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• W2044821615 abstract "Interleukin-1 (IL-1)-induced Ca2+ signaling in fibroblasts is constrained by focal adhesions. This process involves the proteintyrosine phosphatase SHP-2, which is critical for IL-1-induced phosphorylation of phospholipase Cγ1, thereby enhancing IL-1-induced Ca2+ release and ERK activation. Currently, the mechanisms by which SHP-2 modulates Ca2+ release from the endoplasmic reticulum are not defined. We used immunoprecipitation and fluorescence protein-tagged SHP-2 or endoplasmic reticulum (ER)-protein expression vectors, and an ER-specific calcium indicator, to examine the functional relationships between SHP-2, focal adhesions, and IL-1-induced Ca2+ release from the ER. By total internal reflection fluorescence microscopy to image subplasma membrane compartments, SHP-2 co-localized with the ER-associated proteins calnexin and calreticulin at sites of focal adhesion formation in fibroblasts. IL-1β promoted time-dependent recruitment of SHP-2 and ER proteins to focal adhesions; this process was blocked in cells treated with small interfering RNA for SHP-2 and in cells expressing a Y542F SHP-2 mutant. IL-1 stimulated inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the ER subjacent to the plasma membrane that was tightly localized around fibronectin-coated beads and was reduced 4-fold in cells expressing Tyr-542 SHP-2 mutant. In subcellular fractions enriched for ER proteins, immunoprecipitation demonstrated that IL-1-enhanced association of SHP-2 with the type 1 inositol 1,4,5-trisphosphate receptor was dependent on Tyr-542 of SHP-2. We conclude that Tyr-542 of SHP-2 modulates IL-1-induced Ca2+ signals and association of the ER with focal adhesions. Interleukin-1 (IL-1)-induced Ca2+ signaling in fibroblasts is constrained by focal adhesions. This process involves the proteintyrosine phosphatase SHP-2, which is critical for IL-1-induced phosphorylation of phospholipase Cγ1, thereby enhancing IL-1-induced Ca2+ release and ERK activation. Currently, the mechanisms by which SHP-2 modulates Ca2+ release from the endoplasmic reticulum are not defined. We used immunoprecipitation and fluorescence protein-tagged SHP-2 or endoplasmic reticulum (ER)-protein expression vectors, and an ER-specific calcium indicator, to examine the functional relationships between SHP-2, focal adhesions, and IL-1-induced Ca2+ release from the ER. By total internal reflection fluorescence microscopy to image subplasma membrane compartments, SHP-2 co-localized with the ER-associated proteins calnexin and calreticulin at sites of focal adhesion formation in fibroblasts. IL-1β promoted time-dependent recruitment of SHP-2 and ER proteins to focal adhesions; this process was blocked in cells treated with small interfering RNA for SHP-2 and in cells expressing a Y542F SHP-2 mutant. IL-1 stimulated inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the ER subjacent to the plasma membrane that was tightly localized around fibronectin-coated beads and was reduced 4-fold in cells expressing Tyr-542 SHP-2 mutant. In subcellular fractions enriched for ER proteins, immunoprecipitation demonstrated that IL-1-enhanced association of SHP-2 with the type 1 inositol 1,4,5-trisphosphate receptor was dependent on Tyr-542 of SHP-2. We conclude that Tyr-542 of SHP-2 modulates IL-1-induced Ca2+ signals and association of the ER with focal adhesions. IL-1β 3The abbreviations used are: IL-1, interleukin-1; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 2-APB, 2-aminoethoxydiphenyl borate; ERK, extracellular signal-regulated kinase; ER, endoplasmic reticulum; BSA, bovine serum albumin; siRNA, small interfering RNA; PBS, phosphate-buffered saline; GFP, green fluorescent protein; YFP, yellow fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TIRF, total internal reflection fluorescence. 