SH2-Bβ Is a Rac-binding Protein That Regulates Cell Motility
2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês
10.1074/jbc.m111138200
ISSN1083-351X
AutoresMaria Diakonova, David R. Gunter, James Herrington, Christin Carter‐Su,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe Src homology 2 (SH2) domain-containing protein SH2-Bβ binds to and is a substrate of the growth hormone (GH) and cytokine receptor-associated tyrosine kinase JAK2. SH2-Bβ also binds, via its SH2 domain, to multiple activated growth factor receptor tyrosine kinases. We have previously implicated SH2-Bβ in GH and platelet-derived growth factor regulation of the actin cytoskeleton. We extend these findings by establishing a potentiating effect of SH2-Bβ on GH-dependent cell motility and defining regions of SH2-Bβ required for this potentiation. Time-lapse video microscopy, phagokinetic, and/or wounding assays demonstrate reduced movement of cells overexpressing SH2-Bβ lacking an intact SH2 domain because of a point mutation or a C-terminal truncation. An N-terminal proline-rich domain (amino acids 85–106) of SH2-Bβ is required for inhibition of cellular motility by SH2 domain-deficient mutants. Co-immunoprecipitation experiments indicate that Rac binds to this domain. GH is shown to activate endogenous Rac, and dominant negative mutants of SH2-Bβ are shown to inhibit membrane ruffling induced by constitutively active Rac. These findings suggest that SH2-Bβ is an adapter protein that facilitates actin rearrangement and cellular motility by recruiting Rac and potentially Rac-regulating, Rac effector, or other actin-regulating proteins to activated cytokine (e.g. GH) and growth factor receptors. The Src homology 2 (SH2) domain-containing protein SH2-Bβ binds to and is a substrate of the growth hormone (GH) and cytokine receptor-associated tyrosine kinase JAK2. SH2-Bβ also binds, via its SH2 domain, to multiple activated growth factor receptor tyrosine kinases. We have previously implicated SH2-Bβ in GH and platelet-derived growth factor regulation of the actin cytoskeleton. We extend these findings by establishing a potentiating effect of SH2-Bβ on GH-dependent cell motility and defining regions of SH2-Bβ required for this potentiation. Time-lapse video microscopy, phagokinetic, and/or wounding assays demonstrate reduced movement of cells overexpressing SH2-Bβ lacking an intact SH2 domain because of a point mutation or a C-terminal truncation. An N-terminal proline-rich domain (amino acids 85–106) of SH2-Bβ is required for inhibition of cellular motility by SH2 domain-deficient mutants. Co-immunoprecipitation experiments indicate that Rac binds to this domain. GH is shown to activate endogenous Rac, and dominant negative mutants of SH2-Bβ are shown to inhibit membrane ruffling induced by constitutively active Rac. These findings suggest that SH2-Bβ is an adapter protein that facilitates actin rearrangement and cellular motility by recruiting Rac and potentially Rac-regulating, Rac effector, or other actin-regulating proteins to activated cytokine (e.g. GH) and growth factor receptors. growth hormone green fluorescent protein nerve growth factor platelet-derived growth factor Src homology-2 fluorescein isothiocyanate Chinese hamster ovary PAK effector binding domain p21-activated protein kinase guanine nucleotide exchange factor Janus kinase Cell migration is critical for many vital biological functions, including embryonic development, the inflammatory immune response, wound repair, tumor formation and metastasis, and tissue remodeling and growth. The actin cytoskeleton provides both the protrusive and contractile forces required for cell migration via a combination of actin polymerization and depolymerization, actin filament cross-linking, and the interaction of myosin-based motors with actin filaments (1Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3288) Google Scholar). The complexity of cell motility and the fact that it is regulated by many hormones, cytokines, and growth factors, including growth hormone (GH)1 (2Wiedermann C.J. Reinisch N. Braunsteiner H. Blood. 