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• W2019440050 abstract "Integrin-induced cytoskeletal reorganizations are initiated by Cdc42 and Rac1 but little is known about mechanisms by which integrins activate these Rho GTPases. 14-3-3 proteins are adaptors implicated in binding and regulating the function and subcellular location of numerous signaling molecules. In platelets, the 14-3-3ζ isoform interacts with the glycoprotein (GP) Ibα subunit of the adhesion receptor GP Ib-IX. In this study, we show that integrin-induced activation of Cdc42, activation of Rac, cytoskeletal reorganizations, and cell spreading were inhibited in Chinese hamster ovary cells expressing full-length GP Ibα compared with GP Ibα lacking the 14-3-3ζ binding site. Activation of Rho GTPases and cytoskeletal reorganizations were restored by expression of 14-3-3ζ. Spreading in cells expressing truncated GP Ibα was inhibited by co-expressing a chimeric receptor containing interleukin 2 receptor α and GP Ibα cytoplasmic domain. These results identify a previously unrecognized function of 14-3-3ζ, that of mediating integrin-induced signaling. They show that 14-3-3ζ mediates Cdc42 and Rac activation. They also reveal a novel function of platelet GP Ib-IX, that of regulating integrin-induced cytoskeletal reorganizations by sequestering 14-3-3ζ. Signaling across integrins initiates changes in cell behavior such as spreading, migration, differentiation, apoptosis, or cell division. Thus, introduction of the 14-3-3ζ binding domain of GP Ibα into target cells might provide a method for regulating integrin-induced pathways in a variety of pathological conditions. Integrin-induced cytoskeletal reorganizations are initiated by Cdc42 and Rac1 but little is known about mechanisms by which integrins activate these Rho GTPases. 14-3-3 proteins are adaptors implicated in binding and regulating the function and subcellular location of numerous signaling molecules. In platelets, the 14-3-3ζ isoform interacts with the glycoprotein (GP) Ibα subunit of the adhesion receptor GP Ib-IX. In this study, we show that integrin-induced activation of Cdc42, activation of Rac, cytoskeletal reorganizations, and cell spreading were inhibited in Chinese hamster ovary cells expressing full-length GP Ibα compared with GP Ibα lacking the 14-3-3ζ binding site. Activation of Rho GTPases and cytoskeletal reorganizations were restored by expression of 14-3-3ζ. Spreading in cells expressing truncated GP Ibα was inhibited by co-expressing a chimeric receptor containing interleukin 2 receptor α and GP Ibα cytoplasmic domain. These results identify a previously unrecognized function of 14-3-3ζ, that of mediating integrin-induced signaling. They show that 14-3-3ζ mediates Cdc42 and Rac activation. They also reveal a novel function of platelet GP Ib-IX, that of regulating integrin-induced cytoskeletal reorganizations by sequestering 14-3-3ζ. Signaling across integrins initiates changes in cell behavior such as spreading, migration, differentiation, apoptosis, or cell division. Thus, introduction of the 14-3-3ζ binding domain of GP Ibα into target cells might provide a method for regulating integrin-induced pathways in a variety of pathological conditions. The 14-3-3 family of proteins are expressed in all eukaryotic cells. There are at least seven highly conserved isoforms encoded by different gene products. These proteins have molecular weights of 29,000–32,000 and bind numerous cytoplasmic and nuclear signaling molecules including signaling molecules such as Raf, protein kinase C, p130Cas, BAD, and phosphatidylinositol 3-kinase (1Fu H. Xia K. Pallas D.C. Cui C. Conroy K. Narsimhan R.P. Mamon H. Collier R.J. Roberts T.M. Science. 1994; 266: 126-129Crossref PubMed Scopus (243) Google Scholar, 2Wheeler-Jones C.P.D. Learmonth M.P. Martin H. Aitken A. Biochem. J. 1996; 315: 41-47Crossref PubMed Scopus (32) Google Scholar, 3Garcia-Guzman M. Dolfi F. Russello M. Vuori K. J. Biol. Chem. 1999; 274: 5762-5768Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 4Bonnefoy-Berard N. Liu Y.-C. von Willebrand M. Sung A. Elly C. Mustelin T. Yoshida H. Ishizaka K. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10142-10146Crossref PubMed Scopus (133) Google Scholar, 5Zha J. Harada H. Yang E. Jocket J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2257) Google Scholar), proteins such as Cdc25 and Wee1 that are involved in cell cycle control (6Conklin D.S. Galktionov K. Beach D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7892-7896Crossref PubMed Scopus (246) Google Scholar, 7Honda R. Ohba Y. Yasuda H. Biochem. Biophys. Res. Commun. 1997; 230: 262-265Crossref PubMed Scopus (45) Google Scholar), and proteins such as FKHRLl and DAF-16 (8Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 9Cahill C.M. Tzivion G. Nasrin N. Ogg S. Dore J. Ruvkun G. Alexander-Bridges M. J. Biol. Chem. 2001; 267: 13402-13410Abstract Full Text Full Text PDF Scopus (178) Google Scholar) that are involved in regulation of transcription. Interaction of 14-3-3 proteins with signaling molecules can localize, activate, inhibit, or stabilize the target molecules. Because 14-3-3 proteins are dimers, they have been considered as adaptor proteins that recruit and regulate the function of signaling molecules. Whereas they have been implicated in regulation of cell proliferation, cell cycle progression, apoptosis, and differentiation (10Tzivion G. Shen Y.H. Zhu J. Oncogene. 2001; 20: 6331-6338Crossref PubMed Scopus (258) Google Scholar, 11van Hemert M.J. Steensma H.Y. van Heusden G.P. Bioessays. 2001; 23: 936-946Crossref PubMed Scopus (473) Google Scholar, 12Muslin A.J. Xing H. Cell Signal. 2000; 12: 703-709Crossref PubMed Scopus (351) Google Scholar, 13Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1333) Google Scholar), little is known about the possibility that 14-3-3 proteins might serve as adaptors in recruiting and regulating signaling molecules involved in transmission of signals across transmembrane receptors. It is interesting in this regard that one of the first proteins shown to bind 14-3-3 in intact cells was the GP 1The abbreviations used are: GP, glycoprotein; FITC, fluorescein isothiocyanate; CHO, Chinese hamster ovary; RGDS, Arg-Gly-Asp-Ser; TRITC, tetramethylrhodamine isothiocyanate; vWf, von Willebrand factor; HA, hemagglutinin; GST, glutathione S-transferase; IL2Rα, interleukin 2 receptor α; PBD, p21 binding domain of PAK; FACS, fluorescence-activated cell sorter. Ib-IX complex (14Du X. Fox J.E.B. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), an adhesion receptor that is expressed almost exclusively in platelets. The GP Ib-IX complex consists of a disulfide-linked heterodimer GP Ibα/GP Ibβ non-covalently associated with GP IX (15Berndt M.C. Gregory C. Kabral A. Zola H. Fournier D. Castaldi P.A. Eur. J. Biochem. 1985; 151: 637-649Crossref PubMed Scopus (154) Google Scholar) and the complex associates with the leucine-rich repeat protein GPV, which appears to link two GP Ib-IX trimers (16Dong J.-F. Sae-Tung G. Lopez J.A. Blood. 1997; 89: 4355-4363Crossref PubMed Google Scholar, 17Li C.Q. Dong J.-f. Lanza F. Sanan D.A. Sae-Tung G. Lûpez J.A. J. Biol. Chem. 1995; 270: 16302-16307Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The extracellular domain of GP Ibα binds a number of proteins, including the extracellular matrix protein, von Willebrand factor (vWf), and the plasma proteins, thrombin (16Dong J.-F. Sae-Tung G. Lopez J.A. Blood. 1997; 89: 4355-4363Crossref PubMed Google Scholar, 18Okumura T. Hasitz M. Jamieson G.A. J. Biol. Chem. 1978; 253: 3435-3443Abstract Full Text PDF PubMed Google Scholar, 19Ganguly P. Gould N.L. Br. J. Haematol. 1979; 42: 137-145Crossref PubMed Scopus (42) Google Scholar, 20Larsen N.E. Simons E.R. Biochem. J. 1981; 20: 4141-4147Crossref Scopus (25) Google Scholar), factor XI (21Baglia F.A. Badellino K.O. Li C.Q. Lopez J.A. Walsh P.N. J. Biol. Chem. 2002; 277: 1662-1668Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and factor XIIa (22Bradford H.N. Pixley R.A. Colman R.W. J. Biol. Chem. 2000; 275: 22756-22763Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The cytoplasmic domain of GP Ibα interacts with 14-3-3 (14Du X. Fox J.E.B. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Platelets contain high levels of the ζ, β, and γ isoforms and lower levels of the ϵ and η isoforms of 14-3-3 (2Wheeler-Jones C.P.D. Learmonth M.P. Martin H. Aitken A. Biochem. J. 1996; 315: 41-47Crossref PubMed Scopus (32) Google Scholar). The only isoform that has been shown to interact with GP Ibα is 14-3-3ζ. This interaction was first identified when a 29-kDa protein was found to copurify with GP Ib-IX from platelet membrane extracts (23Du X. Harris S.J. Tetaz T.J. Ginsberg M.H. Berndt M.C. J. Biol. Chem. 1994; 269: 18287-18290Abstract Full Text PDF PubMed Google Scholar). Protein sequencing of internal fragments of the protein revealed that it was 14-3-3ζ. Subsequent studies have shown interaction between GP Ibα and 14-3-3ζ in a two-hybrid system (24Calverley D.C. Kavanagh T.J. Roth G.J. Blood. 1998; 91: 1295-1303Crossref PubMed Google Scholar), shown that 14-3-3ζ and GP Ib-IX interact in platelet lysate (14Du X. Fox J.E.B. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and that it coimmunoprecipitates with GP Ibα expressed in Chinese hamster ovary (CHO) cells (25Gu M. Xi X. Englund G.D. Berndt M.C. Du X. J. Cell Biol. 1999; 147: 1085-1096Crossref PubMed Scopus (123) Google Scholar). Studies with CHO cells expressing GP Ib-IX complex with truncated forms of GP Ibα have shown that truncation of the C-terminal 19 amino acids of GP Ibα abolishes 14-3-3ζ binding to the GP Ib-IX complex (14Du X. Fox J.E.B. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 25Gu M. Xi X. Englund G.D. Berndt M.C. Du X. J. Cell Biol. 1999; 147: 1085-1096Crossref PubMed Scopus (123) Google Scholar) and the use of synthetic peptides has located the binding site to the C-terminal five amino acids of GP Ibα (14Du X. Fox J.E.B. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). When GP Ib-IX binds to vWf, signals are induced across the adhesion receptor. One consequence of this signaling is activation of intracellular signaling pathways leading to activation of αIIbβ3, the β3-containing integrin that mediates platelet adhesion and aggregation (25Gu M. Xi X. Englund G.D. Berndt M.C. Du X. J. Cell Biol. 1999; 147: 1085-1096Crossref PubMed Scopus (123) Google Scholar, 26Ruggeri Z.M. Thromb. Haemostasis. 1997; 611: 611-616Google Scholar, 27Savage B. Cattaneo M. Ruggeri Z.M. Curr. Opin. Hematol. 2001; 8: 270-276Crossref PubMed Scopus (112) Google Scholar, 28Zaffran Y. Meyer S.C. Negrescu E. Reddy K.B. Fox J.E.B. J. Biol. Chem. 2000; 275: 16779-16787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The finding that 14-3-3ζ interacted with the cytoplasmic domain of GP Ibα raised the possibility that 14-3-3ζ might serve to recruit signaling molecules involved in transmission of these signals from the cytoplasmic domain of GP Ibα. However, studies in which the GP Ib-IX complex containing full-length or truncated GP Ibα were expressed in CHO cells suggested that the GP Ib/14-3-3ζ interaction is not required for transmission of the signals that convert CHO cell αvβ3 from an inactive form to a form that can bind immobilized vWf (28Zaffran Y. Meyer S.C. Negrescu E. Reddy K.B. Fox J.E.B. J. Biol. Chem. 2000; 275: 16779-16787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Integrins are another family of adhesion receptors that transmit signals upon ligand binding. Evidence from in vitro binding assays has suggested that 14-3-3 may also interact with the cytoplasmic domain of these adhesion receptors. In one study, β1- and β3-integrin subunits bound to 14-3-3β in a two-hybrid screen (29Han D.C. Rodriguez L.G. Guan J.L. Oncogene. 2001; 20: 346-357Crossref PubMed Scopus (82) Google Scholar). In another study, 14-3-3αβ and 14-3-3ζδ in leukocyte extracts bound to phosphopeptides with the sequence of the cytoplasmic domain of the β2-integrin (30Fagerholm S. Morrice N. Gahmberg C.G. Cohen P. J. Biol. Chem. 2002; 277: 1728-1738Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Although there has been no clear evidence that any isoform of 14-3-3 interacts directly with integrin in intact cells, 14-3-3β has been reported to colocalize with β1-integrin in human foreskin fibroblasts spreading on fibronectin (29Han D.C. Rodriguez L.G. Guan J.L. Oncogene. 