3The abbreviations used are: IL-1, interleukin-1; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 2-APB, 2-aminoethoxydiphenyl borate; ERK, extracellular signal-regulated kinase; ER, endoplasmic reticulum; BSA, bovine serum albumin; siRNA, small interfering RNA; PBS, phosphate-buffered saline; GFP, green fluorescent protein; YFP, yellow fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TIRF, total internal reflection fluorescence. is a key pro-inflammatory cytokine that mediates degradation of the extracellular matrix in several common diseases, including rheumatoid arthritis, pulmonary fibrosis, periodontitis, and cancer (1Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar, 2Honig J. Rordorf-Adam C. Siegmund C. Wiedemann W. Erard F. J. Periodontal. Res. 1989; 24: 362-367Crossref PubMed Scopus (172) Google Scholar, 3O'Neill L.A. Dinarello C.A. Immunol. Today. 2000; 21: 206-209Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 4van den Berg W.B. Z. Rheumatol. 1999; 58: 136-141Crossref PubMed Scopus (102) Google Scholar). IL-1 enhances destruction of extracellular matrices by promoting increased expression of proteases from tissue macrophages and fibroblasts. The expression of matrix-degrading genes in these cells is regulated by the amplitude and duration of IL-1-induced signals (5Boyle D.L. Han Z. Rutter J.L. Brinckerhoff C.E. Firestein G.S. Arthritis Rheum. 1997; 40: 1772-1779Crossref PubMed Scopus (20) Google Scholar, 6Bhat-Nakshatri P. Newton T.R. Goulet Jr., R. Nakshatri H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6971-6976Crossref PubMed Scopus (80) Google Scholar) and most notably of signals that activate mitogen-activated protein kinases (7Brauchle M. Gluck D. Di Padova F. Han J. Gram H. Exp. Cell Res. 2000; 258: 135-144Crossref PubMed Scopus (85) Google Scholar, 8Reunanen N. Westermarck J. Hakkinen L. Holmstrom T.H. Elo I. Eriksson J.E. Kahari V.M. J. Biol. Chem. 1998; 273: 5137-5145Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). In cultured fibroblasts IL-1-induced activation of the mitogen-activated protein kinase ERK is dependent on maturation of focal adhesions (9Lo Y.Y. Luo L. McCulloch C.A. Cruz T.F. J. Biol. Chem. 1998; 273: 7059-7065Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Conceivably, focal adhesion-associated proteins such as integrins and the focal adhesion kinase (10Arora P.D. Ma J. Min W. Cruz T. McCulloch C.A. J. Biol. Chem. 1995; 270: 6042-6049Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) may modulate the generation of IL-1-induced signals. Although IL-1 type 1 (signaling) receptors are restricted largely to focal adhesions in cultured fibroblasts (10Arora P.D. Ma J. Min W. Cruz T. McCulloch C.A. J. Biol. Chem. 1995; 270: 6042-6049Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 11Luo L. Cruz T. McCulloch C. Biochem. J. 1997; 324: 653-658Crossref PubMed Scopus (49) Google Scholar, 12Qwarnstrom E.E. Page R.C. Gillis S. Dower S.K. J. Biol. Chem. 1988; 263: 8261-8269Abstract Full Text PDF PubMed Google Scholar) and thus may account for the dependence of IL-1 signaling on these adhesive domains, other proteins associated with focal adhesions that are post-translationally modified may also contribute to this phenomenon. Many signaling systems in cells utilize protein phosphorylation, a post-translational modification that is controlled in turn by the net relative activities of protein-tyrosine kinases and protein-tyrosine phosphatases. Although protein-tyrosine phosphatases can terminate various receptor tyrosine kinases-induced signals, they can also activate signaling cascades. Protein-tyrosine phosphatases such as SHP-2 form part of the signaling cascade triggered in various protein kinase-linked receptors that lead to ERK activation. For example, SHP-2 helps to sustain ERK activity in response to platelet-derived growth factor (13Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar), possibly by modulating signals arising from integrin-associated proteins (14Zhang S.Q. Yang W. Kontaridis M.I. Bivona T.G. Wen G. Araki T. Luo J. Thompson J.A. Schraven B.L. Philips M.R. Neel B.G. Mol. Cell. 2004; 13: 341-355Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). SHP-2 can also enhance IL-1-induced ERK activation; this process is tightly linked to integrin clustering in focal adhesions (15MacGillivray M. Herrera-Abreu M.T. Chow C.W. Shek C. Wang Q. Vachon E. Feng G.S. Siminovitch K.A. McCulloch C.A. Downey G.P. J. Biol. Chem. 2003; 278: 27190-27198Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 16Herrera Abreu M.T. Wang Q. Vachon E. Suzuki T. Chow C.W. Wang Y. Hong O. Villar J. McCulloch C.A. Downey G.P. J. Cell. Physiol. 