1993; 82: 954-960Crossref PubMed Google Scholar) and platelet-derived growth factor (PDGF) (3Seppa H. Grotendorst G. Seppa S. Schiffmann E. Martin G.R. J. Cell Biol. 1982; 92: 584-588Crossref PubMed Scopus (561) Google Scholar), suggest that multiple signaling mechanisms exist to regulate this process. Multiple signaling proteins have been implicated in the regulation of the actin cytoskeleton and cellular motility. Prominent among these are members of the Rho small GTPase family of proteins (4Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (768) Google Scholar, 5Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3744) Google Scholar). Activated Cdc42 has been implicated in the formation of filopodia, long thin extensions containing actin bundles. Activated Rac has been implicated in the formation of lamellipodia and membrane ruffles, broader, weblike extensions. Activated Rho is thought to mediate the formation of stress fibers, elongated actin bundles that traverse the cells and promote cell attachment to the extracellular matrix through focal adhesions. A number of other proteins have been implicated in the regulation of the actin cytoskeleton, including Rho family effector (e.g. PAK, N-WASP (neural Wiskott- Aldrich syndrome protein), ROCK (Rho-associated kinase)) and regulatory proteins (e.g. GEFs (guanine nucleotide exchange factor(s), GTPase-activating proteins, and GDP dissociation inhibitor(s)), adapter or scaffolding proteins (e.g. Nck, Crk, Grb2), and cytoskeleton-regulating/binding proteins (e.g. WASP family, Arp2/3 complex, profilin, ADF (actin-depolymerizing factor)/cofilin, gelsolin) (6Yin H.L. Stull J.T. J. Biol. Chem. 1999; 274: 32529-32530Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 7Machesky L.M. Insall R.H. J. Cell Biol. 1999; 146: 267-272Crossref PubMed Scopus (214) Google Scholar, 8Borisy G.G. Svitkina T.M. Curr. Opin. Cell Biol. 2000; 12: 104-112Crossref PubMed Scopus (404) Google Scholar). However, how all these molecules work together to enable cells to precisely regulate their actin cytoskeleton in response to specific stimuli remains poorly understood. Recently, our laboratory implicated the SH2 domain-containing protein SH2-Bβ in the regulation of the actin cytoskeleton by GH and PDGF (9Herrington J. Diakonova M. Rui L. Gunter D.R. Carter-Su C. J. Biol. Chem. 2000; 275: 13126-13133Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) and in nerve growth factor (NGF)-induced morphological differentiation of PC12 cells into neuronal-like cells (10Rui L. Herrington J.B. Carter-Su C. J. Biol. Chem. 1999; 274: 10590-10594Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). We demonstrated that SH2-Bβ co-localizes with actin in membrane ruffles. Furthermore, introduction into cells of mutant SH2-Bβ lacking a functional SH2 domain inhibited GH- and PDGF-dependent membrane ruffling and pinocytosis and blocked NGF-induced morphological differentiation of PC12 cells. SH2-Bβ is a 670-amino acid, widely expressed, SH2 domain-containing protein that was originally identified by our laboratory in a yeast two-hybrid screen as a binding partner of the tyrosine kinase JAK2 (11Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar). JAK2 binds to multiple members of the cytokine family of receptors, including the GH receptor, and is activated in response to ligand binding to those receptors. This activation of JAK2 is thought to be the initiating step in cytokine receptor signaling (12Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (823) Google Scholar). SH2-Bβ and/or other isoforms (α, γ) of SH2-B have also been shown to bind, via their SH2 domain, to the activated form of multiple receptor tyrosine kinases, including the receptors for PDGF, NGF (TrkA), insulin, and insulin-like growth factor (11Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar, 13Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar, 14Rui L. Gunter D.R. Herrington J. Carter-Su C. Mol. Cell. Biol. 2000; 20: 3168-3177Crossref PubMed Scopus (51) Google Scholar, 15Qian X. Riccio A. Zhang Y. Ginty D.D. Neuron. 