2001; 20: 346-357Crossref PubMed Scopus (82) Google Scholar). The adhesion-induced signals transmitted across integrins induce cytoskeletal reorganizations that lead to cell spreading and migration and regulate pathways involved in development, inflammation, wound repair, cell division, differentiation, and apoptosis (31Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2822) Google Scholar, 32Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1474) Google Scholar). Previous work has shown that the integrin-induced cytoskeletal reorganizations are initiated by the Rho GTPases, Cdc42, and Rac (33Clark E.A. King W.G. Brugge J.S. Symons M. Hynes R.O. J. Cell Biol. 1998; 142: 573-586Crossref PubMed Scopus (535) Google Scholar, 34Price L.S. Leng J. Schwartz M.A. Bokoch G.M. Mol. Biol. Cell. 1998; 9: 1863-1871Crossref PubMed Scopus (529) Google Scholar). Whereas mechanisms by which these GTPases are recruited and activated following transmission of signals across other families of receptors have been well characterized, little is known about the way in which signals transmitted through integrin cytoplasmic domains activate these Rho GTPases. In the present study, we considered the possibility that 14-3-3ζ might be involved in mediating integrin-induced Rho GTPase activation. To investigate this possibility we sequestered endogenous 14-3-3ζ in CHO cells, observed the functional consequences on integrin-induced signaling, and then expressed additional 14-3-3ζ to determine whether normal integrin-induced events were restored. The approach that we used to sequester 14-3-3ζ was to express GP Ib-IX in CHO cells. Normal CHO cells spreading on an integrin substrate extended filopodia and membrane ruffles and eventually formed well spread cells filled with stress fibers. However, if the GP Ib-IX complex was present, integrin-induced cytoskeletal reorganizations were inhibited. Cells expressing GP Ib-IX complex containing a truncated form of GP Ibα, which lacked the 14-3-3ζ binding site, spread normally and normal cytoskeletal reorganizations occurred. Overexpression of 14-3-3ζ in cells in which endogenous 14-3-3ζ had been sequestered by GP Ib-IX restored cell spreading. Sequestration of 14-3-3ζ inhibited integrin-induced Cdc42 and Rac activation; overexpression of 14-3-3ζ restored GTPase activation. These studies describe a previously unrecognized function of 14-3-3 proteins. They show that 14-3-3ζ has a critical role in inducing the cascades of signaling reactions that occur as integrins mediate cell adhesion. They show that 14-3-3ζ mediates step(s) upstream of Cdc42 and Rac activation. In addition, the studies show that the 14-3-3ζ binding domain on the cytoplasmic domain of GP Ibα can inhibit integrin-induced Cdc42 and Rac activation by a mechanism that involves sequestration of 14-3-3ζ. Thus, introduction of the 14-3-3ζ binding domain of GP Ibα into target cells might provide a method for regulating integrin-induced events such as migration, cell division, differentiation, or apoptosis in a variety of pathological conditions. Cell Culture and Transfection—CHO cells were grown in Ham's F-12 medium (Invitrogen) containing 10% fetal bovine serum and a mixture of penicillin/streptomycin antibiotics (Invitrogen). Cells were stably transfected with the cDNA encoding GP Ibβ, GP IX, and full-length or truncated GP Ibα as previously described (35Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The truncated form of GP Ibα was generated by introducing a stop codon at position 591 of the cytoplasmic domain of GP Ibα, utilizing Stratagene's Double Take double-stranded mutagenesis system (35Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The resulting protein lacked the C-terminal 19 amino acids. Expression of GP Ib-IX on the cell surface was analyzed by flow cytometric analysis using a monoclonal antibody against GP Ibα (Immunotech, Westbrook, ME). In some experiments, cells expressing truncated GP Ib-IX were cotransfected with a chimeric receptor consisting of IL2Rα extracellular and transmembrane domains linked to the GP Ibα cytoplasmic domain (kindly provided