2006; 207: 132-143Crossref PubMed Scopus (23) Google Scholar) and is mediated possibly by Src family-related kinase phosphorylation of SHPS-1 and recruitment of SHP-2 (13Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar), or by interaction of SHP-2 with focal adhesion proteins such as paxillin and Gab1 (16Herrera Abreu M.T. Wang Q. Vachon E. Suzuki T. Chow C.W. Wang Y. Hong O. Villar J. McCulloch C.A. Downey G.P. J. Cell. Physiol. 2006; 207: 132-143Crossref PubMed Scopus (23) Google Scholar). Further, SHP-2 regulates cell spreading on fibronectin, focal adhesion formation (17Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), and RhoA activity (18Schoenwaelder S.M. Petch L.A. Williamson D. Shen R. Feng G.S. Burridge K. Curr. Biol. 2000; 10: 1523-1526Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Currently, the mechanisms and the protein-protein interactions that are required for integrins and SHP-2 to mediate IL-1 activation of ERK are not defined. SHP-2 regulates the activity of Src family kinases, and ultimately the activation of ERK, by controlling the recruitment of Csk (14Zhang S.Q. Yang W. Kontaridis M.I. Bivona T.G. Wen G. Araki T. Luo J. Thompson J.A. Schraven B.L. Philips M.R. Neel B.G. Mol. Cell. 2004; 13: 341-355Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). The activation of ERK through Src family kinases is thought to involve phosphorylation of phospholipase Cγ1, a step in the signaling pathway that is required for calcium release from internal stores and the subsequent activation of the Ras-Raf-Mek-ERK cascade (14Zhang S.Q. Yang W. Kontaridis M.I. Bivona T.G. Wen G. Araki T. Luo J. Thompson J.A. Schraven B.L. Philips M.R. Neel B.G. Mol. Cell. 2004; 13: 341-355Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Ras activation may in turn rely on its anchorage to ER membranes (19Chiu V.K. Bivona T. Hach A. Sajous J.B. Silletti J. Wiener H. Johnson 2nd, R.L. Cox A.D. Philips M.R. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (510) Google Scholar) as well as recruitment of SHP-2 to plasma and ER membranes (14Zhang S.Q. Yang W. Kontaridis M.I. Bivona T.G. Wen G. Araki T. Luo J. Thompson J.A. Schraven B.L. Philips M.R. Neel B.G. Mol. Cell. 2004; 13: 341-355Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Currently, the mechanisms by which the various participants in this signaling pathway are marshaled to specific sites within the cell are not known. For many complex signaling systems, including IL-1, anchorage of signaling proteins to specific organelles or organellar membranes is required for effective signal transduction (20Juliano R.L. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 283-323Crossref PubMed Scopus (491) Google Scholar). Because IL-1-induced ERK activation requires the release of Ca2+ from the ER (21Wang Q. Downey G.P. Choi C. Kapus A. McCulloch C.A. Faseb J. 2003; 17: 1898-1900Crossref PubMed Google Scholar), we considered that focal adhesions and the ER may provide spatially discrete staging sites (22Cohen P. Nat. Cell Biol. 2002; 4: E127-E130Crossref PubMed Scopus (747) Google Scholar), enabling SHP-2-dependent activation of ERK by IL-1. Notably, the ER proteins kinectin (23Tran H. Pankov R. Tran S.D. Hampton B. Burgess W.H. Yamada K.M. J. Cell Sci. 2002; 115: 2031-2040Crossref PubMed Google Scholar) and the IP3 receptor (24Sugiyama T. Matsuda Y. Mikoshiba K. FEBS Lett. 2000; 466: 29-34Crossref PubMed Scopus (32) Google Scholar) co-localize with focal adhesions. We have shown earlier that the ER-associated protein calnexin co-localizes with focal adhesions (25Wang Q. Downey G.P. Herrera-Abreu M.T. Kapus A. McCulloch C.A. J. Biol. Chem. 2005; 280: 8397-8406Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and that SHP-2 is critical for IL-1-induced focal adhesion maturation (16Herrera Abreu M.T. Wang Q. Vachon E. Suzuki T. Chow C.W. Wang Y. Hong O. Villar J. McCulloch C.A. Downey G.P. J. Cell. Physiol. 