1998; 21: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 16Wang J. Riedel H. J. Biol. Chem. 1998; 273: 3136-3139Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 17Riedel H. Yousaf N. Zhao Y. Dai H. Deng Y. Wang J. Oncogene. 2000; 19: 39-50Crossref PubMed Scopus (39) Google Scholar), suggesting that SH2-Bβ may play a fundamental role in cell function. SH2-Bβ lacks a known enzymatic domain, but contains multiple protein and/or lipid interacting domains, including a pleckstrin homology domain and three proline-rich regions in addition to its SH2 domain (Fig. 1). This domain structure suggests that SH2-B isoforms may serve as adapter or scaffolding proteins that recruit proteins to activated membrane receptor complexes. Consistent with this, SH2-B isoforms are also phosphorylated on tyrosines in response to GH, PDGF, and NGF (10Rui L. Herrington J.B. Carter-Su C. J. Biol. Chem. 1999; 274: 10590-10594Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 11Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar,15Qian X. Riccio A. Zhang Y. Ginty D.D. Neuron. 1998; 21: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 18Rui L. Carter-Su C. J. Biol. Chem. 1998; 273: 21239-21245Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar); phosphorylated tyrosines often serve as binding sites for proteins containing SH2 or PTB domains (19Kuriyan J. Cowburn D. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 259-288Crossref PubMed Scopus (468) Google Scholar). SH2-Bβ has been shown to activate JAK2 (13Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar) and TrkA (20Qian X. Ginty D.D. Mol. Cell. Biol. 2001; 21: 1613-1620Crossref PubMed Scopus (71) Google Scholar). However, some SH2-Bβ mutants that act as dominant negatives for membrane ruffling and neuronal differentiation do not appear to block kinase activity, suggesting that SH2-Bβ serves a function in cytokine and growth factor signaling in addition to regulation of kinase activity. Taken together, these observations suggest that SH2-Bβ may regulate the actin cytoskeleton by serving as a scaffolding or adapter protein that recruits to activated receptor kinases molecules that regulate the actin cytoskeleton. In this work, we extend our initial findings implicating SH2-Bβ in the regulation of the actin cytoskeleton by providing strong evidence that SH2-Bβ is required for maximal cell motility using time-lapse video microscopy and two motility assays (phagokinetic and wounding). GH is shown to stimulate cell motility, and two regions of SH2-Bβ (the SH2 domain and an N-terminal proline-rich domain (amino acids 85–106)) are implicated in the effect of SH2-Bβ on GH-stimulated cell motility. GH is shown to activate the small GTPase Rac, and Rac is shown to bind to SH2-Bβ; the binding requires amino acids 85–106 of SH2-Bβ. Overexpression of dominant negative mutants of SH2-Bβ block the ability of constitutively active Rac to promote membrane ruffling, consistent with intact SH2-Bβ being required for Rac promotion of membrane ruffling. These results suggest that SH2-Bβ acts as an adapter or scaffolding protein that recruits Rac and potentially Rac-regulating proteins, Rac effector proteins, and/or other actin-regulating proteins to membrane cytokine and growth factor receptors. The stock of murine 3T3-F442A fibroblasts was provided by Dr. H. Green (Harvard University, Cambridge, MA). Recombinant human GH was the gift of Eli Lilly Co. Monoclonal anti-Myc antibody (αMyc; 9E10) and rabbit anti-mouse IgG were obtained from Santa Cruz Biotechnology. Ascites fluid containing 9E10, used for immunoprecipitation experiments (1:200 dilution), was obtained from the Michigan Diabetes Research and Training Center Hybridoma Core Facility. Polyclonal antibodies raised against a glutathione S-transferase fusion protein containing the C-terminal portion of SH2-Bβ has been described previously (11Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar). Monoclonal αGFP (Living Colors A.v. JL-8) was obtained from CLONTECH Laboratories, Inc., and monoclonal αRac (clone 238A8) was from Upstate Biotechnology, Inc. Goat anti-mouse-Cy5 and Texas Red-phalloidin were from Molecular