by Dr. Xiaoping Du, Department of Pharmacology, University of Illinois, College of Medicine, Chicago, IL) along with pSV2-hph. Cells co-transfected with IL2Rα lacking a cytoplasmic domain were used as a control (kindly provided by Dr. Susan LaFlamme, Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY). Membrane expression of IL2Rα was assessed using flow cytometry after staining of the cells with a monoclonal antibody against IL2Rα (Immunotech). A FACScan flow cytometer was used for flow cytometry analysis (BD Biosciences, Advanced Cellular Biology, San Jose, CA) as described previously (36Meyer S.C. Fox J.E.B. J. Biol. Chem. 1995; 270: 14693-14699Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). For transient transfection of an HA-tagged 14-3-3ζ construct encoded in the vector pCMV5 (kindly provided by Dr. Charles Abrams, Hematology-Oncology Division, University of Pennsylvania, Philadelphia, PA), cells were transfected for 3 h with 3 μg of the construct and LipofectAMINE Plus reagent according to the manufacturer's instructions (Invitrogen). Immunofluorescence Microscopy—Cells were incubated with 5 μg/ml botrocetin (Pentapharm, Basel, Switzerland) in serum-free media and plated on Lab-Tek glass chamber slides (Nunc, Inc., Naperville, IL) that had been previously coated with 5 μg/ml vWf (American Diagnostics, Greenwich, CT) and saturated with 3% bovine serum albumin (35Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In some experiments, as described in the text, suspensions of cells were incubated for 30 min at room temperature with 4 mm Arg-Gly-Asp-Ser (RGDS) peptide (Sigma) or 2 mm EDTA, prior to plating. The cells were fixed at various time points, permeabilized with 0.5% Triton X-100, and stained as previously described (35Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 36Meyer S.C. Fox J.E.B. J. Biol. Chem. 1995; 270: 14693-14699Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Actin filaments were stained with 1 μg/ml tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin (Sigma). For analysis of cells transiently expressing HA-tagged 14-3-3ζ, transfected cells were allowed to recover for 48 h in serum-containing medium. Cells were then replated onto vWf-coated dishes for 2 h and subsequently examined by immunofluorescence. Actin filaments were detected with TRITC-labeled phalloidin and HA-tagged 14-3-3ζ detected with monoclonal antibody (clone 12CA5, Roche Diagnostics) against HA epitope followed by secondary antibodies conjugated to Alexa 488 (Molecular Probes, Eugene, OR). Fluorescence microscopy was performed using a Leica TCS-NT laser scanning confocal microscope as described previously (37Meyer S.C. Sanan D.A. Fox J.E.B. J. Biol. Chem. 1998; 273: 3013-3020Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Cell area measurement was performed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Data were presented as the mean ± S.D. and were compared using Student's t test. Biochemical Assay of Activated GTPases in CHO Cell Lysates—GST-p21 binding domain of PAK (PBD) was produced in DH5α cells transformed with pGEX plasmid containing PBD cDNA (kindly provided by Dr. Martin Schwartz, Department of Microbiology, Cardiovascular Research Center, Mellon Prostate Cancer Research Center, University of Virginia, Charlottesville, VA) and subsequently bound to glutathione-coupled agarose beads (Sigma) as previously described (38Ren X. Kiosses W. Schwartz M. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar). CHO cells were plated on vWf in the presence of botrocetin for 45 min, scraped off the dishes, and lysed in an ice-cold buffer containing 50 mm Tris, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mm NaCl, pH 7.2, and a mixture of protease inhibitors (Complete, Roche Diagnostics). Cell lysates were cleared by centrifugation at 16,000 × g for 8 min and supernatants were incubated with GST-PBD beads for 45 min. Beads were washed four times in ice-cold buffer containing 50 mm Tris, protease inhibitors, 1% Triton X-100, and 150 mm NaCl, pH 7.2. Proteins bound to the beads were solubilized by addition of an electrophoresis buffer containing 3% SDS