2006; 207: 132-143Crossref PubMed Scopus (23) Google Scholar), in part by regulating phosphorylation of phospholipase Cγ1. Accordingly, we sought to determine what molecular determinants of SHP-2 are responsible for mediating IL-1 promotion of interactions between focal adhesions and the ER. Materials—Fibronectin, poly-l-lysine, BSA, puromycin, anisomycin, and mouse monoclonal antibodies to vinculin were obtained from Sigma. Rabbit polyclonal and mouse monoclonal anti-SHP-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody to phospho-SHP-2 (tyrosine 542) was from Cell Signaling (Beverly, MA). Mouse monoclonal anti-calnexin and anti-Bip/GRP78 were obtained from BD Biosciences (Mississauga, Ontario, Canada). Rabbit anti-calnexin was obtained from Stressgen (Victoria, British Columbia, Canada). Rabbit polyclonal anti-IP3R-1 was obtained from Affinity BioReagents (Golden, CO). Goat antiintegrin α5β1 was purchased from Chemicon (Temecula, CA). FuGENE 6 transfection reagent and X-tremeGENE siRNA transfection reagent was purchased from Roche Applied Science. Acidified bovine type I collagen (Invitrogen) was purchased from Cohesion Technologies Inc. (Palo Alto, CA). Recombinant human IL-1β was obtained from R&D Systems (Minneapolis, MN). Fluo-4/AM and mag-fluo-4/AM were obtained from Molecular Probes (Eugene, OR). 2-APB, xestospongin C, and swinholide A were obtained from Calbiochem. Cell Culture and Bead Preparations—Human gingival fibroblasts were grown in minimal essential medium containing 10% fetal bovine serum. Rat2 cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum. Murine embryonic fibroblasts (SHP-2-reconstituted murine embryonic fibroblasts and SHP-2 542 tyrosine-phenylalanine mutant (SHP-2 Y542F) were grown in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and antibiotics (100 units/ml penicillin, 100 units/ml streptomycin, and 2 μg/ml puromycin). Cells were used between the 5th and 12th passages as previously described (25Wang Q. Downey G.P. Herrera-Abreu M.T. Kapus A. McCulloch C.A. J. Biol. Chem. 2005; 280: 8397-8406Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 26Araki T. Mohi M.G. Ismat F.A. Bronson R.T. Williams I.R. Kutok J.L. Yang W. Pao L.I. Gilliland D.G. Epstein J.A. Neel B.G. Nat. Med. 2004; 10: 849-857Crossref PubMed Scopus (334) Google Scholar). Latex microbeads were coated with fibronectin (10 μg/ml) or BSA (2 mg/ml). For collagen coating, 3 mg/ml acidified, pepsin-digested bovine type I collagen (Celltrix, Palo Alto, CA) was used. NaOH was added to the solution to a final concentration of 0.1 m to equilibrate the pH to 7.4 and thereby facilitate collagen fibril assembly on the beads. Bead suspensions were incubated at 37 °C for 20 min. Beads were washed three times, re-suspended in PBS, and sonicated for 10 s (output setting 3 and power 15%, Branson). Isolation of Focal Adhesions—Cells were grown to 80-90% confluence on 60-mm tissue culture dishes and cooled to 4 °C prior to the addition of collagen-, BSA-, or anti-integrin α5β1-coated magnetite beads. Focal adhesion complexes were isolated from cells after specific incubation time periods as described previously (27Plopper G. Ingber D.E. Biochem. Biophys. Res. Commun. 1993; 193: 571-578Crossref PubMed Scopus (149) Google Scholar). In brief, cells were washed three times with ice-cold PBS to remove unbound beads and scraped into ice-cold cytoskeleton extraction buffer (CKSB, 0.5% Triton X-100, 50 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 20 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, 10 mm PIPES, pH 6.8). The cell-bead suspension was sonicated for 10 s (output setting 3 and power 15%, Branson), and the beads were isolated from the lysate using a magnetic separation stand. The left lysates were used for non-focal adhesion fraction. The beads were re-suspended in fresh ice-cold CKSB, homogenized with a Dounce homogenizer (20 strokes), and re-isolated magnetically. The beads were washed in CSKB, sedimented with a microcentrifuge, re-suspended in Laemmli sample buffer, and placed in a boiling water bath for 5 min to allow the collagen-associated complexes to dissociate from the beads. The beads were sedimented, and lysates were collected for analysis. Subcellular Fractionation—Cells were harvested, resuspended in an isotonic buffer (10 mm Tris, pH 7.6, 100 mm CaCl2, 200 mm sucrose), and disrupted by Dounce homogenization followed by 20 strokes. The homogenate was spun at 800 × g for 10 min, and the supernatant was recovered and further centrifuged for 10 min at 8,000 × g. The resulting supernatant was further centrifuged for 1.5 h at 28,000 × g. The resulting pellet constituted the microsomal ER fraction (28Oakes S.A. Scorrano L. Opferman J.T. Bassik M.C. Nishino M. Pozzan T. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 105-110Crossref PubMed Scopus (383) Google Scholar). The authenticity of the ER fraction was confirmed by immunoblotting for the ER-specific protein calnexin. Immunoblotting—The protein concentrations of cell lysates were determined by Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded onto SDS-polyacrylamide gels (10% acrylamide), resolved by electrophoresis, and transferred to nitrocellulose membranes. Membranes were incubated for 1 h at room temperature in Tris-buffered saline solution with 5% milk or 0.2% BSA to block nonspecific binding sites. Membranes were incubated with the primary antibodies overnight at 4 °C in Tris-buffered saline with 0.1% Tween 20. Horseradish peroxidase secondary antibodies were incubated for 1 h at room temperature in Tris-buffered saline with 0.1% Tween 20 and 5% milk or 0.2% BSA. Labeled proteins were visualized by chemiluminescence as per the manufacturer's instructions (Amersham Biosciences). Immunoprecipitation—Cells at 80-90% confluence on 100-mm tissue culture dishes in normal growth medium were treated with IL-1 and washed three times in ice-cold PBS. Triton X-100 lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.5% Triton X-100, 20 μg/ml leupeptin, 20 μg/ml aprotinin, 50 mm NaF, 10 mm NaPPi, 1 mm Na3VO4) was added to each well. The cell lysates were scraped into a microcentrifuge tube and rotated for 20 min at 4 °C. Pansorbin A was used to block nonspecific binding in the lysates (1 h at 4 °C) and after centrifugation for 15 min at 4 °C, the supernatants were transferred to new tubes. The lysates were incubated with 50 μl agarose-conjugated rabbit polyclonal SHP-2 (5 μl of antibody/50 μl of agarose beads) overnight at 4 °C on a rotating wheel. The pellet of beads was washed three times with Triton X-100 lysis buffer, re-suspended in 2× SDSPAGE sample buffer, and boiled for 10 min. Equal amounts of the eluted proteins were analyzed by SDS-PAGE followed by immunoblotting. Before immunoprecipitation, ER fractions were solubilized in 1% CHAPS-containing buffer (5 mm sodium phosphate, pH 7.4, 2.5 mm EDTA, 100 mm NaCl, 1 mm NaF, 1 mm sodium orthovanadate) and incubated with anti-SHP-2 antibody bound to protein A/G-agarose beads (Santa Cruz Biotechnology). Protein complexes were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed for the indicated proteins. Transfection—Cells were seeded in 6-well plates at a density of 1 × 105/well 24 h before transfection to yield a 30-40% confluent culture on the day of transfection. Transient transfections were performed using FuGENE 6 transfection reagent (Roche Applied Science), according to the manufacturer's protocol. Briefly, cells were incubated with DNA-FuGENE 6 reagent (1:3) complexes for 5-7 h. Within 48 h after transfection, cells were subjected to further experiments. Short Interfering RNA—Specific inhibition of SHP2 was conducted with siRNA (sequence: ag-cccaaaaagaguuacauugcc-dtt (1080-1100 of the SHP-2 sequence). Rat2 cells grown in 6-plate wells were transfected with SHP-2-siRNA or GFP-siRNA (control) using X-tremeGENE siRNA transfection reagent (Roche Applied Science) according to the manufacturer's specifications. Measurement of the gene-knockdown was preformed in 24-72 h. Cells were washed in PBS and lysed with SDS-lysis buffer. Lysates were collected for immunoblotting. Fluorescence Microscopy—Chamber slides (2- and 4-well, Lab-Tek) were coated with poly-l-lysine (100 μg/ml in PBS) and fibronectin- or BSA-coated latex microbeads. Cells were plated for 3-4 h at 37 °C. Prior to immunostaining, cell were stimulated with IL-1 (20 ng/ml for 20 min), fixed (4.0% formaldehyde in PBS for 10 min at room temperature), blocked, and permeabilized in PBS with 0.2% Triton X-100 for 15 min at room temperature. Antibodies were diluted in PBS with 1% BSA. Immunofluorescence staining was performed with rabbit anti-SHP-2 antibody (1:100) and mouse anti-calnexin (1:150) for 1 h at room temperature. Slides were washed with PBS, incubated