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• W2017012788 abstract "Cadherins are transmembrane receptors that mediate cell-cell adhesion. They play an essential role in embryonic development and maintenance of tissue architecture. The Rho family small GTPases regulate actin cytoskeletal dynamics in different cell types. The function of two family members, Rho and Rac, is required for the stability of cadherins at cell-cell contacts. Consistent with the published data we have found that Rac is activated upon induction of intercellular adhesion in epithelial cells. This activation is dependent on functional cadherins (Nakagawa, M., Fukata, M., Yamaga, M., Itoh, N., and Kaibuchi, K. (2001) J. Cell Sci. 114, 1829–1838; Noren, N. K., Niessen, C. M., Gumbiner, B. M., and Burridge, K. (2001) J. Biol. Chem. 276, 3305–3308). Here we show for the first time that clustering of cadherins using antibody-coated beads is sufficient to promote Rac activation. In the presence of Latrunculin B, Rac can be partially activated by antibody-clustered cadherins. These results suggest that actin polymerization is not required for initial Rac activation. Contrary to what has been described before, phosphatidylinositol 3-kinases are not involved in Rac activation following cell-cell adhesion in keratinocytes. Interestingly, inhibition of epidermal growth factor receptor signaling efficiently blocks the increased Rac-GTP levels observed after contact formation. We conclude that cadherin-dependent adhesion can activate Rac via epidermal growth factor receptor signaling. Cadherins are transmembrane receptors that mediate cell-cell adhesion. They play an essential role in embryonic development and maintenance of tissue architecture. The Rho family small GTPases regulate actin cytoskeletal dynamics in different cell types. The function of two family members, Rho and Rac, is required for the stability of cadherins at cell-cell contacts. Consistent with the published data we have found that Rac is activated upon induction of intercellular adhesion in epithelial cells. This activation is dependent on functional cadherins (Nakagawa, M., Fukata, M., Yamaga, M., Itoh, N., and Kaibuchi, K. (2001) J. Cell Sci. 114, 1829–1838; Noren, N. K., Niessen, C. M., Gumbiner, B. M., and Burridge, K. (2001) J. Biol. Chem. 276, 3305–3308). Here we show for the first time that clustering of cadherins using antibody-coated beads is sufficient to promote Rac activation. In the presence of Latrunculin B, Rac can be partially activated by antibody-clustered cadherins. These results suggest that actin polymerization is not required for initial Rac activation. Contrary to what has been described before, phosphatidylinositol 3-kinases are not involved in Rac activation following cell-cell adhesion in keratinocytes. Interestingly, inhibition of epidermal growth factor receptor signaling efficiently blocks the increased Rac-GTP levels observed after contact formation. We conclude that cadherin-dependent adhesion can activate Rac via epidermal growth factor receptor signaling. lysophosphatidic acid extracellular signal-regulated kinase epidermal growth factor phosphatidylinositol 3-kinase guanine nucleotide exchange factor Ccd42/Rac interactive binding p21-activated kinase GTPase activating protein mitogen-activated protein kinase/ERK kinase bovine serum albumin vascular endothelial Cadherins are transmembrane receptors that mediate calcium-dependent cell-cell adhesion. They play an essential role in embryonic development and maintenance of tissue architecture in adults (1Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2923) Google Scholar). Cadherins interact in an anti-parallel fashion with the same type of receptors on adjacent cells (known as homophilic binding), thereby mediating formation of cell-cell contacts. Intercellular adhesion is strengthened by the clustering of cadherin receptors at junctions and the association between cadherin complexes and the actin cytoskeleton (2Adams C.L. Nelson W.J. Curr. Opin. Cell Biol. 1998; 10: 572-577Crossref PubMed Scopus (237) Google Scholar, 3Yap A.S. Brieher W.M. Gumbiner B.M. Ann. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (686) Google Scholar). Cadherin-dependent adhesion is subject to regulation by cytoplasmic proteins, and recent work has focused on modulation by the Rho family of small GTPases. Members of this family of GTP-binding proteins regulate actin cytoskeletal dynamics and focal complex formation in different cell types (4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar). The function of two family members, Rho and Rac, is required for the stability of cadherins at cell-cell contacts (5Braga V. Exp. Cell Res. 2000; 261: 83-90Crossref PubMed Scopus (107) Google Scholar). The exact mechanisms by which Rho and Rac operate are unclear, but we and others have shown that Rac plays a role in actin recruitment to junctions (6Takaishi K. Sasaki T. Kotani H. Nishioka H. Takai Y. J. Cell Biol. 1997; 139: 1047-1059Crossref PubMed Scopus (474) Google Scholar) and to clustered cadherin receptors (7Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (654) Google Scholar, 8Lambert M. Choquet D. Mege R.-M. J. Cell Biol. 2002; 157: 469-479Crossref PubMed Scopus (109) Google Scholar). Rho and Rac function in signal transduction cascades downstream of a variety of cell surface receptors. For example, Rho mediates stress fiber formation in response to lysophosphatidic acid (LPA)1 stimulation, whereas Rac is required for growth factor-induced membrane ruffling (9Kjoller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Crossref PubMed Scopus (342) Google Scholar). More recently it has been directly demonstrated that ligand binding to cell surface receptors can activate Rho and Rac (10Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1361) Google Scholar, 11Bernard V. Bohl B. Bokoch G. J. Biol. Chem. 1999; 274: 13196-13204Abstract Full Text Full Text PDF Scopus (83) Google Scholar). In addition, the regulation of the activity of Rho, Rac, and Cdc42 by integrin engagement has been determined and shown to be dependent on time, matrix composition, and matrix concentration (12Adams J.C. Schwartz M.A. J. Cell Biol. 2000; 150: 807-822Crossref PubMed Scopus (107) Google Scholar, 13del Pozo M.A. Price L.S. Alderson N.B. Ren X.D. Schwartz M.A. EMBO J. 2000; 19: 2008-2014Crossref PubMed Scopus (405) Google Scholar, 14Cox E.A. Sastry S.K. Huttenlocher A. Mol. Biol. Cell. 2001; 12: 265-277Crossref PubMed Scopus (250) Google Scholar, 15Wenk M.B. Midwood K.S. Schwarzbauer J.E. J. Cell Biol. 2000; 150: 913-919Crossref PubMed Scopus (106) Google Scholar). Lately, it has become apparent that cadherin-mediated adhesion can trigger intracellular signaling events including activation of phosphatidylinositol 3-kinases (PI3-kinases) and the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway (16Kim S.H., Li, Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 17Pece S. Chiariello M. Murga C. Gutkind J.S. J. Biol. Chem. 1999; 274: 19347-19351Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 18Pece S. Gutkind J.S. J. Biol. Chem. 2000; 275: 41227-41233Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). In addition, the epidermal growth factor (EGF) receptor has been coimmunoprecipitated with the cadherin adhesion complex from epithelial cells and is activated upon cell-cell contact formation (18Pece S. Gutkind J.S. J. Biol. Chem. 2000; 275: 41227-41233Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 19Hoschuetzky H. Aberle H. Kemler R. J. Cell Biol. 1994; 127: 1375-1380Crossref PubMed Scopus (672) Google Scholar). Furthermore, recent work has demonstrated that cell-cell contact formation can activate Rac in Madine-Darby canine kidney and Chinese hamster ovary cells transfected with C-cadherin (20Nakagawa M. Fukata M. Yamaga M. Itoh N. Kaibuchi K. J. Cell Sci. 2001; 114: 1829-1838Crossref PubMed Google Scholar,21Noren N.K. Niessen C.M. Gumbiner B.M. Burridge K. J. Biol. Chem. 2001; 276: 3305-3308Abstract Full Text Full Text PDF Scopus (355) Google Scholar). Spreading of E-cadherin-expressing Chinese hamster ovary cells on immobilized E-cadherin ectodomain also induces Rac activation (22Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). In addition, levels of GTP-bound Rac are higher in VE-cadherin-expressing endothelial cells than in VE-cadherin-null cells (23Lampugnani M.G. Zanetti