Probes, Inc. cDNAs encoding GFP-tagged wild-type SH2-Bβ, SH2-Bβ(R555E), SH2-Bβ(1–555), and SH2-Bβ(504–670) have been described previously (13Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar, 14Rui L. Gunter D.R. Herrington J. Carter-Su C. Mol. Cell. Biol. 2000; 20: 3168-3177Crossref PubMed Scopus (51) Google Scholar). cDNAs encoding GFP-tagged (1–85, 1–106, and 504–670 (R555E)) or Myc-tagged (61–670, 118–670, and 118–670 (R555E)) truncation mutants of SH2-Bβ were made in our laboratory by introducing appropriate restriction sites or stop codons using a Stratagene QuikChange kit and SH2-Bβ or SH2-Bβ(R555E) as a template. 2D. R. Gunter, H. Chong, and C. Carter-Su, manuscript in preparation. cDNAs encoding mutant Rac N17, V12, and L61 (21Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3081) Google Scholar) were used with the permission of A. Hall (University College, London, United Kingdom). cDNA encoding GST-PAK effector binding domain (PBD) of PAK1 was a gift of G. Bokoch (Scripps Research Institute, La Jolla, CA). cDNA encoding rat GH receptor (22Emtner M. Mathews L.S. Norstedt G. Mol. Endocrinol. 1990; 4: 2014-2020Crossref PubMed Scopus (43) Google Scholar) was provided by G. Norstedt (Karolinska Institute, Stockholm, Sweden). cDNA encoding JAK2 was a gift of J. Ihle (St. Jude Children's Research Hospital, Memphis, TN). 3T3-F442A and 293T cells were cultured as described (18Rui L. Carter-Su C. J. Biol. Chem. 1998; 273: 21239-21245Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). 3T3-F442A cells were plated on coverslips and transfected with 2.5 μg (total) of cDNA using Transfast (Promega). 293T cells plated in 100-mm dishes were transfected with 10 μg of cDNA using calcium phosphate precipitation. 36 h after transfection, 3T3-F442A cells on coverslips were incubated in serum-free medium overnight. Cells were observed by phase-contrast optics in a Nikon TE300 inverted microscope with shutter-controlled illumination (Uniblitz, Rochester, NY). Cells were recorded for 2 min, treated with GH as indicated, and recorded further. Images were collected using a cooled, digital CCD camera (Quantix, Photometrics, Tucson, AZ) and were recorded (1 frame/15 s) and stored using MetaMorph 2.0 image analysis software (Universal Imaging, West Chester, PA). To quantify lamellipodia extension and retraction, individual transfected cells were located with a FITC filter set. Binary images of the cell profile were generated from the video frames using MetaMorph. Subtracting a binary image from the subsequent one produced a binary image of areas of extension, and subtracting the second binary image from the first produced a binary image of areas of retraction (23Bershadsky A.D. Vaisberg E.A. Vasiliev J.M. Cell Motil. Cytoskel. 1991; 19: 152-158Crossref PubMed Scopus (92) Google Scholar). The area of extension (retraction) was normalized to the cross-sectional area of the cell. Six cells expressing GFP alone or GFP-SH2-Bβ(R555E), five cells expressing GFP-SH2-Bβ, and three cells expressing GFP-SH2-Bβ(1–555) (data not shown) were videotaped and analyzed. For the phagokinetic assay (24Albrecht-Buehler G. Cell. 1977; 11: 395-404Abstract Full Text PDF PubMed Scopus (384) Google Scholar), cells were plated on colloid gold-covered coverslips 48 h after transfection. After 2 or 24 h, the coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature and mounted on slides. Individual transfected cells expressing GFP-tagged forms of SH2-Bβ were located with a FITC filter set using a Nikon TE200 microscope. Phase-contrast images were collected and analyzed by NIH Image software. The particle-free area indicating where cells moved was measured for 10 cells each in three (3T3-F442A cells) or four (293T cells) independent experiments. The phagokinetic index was calculated as a ratio of the particle-free area to the cross-sectional area of the cell. Migration of cells into a wound was assessed as described (25Joberty G. Perlungher R.R. Macara I.G. Mol. Cell. Biol. 1999; 19: 6585-6597Crossref PubMed Scopus (106) Google Scholar, 26Sells M.A. Pfaff A. Chernoff J. J. Cell Biol. 2000; 151: 1449-1458Crossref PubMed Scopus (134) Google Scholar). 