with goat anti-rabbit fluorescein isothiocyanate-conjugated antibody or goat anti-mouse Texas Red conjugate antibody for 1 h, washed, and sealed with coverslips. The slides were viewed by total internal reflection microscopy. Total Internal Reflection Microscopy—We examined the spatial relationships between SHP-2, the ER, and focal adhesions following IL-1 treatment using green or yellow fluorescent protein-tagged SHP-2 and ER-protein expression vectors combined with total internal reflection microscopy (also called evanescent wave microscopy). TIRF generates fluorescence excitation within a narrow zone (100-150 nm) from the coverslip, which excites fluorescent proteins only on the ventral cell surface and immediately below the plasma membrane. Light from an argon laser (488/543 nm) was introduced into an inverted microscope through a single mode fiber and two illumination lenses (Nikon). The light was focused at the back focal plane of a high aperture objective lens (×60, numerical aperture 1.65, oil immersion). To observe green fluorescent protein fluorescence, we used a 488 nm laser line for excitation and a 515/15 nm bandpass filter. For fluorescence from orange/yellow fluorescent proteins (YFP), we used 543 nm excitation and a long-pass 590 nm filter. Comparisons of GFP- and YFP-transfected cells showed very low levels of cross-over emission into the yellow and green channels, respectively. To monitor the localization of the single SHP-2 molecular or ER organelle, the GFP-tagged SHP-2 (GFP-SHP-2) or ER-specific protein (YFP-calnexin, GFP-calreticulin, and YFP-KDEL/calreticulin)-transfected cells were cultured in chamber slides previously coated with poly-l-lysine and fibronectin or BSA beads. Cells were incubated for 3-4 h at 37 °C in normal medium and then stimulated with IL-1. Nikon immersion oil (refractive index = 1.515) was used to establish optical contact between the objective lens and the coverslip. Using fluorescence calibration beads, the measured penetration depth of excitation light was ∼150 nm. Electron Microscopy—Rat2 cells plated on poly-l-lysine (200 ng/ml)-coated coverslips for 3-4 h were incubated with fibronectin-coated latex microbeads for 1 h and then treated with IL-1 (20 ng/ml) at 37 °C for 30 min. Cells were fixed with in 2% glutaraldehyde, 1% tannic acid, 0.1 m sodium cacodylate at pH 7.4 for 2 h at room temperature, washed three times in 0.1 m sodium cacodylate, pH 7.4, and then post-fixed in 1% OsO4 at room temperature in the same buffer. All samples were washed and dehydrated by stepwise exposure to increasing concentrations of ethanol (25, 50, 75, 95, and 100%, v/v) before embedding in Spurrs resin. Sections (80 nm thick) were cut on a Reichert Ultracut E microtome and stained with uranyl acetate and sodium bismuth. The sections were examined with a Hitachi 7000 scanning transmission electron microscope. Electron micrographs are representative of data obtained in two experiments. Imaging of Ca2+ in Sub-membrane Cytosolic and ER Stores—Cells were incubated with 3-4 μm fluo-4/AM and 0.01% pluronic acid for 30-40 min, or 4-5 μm mag-fluo-4/AM, and 0.01% pluronic acid for 30-40 min at 37 °C. The nominally calcium-free buffer consisted of a bicarbonate-free medium containing 150 mm NaCl, 5 mm KCl, 10 mm d-glucose, 1 mm MgSO4, 1 mm Na2HPO4, and 20 mm HEPES at pH 7.4 with an osmolarity of 291 mosm. For experiments requiring external Ca2+, 2 mm CaC12 was added to the buffer; for experiments requiring chelation of external Ca2+, 1 mm EGTA was added. Imaging and fluorescence intensity of sub-membrane cytosolic and ER calcium were obtained with TIRF microscopy using C·IMAGING SYSTEMS-Simple PCI software (Compix, Inc., Cranberry Twp., PA) with an excitation wavelength of 488 nm and an emission bandpass filter of 515/15 nm. Data Analysis—Means ± S.E. were calculated for calcium measurements, including baseline fluo-4/mag-fluo-4 fluorescence intensity and the net change of fluorescence intensity above baseline. For all continuous variables, means ± S.E. were computed and, when appropriate, comparisons between two groups were made with the unpaired Student's t test or, for multiple samples, with analysis of variance. Post hoc testing was done with Tukey's test. Statistical