A. Breviario F. Balconi G. Orsenigo F. Corada M. Spagnuolo R. Betson M. Braga V. Dejana E. Mol. Biol. Cell. 2002; 13: 1174-1189Crossref Scopus (204) Google Scholar). Although these studies showed that Rac activation is dependent on cadherin function, they did not establish the functional significance of Rac activation upon cell-cell contact formation, nor did they address the question of whether cadherin clustering is sufficient to enhance GTP loading on Rac. Although two studies found that PI3-kinase activity was required for full Rac activation, inhibition of PI3-kinase function did not reduce Rac activity to basal levels (20Nakagawa M. Fukata M. Yamaga M. Itoh N. Kaibuchi K. J. Cell Sci. 2001; 114: 1829-1838Crossref PubMed Google Scholar, 22Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), suggesting that other signaling pathways may be involved in Rac activation downstream of cadherin-mediated adhesion. In light of these results, we were interested to determine whether cadherin-mediated adhesion could also activate Rac in keratinocytes. Normal keratinocytes provide the ideal system to look at Rac activation on induction of cell-cell adhesion. They can be grown to confluence in the absence of cadherin-dependent contacts (in low calcium medium), and then cell-cell adhesion can be induced by addition of calcium ions (known as the calcium switch) (24Hodivala K.J. Watt F.M. J. Cell Biol. 1994; 124: 589-600Crossref PubMed Scopus (205) Google Scholar). Because cells are confluent and touching each other, they do not need to migrate to form junctions, and contact formation occurs rapidly. Using this system, we investigated the requirements for Rac activation following cell-cell contact formation in terms of (a) clustering of cadherin receptors, (b) actin polymerization, and (c) PI3-kinase and EGF receptor signaling. Our results shed light on the mechanisms via which cadherin receptors are functionally coupled to Rac activation. Normal human keratinocytes (strains Kb and Sa, passages 3–7) were cultured as described previously (25Braga V.M. Betson M., Li, X. Lamarche-Vane N. Mol. Biol. Cell. 2000; 11: 3703-3721Crossref PubMed Scopus (131) Google Scholar). Cells were grown to confluence in low calcium medium (25Braga V.M. Betson M., Li, X. Lamarche-Vane N. Mol. Biol. Cell. 2000; 11: 3703-3721Crossref PubMed Scopus (131) Google Scholar). To induce intercellular adhesion calcium chloride was added to 2 mm. Microinjection experiments and recombinant protein production were performed as described previously (7Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (654) Google Scholar). Immunofluorescence was performed as described previously (7Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (654) Google Scholar). Primary monoclonal antibodies used were: anti-E-cadherin (HECD-1, mouse and ECCD-2, rat) (26Hirai Y. Nose A. Kobayashi S. Takeichi M. Development. 1989; 105: 271-277PubMed Google Scholar); anti-P-cadherin (NCC-CAD-299, mouse) (27Shimoyama Y. Yoshida T. Terada M. Shimosato Y. Abe O. Hirohashi S. J. Cell Biol. 1989; 109: 1787-1794Crossref PubMed Scopus (101) Google Scholar); anti-α3β1-integrin (VM-2, mouse) (28Kaufmann R. Frosch D. Westphal C. Weber L. Klein C.E. J. Cell Biol. 1989; 109: 1807-1815Crossref PubMed Scopus (109) Google Scholar); anti-Rac (23A8, mouse); anti-activated EGF receptor from Transduction Laboratories (clone 74, mouse); and anti-diphosphorylated ERK1/2 from Sigma (mouse). Rabbit polyclonal antibodies used were: anti-α-catenin (VB1) (29Braga V.M.M. Hodivala K.J. Watt F.M. Cell Adhes. Commun. 1995; 3: 201-215Crossref PubMed Scopus (58) Google Scholar); anti-phosphoSer473-Akt (PW66); anti-Akt (PW56) (30Watton S.J. Downward J. Curr. Biol. 1999; 9: 433-436Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar); anti-phosphoTyr1173-EGF receptor (R42/pY1173) (31Oksvold M.P. Skarpen E. Lindeman B. Roos N. Huitfeldt H.S. J. Histochem. Cytochem. 