293T cells were transfected with cDNAs encoding epitope-tagged forms of SH2-Bβ, GFP, or Myc-tagged Lin-7, with or without cDNA encoding rat GH receptor. Cells were re-plated at both high (5 × 105 cells/dish) and low (1.5 × 105 cells/dish) density onto 100-mm diameter dishes. After 2 days, when cells in the high density dish reached a monolayer, a plastic pipette tip was drawn across the center of some of the plate several times to produce a clean 1-mm-wide wound area of some of the culture (Fig. 5). After an additional 24-h incubation to permit migration of cells into the empty area, migration of the cells was examined using a phase-contrast microscope and the GFP-tagged cells visualized by fluorescence microscopy. In other cells (70–80% confluent) (Fig. 6), cultures were serum-deprived for 24 h before being wounded. Those cells were rinsed with phosphate-buffered saline and incubated with Dulbecco's modified Eagle's medium + 0.5% calf serum with GH as indicated. After another 24-h incubation to permit migration of cells into the empty area, migration of the cells was examined using a phase-contrast microscope with a calibrated eyepiece. The distance between the leading edge of the migrating cells from the edge of the wound was measured. The number of tag-positive and tag-negative cells in the wound area were counted (100 cells total/wound). The number of tag-positive and negative cells (100 cells total) were also counted in the low density, non-wounded plate. For Myc-tagged plasmids, cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100, and stained with αMyc. To take into account the efficiency of transfection, a motility index was calculated as a ratio of percentage of tag-positive cells migrating into the wound area to the percentage of tag-positive cells in the low density dish. To confirm that differences in the number of cells present in the wound were the result of differences in motility rather than in cell proliferation, serum-deprived, wounded, and GH-treated (500 ng/ml GH) cells expressing GFP alone, GFP-SH2-Bβ, and GFP-SH2-Bβ(R555E) were analyzed by fluorescence-activated cell sorting (Elite, ESP) in parallel with non-wounded but serum-deprived and GH-treated cells. The percentage of GFP positive cells was similar for wounded, GH-treated plates and non-wounded, non-hormone-treated plates. Furthermore, the majority (60–70%) of the cells in all plates were stopped in the G1 phase of the cell cycle (data not shown) in contrast to cells maintained in serum, in which >60% of the cells were in S phase as determined by fluorescence-activated cell sorting of DNA stained cells (propidium iodide, Hoechst, or 4,6-diamidino-2-phenylindole). Ten wounds were sampled for each type of transfection. Each experiment was repeated at least three times with similar results.Figure 6Maximal GH-dependent migration of cells into wound requires the SH2 domain and a proline-rich region of SH2-Bβ. 293T cells were transfected with cDNA encoding the indicated forms of epitope-tagged SH2-Bβ.A, cell movement into a wound was assessed as an average migration distance of the leading edge. B, a motility index was calculated as a ratio of % transfected cells migrating into the wound to the % transfected cells. Bars represent mean ± S.E., n = 30. *, p < 0.05 compared with cells expressing vehicle with the same GH treatment; +,p < 0.05 compared with vehicle without GH.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Activated Rac was detected using the PBD (27Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). 