significance was set at p < 0.05. For all experiments, n ≥ 3 independent cultures were used. For calcium measurements, only a single cell in each culture was measured. Sub-membrane Co-localization of SHP-2 to Focal Adhesions and the ER—To examine the spatial relationships between SHP2, focal adhesions, and the ER, Rat2 fibroblasts were cotransfected with one of either GFP-SHP2, YFP-calnexin, GFP-calreticulin, or YFP-calreticulin fusions. Transfected cells were re-plated on poly-l-lysine-coated coverslips on which either fibronectin or BSA-coated microbeads had been previously attached. Cells were incubated with IL-1 or vehicle. TIRF imaging was used to assess protein recruitment into nascent focal adhesion-like structures that were induced by the matrix ligand-coated beads attached to the coverslip (Fig. 1A). Under these conditions, SHP-2, calnexin, and calreticulin co-localized with fibronectin-coated but not with BSA-coated beads. Staining intensity around fibronectin bead-associated focal adhesions for each of these proteins was further enhanced by IL-1 treatment. Fluorescence microscopic studies of transfected proteins were complemented by examination of endogenous focal adhesion and ER proteins that were associated with collagen-coated beads (Fig. 1B). In these experiments collagen magnetite beads were bound to the dorsal surfaces of cells as previously described (29Arora P.D. Fan L. Sodek J. Kapus A. McCulloch C.A. Exp. Cell Res. 2003; 286: 366-380Crossref PubMed Scopus (20) Google Scholar) and, following IL-1 or vehicle control treatments, the bead-associated proteins were assessed by immunoblot analysis. For the focal adhesion protein vinculin, as well as SHP-2 and the ER-associated proteins calnexin and BiP, IL-1 treatment increased the relative abundance of proteins associated with collagen-coated beads but not with BSA-coated beads. For those proteins that remained in the lysis buffer after preparation of focal adhesions (i.e. the non-focal adhesion fraction), there was no IL-1-induced enhancement of vinculin, SHP-2, calnexin, or BiP. For all experiments, equal amounts of proteins were loaded in each lane. GAPDH, a control, non-ER and non-focal adhesion cytoplasmic protein did not associate with beads, and its relative abundance in focal adhesion preparations was not enhanced by IL-1 treatment. In cells treated with swinholide, an actin severing toxin that disrupts focal adhesions (30MacGillivray M.K. Cruz T.F. McCulloch C.A. J. Biol. Chem. 2000; 275: 23509-23515Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), there were no significant amounts of ER or focal adhesion proteins detected in the pool of bead-associated proteins. We next sought to determine if beads coated with antibody to the fibronectin receptor could induce focal adhesions and if focal adhesion proteins were recruited by IL-1. Magnetite or latex beads were coated with antibody to α5β1 integrin and then incubated with cells (Fig. 1, B and C). Under these conditions, IL-1 did not induce recruitment of vinculin, SHP-2, calnexin, or BiP to beads indicating that clustering of integrins without concomitant activation was not sufficient to induce mature focal adhesions. As analyses of bead-associated proteins indicated associations between focal adhesions around beads and the endoplasmic reticulum, we incubated fibronectin-coated beads with cells and examined them by electron microscopy (Fig. 1D). This analysis showed that, in beads attached to cells, endoplasmic reticulum cisternae were immediately subjacent to beads while in cells without contacting beads, there was no obvious recruitment of endoplasmic reticulu" @default.
• W2044821615 created "2016-06-24" @default.
• W2044821615 creator A5051002562 @default.
• W2044821615 creator A5062765426 @default.
• W2044821615 creator A5065243448 @default.
• W2044821615 creator A5069546196 @default.
• W2044821615 creator A5079462313 @default.
• W2044821615 date "2006-10-01" @default.
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• W2044821615 title "Phosphorylation of SHP-2 Regulates Interactions between the Endoplasmic Reticulum and Focal Adhesions to Restrict Interleukin-1-induced Ca2+ Signaling" @default.
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