2000; 48: 21-33Crossref PubMed Scopus (47) Google Scholar); anti-EGF receptor (Cell Signaling Technology); and anti-ERK2 (Santa-Cruz). Secondary antibodies and Dextran-Texas Red were from Jackson Immuno Research Laboratories (Stratech Scientific). Rabbit anti-mouse IgG and fluorescein isothiocyanate-phalloidin were bought from Sigma. Pull-down assays, using a GST-PAK-CRIB fusion protein, were performed essentially as described (32Sander E.E. van Delft S. Klooster J.P. Reid T. van der Kammen R.A. Michaelis F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (586) Google Scholar). One 9-cm dish of confluent keratinocytes was used per data point. Cells were lysed in lysis buffer (1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 mm Tris-HCl pH 7.5, 150 mmsodium chloride, 10 mm magnesium chloride and 1 μg/ml leupeptin, 1 μg/ml pepstatin, 0.1 mm phenylmethylsulfonyl fluoride, and 0.1 mm pefabloc). Lysates were incubated with PAK-CRIB beads (20 μg/dish) for 45 min, and beads were washed three times with wash buffer (1% Triton X-100, 50 mm Tris-HCl pH 7.5, 150 mm sodium chloride, and 10 mmmagnesium chloride). Precipitated proteins and 2% of each lysate were separated by SDS-PAGE, blotted, and probed for Rac. For antibody blocking experiments, cells were incubated with 5 μg/ml HECD-1 and 2 μg/ml NCC-CAD-299 for 30 min at 37 °C prior to addition of calcium. As a control, cells were preincubated with 7 μg/ml rabbit anti-mouse IgG. For clustering experiments, keratinocytes grown in low calcium medium were incubated for 15 min on ice with 5 μg/ml HECD-1 and 2 μg/ml NCC-CAD-299. Anti-mouse IgG was added to 35 μg/ml, and cells were incubated for 30 min at 37 °C. For bead experiments, 15 μm latex beads (Polysciences) were coated with HECD-1 described as previously (7Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (654) Google Scholar). Latrunculin B (Calbiochem) was titrated to 0.3 μm, which prevented actin cytoskeleton remodeling in keratinocytes without causing cellular retraction. Cells were treated with Latrunculin B or Me2SO control for 10 min and incubated with HECD-1-coated beads (2.4 × 107 beads per dish) for 10 min before lysis. In inhibition experiments, cells were incubated with 30 μmLY294002 (Sigma), 316 nm tyrphostin AG1478 (Calbiochem) (18Pece S. Gutkind J.S. J. Biol. Chem. 2000; 275: 41227-41233Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar), 35 μm PD98508 (Calbiochem), or Me2SO vehicle for 30 min prior to stimulation with 2 mm calcium for 5 min. In EGF stimulation experiments, cells were incubated with LY294002 or Me2SO vehicle as described above before stimulation with 10 nm EGF for 10 min. To probe for Akt phosphorylation, cells were lysed in lysis buffer (1% Triton X-100, 20 mmβ-glycerophosphate, 20 mm sodium fluoride, 2 mm EDTA, 0.2 mm sodium vanadate, 10 mm benzamidine, 2.5 μg/ml microcystin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 0.1 mm phenylmethylsulfonyl fluoride, and 0.1 mm pefabloc). Lysates were separated by SDS-PAGE, blotted, and probed using antibodies specific for total Akt (PW66) or Akt phosphorylated on serine 473 (PW56) (30Watton S.J. Downward J. Curr. Biol. 1999; 9: 433-436Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Cells were lysed in lysis buffer (1% Triton X-100, 50 mm Tris-HCl pH 7.5, 150 mm sodium chloride, 2 mm EDTA, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm pefabloc, 20 mmβ-glycerophosphate, 20 mm sodium fluoride, 0.2 mm sodium vanadate, and 1 mm sodium molybdate). Lysates were run on SDS-PAGE gels, blotted, and probed for total EGF receptor, activated EGF receptor (clone 74), or EGF receptor phosphorylated on tyrosine 1173 (R42/pY1173) (31Oksvold M.P. Skarpen E. Lindeman B. Roos N. Huitfeldt H.S. J. Histochem. Cytochem. 2000; 48: 21-33Crossref PubMed Scopus (47) Google Scholar). A number of studies have shown that a dominant negative version of Rac (N17Rac) inhibits the localization of cadherin receptors at sites of cell-cell contact (6Takaishi K. Sasaki T. Kotani H. Nishioka H. Takai Y. J. Cell Biol. 1997; 139: 1047-1059Crossref PubMed Scopus (474) Google Scholar, 7Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (654) Google Scholar). Dominant negative mutants are presumed to function by sequestering upstream activators for the small GTPases, the guanine nucleotide exchange factors (GEFs) (reviewed in Ref. 33Feig L.A. Nat. Cell. Biol. 1999; 1: E25-E27Crossref PubMed Scopus (342) Google Scholar). However, there appears to be more than 46 