and 2% 2-mercaptoethanol and Western blots were prepared. Western Blot Analysis—For analysis of 14-3-3 isoforms normally expressed in CHO cells, cells were solubilized in an electrophoresis buffer containing 3% SDS and 2% 2-mercaptoethanol, boiled for 10 min, electrophoresed through SDS-polyacrylamide gels containing 10% polyacrylamide, and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA) by standard Western blotting techniques (39Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44938) Google Scholar). Western blots were probed with polyclonal antibodies against 14-3-3 α, β, γ, and ζ (Santa Cruz Biotechnology, Santa Cruz, CA) followed by anti-rabbit horseradish peroxidase-coupled antibody (Amersham Biosciences). To quantitate the amount of HA-tagged 14-3-3ζ in CHO cells transiently expressing this protein, Western blots of whole cell extracts were probed with a monoclonal antibody against HA epitope (Roche Diagnostics) and anti-mouse horseradish peroxidase-coupled antibody (Amersham Biosciences). For analysis of Cdc42 or Rac in whole cell extracts or activated Cdc42 or Rac bound to GST-coupled beads, Western blots were probed with monoclonal Cdc42 or Rac antibodies (Transduction Laboratories, San Diego, CA) and anti-mouse horseradish peroxidase-coupled antibody. Antigen-antibody complexes on Western blots were detected by incubating the membranes with ECL reagent (Amersham Biosciences) and exposing them to Kodak X-Omat films (Eastman Kodak, Rochester, NY). Quantitation of antigen-antibody complexes was performed by NIH Image 1.62 software. Western Blots to Identify 14-3-3 Isoforms Expressed in CHO Cells—To determine which isoforms of 14-3-3 were expressed in the CHO cells used for the studies, Western blots of whole cell extracts were probed with antibodies against 14-3-3 β, γ, and ζ. Examination of the blots showed that 4-3-3β, 14-3-3γ, and 14-3-3ζ were present (data not shown). Effect of the Cytoplasmic Domain of GP Ib-IX on Integrin-induced Signaling—GP Ibα interacts with 14-3-3ζ in platelets; it also interacts with CHO cell 14-3-3ζ when transfected into these cells (23Du X. Harris S.J. Tetaz T.J. Ginsberg M.H. Berndt M.C. J. Biol. Chem. 1994; 269: 18287-18290Abstract Full Text PDF PubMed Google Scholar, 25Gu M. Xi X. Englund G.D. Berndt M.C. Du X. J. Cell Biol. 1999; 147: 1085-1096Crossref PubMed Scopus (123) Google Scholar). A form that lacks 19 C-terminal amino acids (m591) (35Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) retains its interaction with its other known binding partner, actin-binding protein (35Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), but fails to interact with 14-3-3ζ (25Gu M. Xi X. Englund G.D. Berndt M.C. Du X. J. Cell Biol. 1999; 147: 1085-1096Crossref PubMed Scopus (123) Google Scholar). To gain insight into the function of 14-3-3ζ in CHO cells, we transfected cells with cDNA for full-length GP Ibα or m591. Because GP Ibα is not efficiently expressed in the membrane of CHO cells unless all three subunits of the GP Ib-IX complex are present (40Lopez J.A. Leung B. Reynolds C.C. Li C.Q. Fox J.E.B. J. Biol. Chem. 1992; 267: 12851-12859Abstract Full Text PDF PubMed Google Scholar), we also transfected the cells with cDNA for GP Ibβ and GP IX. FACS analysis revealed that comparable amounts of full-length and truncated protein were expressed (Fig. 1). Many integrins cannot bind extracellular ligands until intracellular signals “activate” the integrins by inducing clustering or conformational changes (41Woodside D.G. Liu S. Ginsberg M.H. Thromb. Haemostasis. 2001; 86: 316-323Crossref PubMed Scopus (65) Google Scholar). Activation can be induced by signals transmitted across a variety of cytokine or adhesion receptors (41Woodside D.G. Liu S. Ginsberg M.H. Thromb. Haemostasis. 2001; 86: 316-323Crossref PubMed Scopus (65) Google Scholar). In platelets, activation is induced by signals transmitted as GP Ib-IX binds vWf (42De Marco L. Girolami A. Zimmerman T.S. Ruggeri Z.