LFA-1 adhesion pathway. p110* was partially inhibited, probably because the consequences of expression of p110* expression cannot fully be reversed when the indicated concentrations of inhibitors and short incubation times are used. Although prolonged incubation (i.e. several hours, data not shown) with Ly294002 or wortmannin reduced the adhesion levels induced by p110* dramatically, we found that all responses, including adhesion induced by phorbol ester and Mn2+ were strongly affected in this case, probably attributable to some rather nonspecific, global effect. LFA-1-dependent adhesion mediated by overexpression of cytohesin-1 was partially affected, suggesting that this phenotype is at least in part independent of PI 3-kinase, consistent with the idea that cytohesin-1 may be located downstream of PI 3-kinase. Taken together, these findings indicate that a constitutively PI 3-kinase is sufficient for the up-regulation of β2 integrin-mediated cell adhesiveness. How is PI 3-kinase coupled to the regulation of integrin activity? We postulated that the PH domain of cytohesin-1 may provide a link between the activation of cell adhesion mediated by PI 3-kinase and the recruitment of cytohesin-1 to the plasma membrane. Therefore, membrane recruitment of cytohesin-1 in the Jurkat cell line was assessed biochemically by the separation of crude cell extracts. Localization of either overexpressed cytohesin-1 or the isolated PH domain in the cytosolic or the particulate fractions was detected by Western blot analysis. We found that a substantial fraction of both full-length cytohesin-1 and the isolated PH domain was associated with membranes (Fig. 2, A andB ). Is the membrane recruitment mediated by the cytohesin-1 PH domain associated with its cellular function? To answer this question, we generated a point mutant (R281C) of the PH domain, which corresponds to a residue in the PH domain of Btk that had previously been shown to important for PIP3 binding and cellular function (39Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. G
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