GEFs for Rho family GTPases, many of which are rather promiscuous in exchange activity (reviewed in Ref. 34Zheng Y. Trends Biochem. Sci. 2002; 26: 724-732Abstract Full Text Full Text PDF Scopus (350) Google Scholar). Therefore, it is possible that N17Rac blocks cadherin-mediated adhesion by inhibition of a Rho GTPase other than Rac. To determine whether Rac does indeed play a role in regulation of cadherin localization at intercellular junctions, we investigated whether an activated version of Rac (L61Rac) can rescue the inhibitory effect of N17Rac on cell-cell contacts. Normal human keratinocytes cultured in the absence of intercellular contacts were injected with recombinant dominant negative Rac protein, and cell-cell adhesion was induced for 1 h. Cells were fixed and stained for E-cadherin (Fig.1, A and B). As reported previously (7Braga V.M.M. Machesky L.M. Hall A. Hotchin N.A. J. Cell Biol. 1997; 137: 1421-1431Crossref PubMed Scopus (654) Google Scholar), N17Rac inhibited localization of cadherin receptors at boundaries between injected cells (Fig. 1, Aand B). Microinjection of L61Rac alone had no effect on cadherin localization at junctions after incubation for 1 h (Fig.1, C and D). Interestingly, co-injection of L61Rac and N17Rac completely rescued the inhibitory effect of N17Rac on cadherin-mediated adhesion (Fig. 1, E and F). This effect is specific for L61Rac because L61Cdc42 cannot restore junctional cadherin staining in N17Rac-injected cells. 2J. K. Zhang, unpublished observations. Together these results provide strong evidence that N17Rac blocks localization of cadherin receptors at junctions by inhibiting Rac function. Thus, Rac plays an important role in the regulation of cadherin-mediated adhesion. Because Rac activity is required for the stability of cadherin receptors at junctions, is Rac activated upon cell-cell contact formation in keratinocytes? To evaluate Rac activation, we performed pull-down assays utilizing the Cdc42/Rac interactive binding (CRIB) domain of the Rac effector p21-activated kinase (PAK) (32Sander E.E. van Delft S. Klooster J.P. Reid T. van der Kammen R.A. Michaelis F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (586) Google Scholar). We confirmed the specificity of the assay under our conditions by loading keratinocyte lysates with GTPγS or GDP and assessing Rac activity (Fig. 2 a). To investigate whether cell-cell adhesion can activate the small GTPase Rac, we grew keratinocytes in low calcium medium and induced cell-cell contacts for various time periods before measuring Rac activation. We found that endogenous Rac was activated 2–4-fold within 5 min of addition of calcium, and this activation was sustained up to 120 min (Fig. 2, b and c). It is possible that the up-regulation of Rac described above was caused by stimulation of calcium-dependent intracellular signaling pathways rather than induction of cadherin-dependent adhesion. To distinguish between these two possibilities, we inhibited cadherin function during the calcium switch. Keratinocytes express at least two members of the cadherin family (E- and P-cadherin) (26Hirai Y. Nose A. Kobayashi S. Takeichi M. Development. 1989; 105: 271-277PubMed Google Scholar). Cells were preincubated with anti-E- and anti-P-cadherin antibodies at concentrations known to block cadherin function in keratinocytes (24Hodivala K.J. Watt F.M. J. Cell Biol. 1994; 124: 589-600Crossref PubMed Scopus (205) Google Scholar). To confirm that the antibodies blocked cadherin function, we demonstrated that, in the presence of the inhibitory antibodies, no cadherin clustering was found at intercellular boundaries (Fig. 2 d). Incubation with anti-cadherin antibodies prevented Rac up-regulation upon addition of calcium ions, whereas control IgG did not (Fig. 2 e). Thus, the Rac activation is dependent on functional cadherin receptors, and addition of calcium ions per se is not sufficient to trigger Rac activation. These results demonstrated that cell-cell adhesion mediated by cadherins could activate Rac in keratinocytes. Because junction formation is accompanied by the clustering of cadherin receptors (3Yap A.S. Brieher W.M. Gumbiner B.M. Ann. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (686) Google Scholar), we investigated whether clustering of cadherins with antibodies was sufficient to activate Rac. Keratinocytes grown in low calcium medium were incubated with anti-E- and anti-P-cadherin antibodies (mouse monoclonals). Clustering of the receptors was then induced by addition of anti-mouse IgG, before assaying for Rac activity. Under these conditions, we could observe clusters of cadherins on the cell surface by immunofluorescence (Fig.3 A). We found that clustering induced a modest increase in Rac activity relative to samples where the anti-cadherin antibodies were omitted (Fig. 3 B). Quantification of the relative Rac activation in several experiments revealed that treatment of cells with anti-cadherin antibodies alone only induced a 1.2-fold increase in Rac activation relative to cells treated with anti-mouse IgG alone. Clustering of surface cadherins with two layers of antibodies induced a larger increase in Rac activation (1.7-fold; Fig. 3, B and F). However, the degree of Rac activation after antibody clustering is lower than the activation observed following cell-cell contact formation (2–4-fold). We hypothesized that this weak Rac activation may be due to the small number of cadherin molecules per cluster formed. To overcome this problem, we incubated cells for 10 min in low calcium medium with beads coated with anti-E-cadherin antibodies. Immunofluorescence of cells treated with these beads revealed binding of anti-E-cadherin beads to the cell surface and recruitment of α-catenin and F-actin to the beads (Fig. 3 C,arrowheads). In contrast, few beads coated with BSA alone bound to the cell surface, and those that did recruited little α-catenin or F-actin (Fig. 3 C, arrows,top panels). We found that cells incubated with anti-E-cadherin beads showed increased Rac activation in comparison with cells treated with BSA beads (Fig. 3 D). Quantification of the results of three experiments revealed that anti-E-cadherin beads enhanced Rac activation by a factor of 2.1 compared with BSA beads (Fig. 3 G), suggesting that clustering of cadherins is sufficient to activate Rac. To determine whether new actin polymerization is required for Rac activation by clustered cadherin molecules, we treated cells with Latrunculin B prior to receptor clustering. Latrunculin B inhibits actin polymerization by sequestering actin monomers (36Coue M. Brenner S.L. Spector I. Korn E.D. FEBS Lett. 1987; 213: 316-318Crossref PubMed Scopus (642) Google Scholar, 37Spector I. Shochet N.R. Blasberger D. Kashman Y. Cell Motil. Cytoskeleton. 1989; 13: 127-144Crossref PubMed Scopus (485) Google Scholar). Treatment of cells with Latrunculin B for 10 min before adding anti-E-cadherin beads still allowed binding of beads to the cell surface and recruitment of α-catenin but blocked actin recruitment (Fig.3 C, arrows, bottom panels). Upon assaying for Rac activation, we found that Latrunculin B treatmentper se caused an increase in Rac activation. Incubation with E-cadherin beads in the presence of Latrunculin B induced a further 1.4-fold increase in Rac activation (Fig. 3, D andG). Thus, Rac activation can occur in the absence ofde novo actin polymerization, but full induction of Rac activity does require formation of new actin filaments. However, the use of beads to artificially cluster cadherin receptors may resemble the phagocytotic process. Rac activity is necessary for Fcγ-mediated phagocytosis (38Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (805) Google Scholar). Therefore, the possibility exists that anti-E-cadherin beads may induce Rac activation by triggering early events in phagocytosis. As a control to confirm that this was not the case, we treated cells with beads coated with antibodies against another cell surface receptor, α3β1-integrin. As expected, these beads showed little staining for α-catenin but did recruit F-actin (Fig.3 C, asterisks). However, anti-α3β1-integrin beads did not induce increased Rac activity relative to BSA control beads (Fig.3 E). Thus, the clustering of cadherin receptors can specifically activate Rac. We next explored which signaling pathways operate downstream of cadherin-dependent contact