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7424-7428Crossref PubMed Scopus (96) Google Scholar). Previously, we have shown that binding of vWf to GP Ib-IX expressed in CHO cells also induces signals that activate endogenous CHO cell integrins, converting them from a form that cannot interact with vWf to one that can (28Zaffran Y. Meyer S.C. Negrescu E. Reddy K.B. Fox J.E.B. J. Biol. Chem. 2000; 275: 16779-16787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). These signals are transmitted across GP Ib-IX whether the GP Ibα cytoplasmic domain contains the 14-3-3ζ binding site or not (35Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Interaction of activated integrin with vWf activates pathways that induce cytoskeletal reorganizations, changes in cell shape, and cell spreading (28Zaffran Y. Meyer S.C. Negrescu E. Reddy K.B. Fox J.E.B. J. Biol. Chem. 2000; 275: 16779-16787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In the present study, we investigated the possibility that 14-3-3ζ is involved in integrin-induced signal transmission by determining whether integrin-induced cytoskeletal reorganizations were different in cells expressing full-length GP Ibα (which sequesters CHO cell 14-3-3ζ) compared with cells expressing GP Ibα that lacked the 14-3-3ζ binding site. Cells were plated on vWf and allowed to spread for up to 2 h. As reported previously (28Zaffran Y. Meyer S.C. Negrescu E. Reddy K.B. Fox J.E.B. J. Biol. Chem. 2000; 275: 16779-16787Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), the initial adhesion of the cells was induced by GP Ib-IX as shown by inhibition of adhesion by antibodies against GP Ibα, whereas the spreading resulted from activation of integrins and signaling across ligand-occupied integrin, because RGDS, EDTA, and antibodies against αvβ3 inhibited cell spreading. Spreading was markedly inhibited in cells expressing full-length GP Ibα as compared with truncated GP Ibα (Fig. 2). The area of cells expressing full-length GP Ibα was 59% of the area of the cells expressing truncated GP Ibα after 20 min of spreading, 62% after 40 min, and 45.7% after 120 min of spreading (Fig. 3). The area of 100 cells was measured for each cell line and each time point. Typically, spreading of cells on integrin substrates leads to actin polymerization and formation of various actin filament organizations. The earliest changes are induced by Cdc42, which causes localized polymerization of filaments that push membranes outwards in fine filopodia (43Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3746) Google Scholar). Activation of Rac subsequently causes the formation of networks of submembranous filaments and the extension of lamellipodia (43Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3746) Google Scholar, 44Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3083) Google Scholar). At later times, activation of RhoA leads to the formation of stress fibers that mediate the stable attachment of spread cells (43Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3746) Google Scholar, 45Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar).Fig. 3Comparison of the cell area during the spreading of CHO cells expressing GP Ib-IX containing full-length or truncated GP Ibα (m591). Cells transfected with the cDNAs for GP Ibβ and GP IX together with either full-length GP Ibα (black bars) or truncated GP Ibα (m591) (dotted bars) were allowed to spread on vWf-coated slides in the presence of botrocetin for the indicated times. Cells we" @default.
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• W2019440050 cites W2019326922 @default.
• W2019440050 cites W2021528141 @default.
• W2019440050 cites W2021538852 @default.
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• W2019440050 cites W2032961622 @default.
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• W2019440050 cites W2043083034 @default.
• W2019440050 cites W2046943799 @default.
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• W2019440050 cites W2048168473 @default.
• W2019440050 cites W2066367871 @default.
• W2019440050 cites W2069576969 @default.
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• W2019440050 cites W2093226717 @default.
• W2019440050 cites W2093628776 @default.
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