293T cells overexpressing GH receptor or 3T3-F442A cells were deprived overnight and treated with 500 ng/ml GH. Cells were lysed and solubilized proteins incubated with GST-PBD and glutathione-agarose beads (Sigma-Aldrich). Bound proteins were immunoblotted with αRac (1:1 000 dilution) and analyzed using enhanced chemiluminescence (ECL) (Amersham Biosciences, Inc.). Constitutively active Rac L61 expressed in 293T cells was used as a positive control (data not shown). For experiments investigating interaction between forms of GFP-SH2-Bβ and Myc-Rac N17, Myc-Rac V12, or Myc-Rac L61, Rac was immunoprecipitated with αMyc using rabbit anti-mouse and protein A-agarose. SH2-Bβ was visualized by immunoblotting with αGFP. 3T3-F442A cells on coverslips were co-transfected with cDNAs encoding different forms of GFP-tagged SH2-Bβ and Myc-tagged Rac. 48 h later, cells were fixed for 15 min in 4% paraformaldehyde in intracellular buffer consisting of 30 mm HEPES, pH 7.4, 10 mm EGTA, 0.5 mm EDTA, 5 mm MgSO4, 33 mm potassium acetate, 5% polyethylene glycol 400, and 0.02% NaN3. Cells were permeabilized with 1% Triton X-100 in intracellular buffer containing 4% paraformaldehyde for 15 min (28Swanson J.A. Johnson M.T. Beningo K. Post P. Mooseker M. Araki N. J. Cell Sci. 1999; 112: 307-316Crossref PubMed Google Scholar). Coverslips were incubated with αMyc followed by goat anti-mouse-Cy5. F-actin was stained with Texas Red-phalloidin. Confocal imaging was performed with a Noran Oz laser scanning confocal microscope (Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center). The images presented in the figures are representative of at least three separate experiments. Data from at least three separate transfections were pooled and analyzed using a two-tailed unpairedt test. When individual experiments were analyzed, the results were indistinguishable from those obtained from the pooled data. Differences were considered to be statistically significant atp < 0.05. Results are expressed as the mean ± S.E. In all imaging experiments, measurements were taken before determining the identity of the cells on the plate/coverslip. To examine the role of SH2-Bβ in cell motility, we first made a time-lapse recording of 3T3-F442A fibroblasts overexpressing GFP-tagged wild-type SH2-Bβ or an SH2 domain-deficient version of this protein (SH2-Bβ(R555E)). 3T3-F442A cells are highly responsive to GH (29Nixon T. Green H. J. Cell. Physiol. 1983; 115: 291-296Crossref PubMed Scopus (43) Google Scholar,30Carter-Su C. Smit L.S. Recent Prog. Hormone Res. 1997; 53: 61-83Google Scholar). In SH2-Bβ(R555E), the critical Arg (R) within the FLVR motif of the SH2 domain is mutated to Glu (E). This mutant exhibits substantially reduced binding to JAK2 and fails to serve as a substrate of JAK2 (13Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar). Transfected cells were incubated overnight in serum-free medium. GFP-positive cells were located using a FITC filter set. Recording was initiated, and then GH was added. Because these cells did not show directional movement during the time of observation, our study assessed lamellipodia activity. The term “lamellipodia” is used here to refer to dynamic actin-rich extensions at the periphery of the cell (31Small J.V. Electron Microsc. Rev. 1988; 1: 155-174Crossref PubMed Scopus (144) Google Scholar). Lamellipodia activity was quantified by measuring the percentage of cell retraction and extension. Addition of 500 ng/ml GH resulted within 10 min in high lamellipodia activity of untransfected cells (Fig.2 A, top two rows) and cells overexpressing GFP (Fig.2 A, top row) or GFP-SH2-Bβ (data not shown) (transfected cells are indicated in the leftmost panels). Lamellipodia activity was often accompanied by the appearance and centripetal movement of ruffles. In contrast, cells expressing SH2-Bβ(R555E) (Fig. 2 A, second row panels) exhibited very little lamellipodia activity. When cells were treated with 25 ng/ml GH (Fig. 2 A,bottom two rows), a concentration of GH reported previously to cause minimal ruffling in untransfected cells and cells overexpressing GFP (Ref. 9Herrington J. Diakonova M. Rui L. Gunter D.R. Carter-Su C. J. Biol. Chem. 2000; 275: 13126-13133Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), only minimal lamellipodia activity was detected in untransfected cells or cells overexpressing GFP (Fig. 2 A, bottom rows). In contrast, substantial lamellipodia activity was detected in cells overexpressing full-length SH2-Bβ (Fig. 2 A,bottom row). A plot of lamellipodia activity over time is shown (Fig. 2 B) for single representative cells treated with the higher