formation to induce Rac activity. We investigated three different classes of signaling molecule: PI3-kinases, the EGF receptor, and ERK1/2. We made use of the compounds LY294002, tyrphostin AG1478, and PD98059, specific inhibitors of PI3-kinases, the EGF receptor, and mitogen-activated kinase or ERK kinase-1 (MEK1), respectively (39Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar, 40Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1618) Google Scholar, 41Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3254) Google Scholar). We confirmed that the inhibitors were functional under our experimental conditions by probing lysates for Akt phosphorylated on serine 473 (for LY294002; Fig. 4 A) or activated ERK1/2 (AG1478 and PD98059; Fig. 4, B andC). As expected, phosphorylation and activation of these molecules were blocked. Inhibition of keratinocytes with LY294002 increased Rac activity (compare with low calcium sample; Fig. 4 D) but did not inhibit Rac activation induced by cell-cell adhesion. We therefore concluded that PI3-kinases do not play a major role in Rac activation downstream of cadherin adhesion in keratinocytes. Similarly to LY294002, treatment of keratinocytes with tyrphostin AG1478 increased Rac activity (Fig. 4 D). However, the drug dramatically decreased Rac activation upon induction of cell-cell adhesion. Therefore we concluded that EGF receptor function participates in cadherin-dependent Rac stimulation (Fig.4 D). Consistent with this conclusion, calcium-induced cell-cell adhesion induces EGF receptor activation in normal keratinocytes 3M. Betson, E. Lozano, and V. M. M. Braga, unpublished observations. as has been observed in HaCat cells (18Pece S. Gutkind J.S. J. Biol. Chem. 2000; 275: 41227-41233Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Rac activation in these experiments was quantified relative to the control low calcium cultures treated with Me2SO (Fig.4 G; −calcium, DMSO). However, there was some variability in the degree of Rac activation induced by drug treatment per se. For this reason Rac activity was also calculated relative to the control low calcium cultures treated with each inhibitor and was arbitrarily set at 1 (Fig. 4 I; −calcium). The inhibitors Latrunculin B, AG1478, or LY294002 can all activate Rac in keratinocytes. To investigate whether this Rac activation is a nonspecific consequence of treatment with different inhibitors, we incubated cells with the MEK1 inhibitor PD98059. Treatment with PD98059 compound did not increase the basal level of Rac activity in low calcium medium, nor did it inhibit activation of Rac induced by cell-cell contact formation (Fig. 4 E). Quantification of these experiments is shown in Fig. 4, H and I. Thus, blocking signaling pathways in general does not lead to Rac activation. In addition, these results suggested that the ERK/mitogen-activated kinase cascade is not involved in induction of GTP loading on Rac downstream of cadherin-dependent adhesion. EGF-stimulated activation of Rac is PI3-kinase-dependent in fibroblasts (42Ridley A.J. Paterson H.F. Johnston C.L. Diekman D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3071) Google Scholar). Given our results, we were interested to determine whether a similar pathway exists in keratinocytes. Cells grown in the absence of cell-cell contacts were incubated with the PI3-kinase inhibitor LY294002 and stimulated with EGF for 10 min. Whereas in untreated cells EGF stimulation induces a strong Rac activation, this is blocked by inhibition of PI3-kinase (Fig. 4 F). Thus, as in fibroblasts, EGF-induced Rac activation is PI3-kinase-dependent in keratinocytes. Our results demonstrate for the first time that" @default.
• W2017012788 created "2016-06-24" @default.
• W2017012788 creator A5017539310 @default.
• W2017012788 creator A5020112282 @default.
• W2017012788 creator A5025268046 @default.
• W2017012788 creator A5056769047 @default.
• W2017012788 date "2002-10-01" @default.
• W2017012788 modified "2023-10-02" @default.
• W2017012788 title "Rac Activation upon Cell-Cell Contact Formation Is Dependent on Signaling from the Epidermal Growth Factor Receptor" @default.
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