concentration of GH (500 ng/ml). GH stimulated similar levels of lamellipodia activity in cells expressing GFP alone (Fig. 2 B,top panel) or GFP-SH2-Bβ (Fig. 2 B,middle panel); activity was maximal 10 min after GH addition. In contrast, GH stimulated only minimal lamellipodia activity in cells expressing SH2-Bβ(R555E) (Fig. 2 B,bottom panel). The data in Fig. 2 (Aand B) show that overexpressing SH2-Bβ enhances and overexpressing SH2-Bβ(R555Ε) inhibits GH-induced lamellipodia activity, suggesting that SH2-Bβ with an intact SH2 domain is required for maximal GH-induced lamellipodia activity. To examine further the effect of SH2-Bβ on cell motility, we performed a gold particle motility (or phagokinetic) assay. This assay measures the disappearance of gold particles from a coverslip, a process requiring both phagocytosis and locomotion. Cells transfected with the desired cDNA were plated on coverslips coated with colloidal gold particles and incubated at 37 °C for either 2 h (Fig.3, A–H) or 24 h (Figs.3 I and 4). Cells remove particles while they move, thereby producing areas that are free of colloidal gold. These areas were quantified using NIH Image analysis software.Figure 4Epithelial derived cells, like fibroblasts, exhibit phagokinesis. A–D, 3T3-F442A fibroblasts (A), COS cells (B), CHO (C), and 293T cells (D) were tested for their ability to phagocytose colloid gold. Representative tracks after 24 h plating on colloid gold are shown. Scale bar, 25 μm. E, the average phagokinetic index was calculated for 20 cells for each cell line at various times after plating. Bars represent mean ± S.E. F, 293T cells expressing GFP alone or GFP-tagged SH2-Bβ, SH2-Bβ(1–555), SH2-Bβ(R555E), or SH2-Bβ(504–670) were treated and analyzed after 24 h as described for Fig. 3I. Bars represent mean ± S.E.,n = 40. *, p < 0.05 compared with cells expressing GFP.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Initially we measured the motility of 3T3-F442A fibroblasts. Fibroblasts are known to be very motile (32Varani J. Orr W. Ward P.A. Am. J. Pathol. 1978; 90: 159-171PubMed Google Scholar). 3T3-F442A fibroblasts expressing GFP alone (Fig. 3 A) or GFP-SH2-Bβ (Fig.3 C), and identified as GFP-positive cells using a FITC filter set, removed gold particles from a portion of the coverslip during 2 h. Cells expressing GFP-SH2-Bβ(1–555) lacking a functional SH2 domain removed gold particles from a substantially smaller area (Fig. 3 E), as predicted from the video microscopy experiments. Like the SH2 domain-deficient mutants of SH2-Bβ, the C terminus of SH2-Bβ (SH2-Bβ(504–670)) was shown previously to inhibit GH-induced membrane ruffling (9Herrington J. Diakonova M. Rui L. Gunter D.R. Carter-Su C. J. Biol. Chem. 2000; 275: 13126-13133Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The difference in phagokinetic properties between cells transfected with SH2-Bβ(504–670) and nontransfected cells is clearly seen in Fig.3 G, which contains both a transfected and nontransfected cell. The GFP-positive cell (right side) removed very few gold particles whereas the nontransfected cell (left side) produced clear gold-free areas. The area lacking gold particles after 24 h was quantified and normalized for multiple cells. The phagokinetic index was calculated and plotted in Fig. 3 I. Cells expressing either GFP-SH2-Bβ(1–555) or GFP-SH2-Bβ(504–670) demonstrated reduced phagokinesis. To determine whether SH2-Bβ is important for cell motility in non-fibroblast cells, we tested cultured human embryonic kidney (293T) cells, monkey embryonic kidney (COS) cells, and Chinese hamster ovary (CHO) cells for their ability to phagocytose colloid gold during 24 h (Fig. 4). These three cell lines are all epithelial cell-derived and are more amenable to transfection than 3T3-F442A cells. Although phagokinesis of these three cell types was significantly less than that observed for 3T3-F442A fibroblasts (Fig.4 E), all formed visible areas that could be measured (Fig.4, A–D). As predicted from the studies using 3T3-F442A fibroblasts, 293T cells expressing GFP alone o
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