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• W2153164047 abstract "Interleukin-1 (IL-1) signaling is dependent on focal adhesions, structures that are enriched with tyrosine kinases and phosphatases. Because the non-receptor tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is enriched in focal adhesions and IL-1-induced ERK activation requires increased Ca2+, we determined whether SHP-2 modulates IL-1-induced Ca2+ signaling. In SHP-2-deficient fibroblasts, IL-1-induced Ca2+ signaling and ERK activation were markedly diminished compared with cells expressing SHP-2. IL-1-induced Ca2+ release from the endoplasmic reticulum occurred in the vicinity of focal adhesions and was strongly inhibited by the blockage of phospholipase C (PLC) catalytic activity. Immunoprecipitation and immunostaining showed that SHP-2, the endoplasmic reticulum-specific protein calnexin, and PLCγ1 were associated with focal adhesions; however, these associations and IL-1-induced ERK activation dissipated after cells were plated on non-integrin substrates. IL-1 promoted phosphorylation of SHP-2 and PLCγ1. IL-1-induced phosphorylation of PLCγ1 was diminished in SHP-2-deficient cells but was restored by stable transfection with SHP-2. BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)) blocked IL-1-induced phosphorylation of SHP-2 and PLCγ1, indicating mutually dependent interactive roles for Ca2+, SHP-2, and PLCγ1 in IL-1 signaling. We conclude that SHP-2 is critical for IL-1-induced phosphorylation of PLCγ1 and thereby enhances IL-1-induced Ca2+ release and ERK activation. Focal adhesions co-localizing with the endoplasmic reticulum may provide molecular staging sites required for ERK activation. Interleukin-1 (IL-1) signaling is dependent on focal adhesions, structures that are enriched with tyrosine kinases and phosphatases. Because the non-receptor tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is enriched in focal adhesions and IL-1-induced ERK activation requires increased Ca2+, we determined whether SHP-2 modulates IL-1-induced Ca2+ signaling. In SHP-2-deficient fibroblasts, IL-1-induced Ca2+ signaling and ERK activation were markedly diminished compared with cells expressing SHP-2. IL-1-induced Ca2+ release from the endoplasmic reticulum occurred in the vicinity of focal adhesions and was strongly inhibited by the blockage of phospholipase C (PLC) catalytic activity. Immunoprecipitation and immunostaining showed that SHP-2, the endoplasmic reticulum-specific protein calnexin, and PLCγ1 were associated with focal adhesions; however, these associations and IL-1-induced ERK activation dissipated after cells were plated on non-integrin substrates. IL-1 promoted phosphorylation of SHP-2 and PLCγ1. IL-1-induced phosphorylation of PLCγ1 was diminished in SHP-2-deficient cells but was restored by stable transfection with SHP-2. BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)) blocked IL-1-induced phosphorylation of SHP-2 and PLCγ1, indicating mutually dependent interactive roles for Ca2+, SHP-2, and PLCγ1 in IL-1 signaling. We conclude that SHP-2 is critical for IL-1-induced phosphorylation of PLCγ1 and thereby enhances IL-1-induced Ca2+ release and ERK activation. Focal adhesions co-localizing with the endoplasmic reticulum may provide molecular staging sites required for ERK activation. Interleukin-1 (IL-1) 1The abbreviations used are: IL-1, interleukin-1; ERK, extracellular signal-regulated kinase; SHP, Src homology 2 domain-containing protein tyrosine phosphatase; PLC, phospholipase C; BSA, bovine serum albumin; fura-2/AM, fura-2 pentaacetoxymethyl ester; U-73122, 1-(6-[(17β-3-methoxestra-1,3,5(10)trine-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione; U-73343, 1-(6-[(17β-3-methoxestra-1,3,5(10)trine-17-yl)amino]-hexyl)-2,5-pyrrolidine-dione; 2-APB, 2-aminoethoxydiphenyl borate; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; ER, endoplasmic reticulum; siRNA, short interfering RNA; GFP, green fluorescent protein; InsP3, inositol 1,4,5-trisphosphate; HGF, human gingival fibroblast; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester).1The abbreviations used are: IL-1, interleukin-1; ERK, extracellular signal-regulated kinase; SHP, Src homology 2 domain-containing protein tyrosine phosphatase; PLC, phospholipase C; BSA, bovine serum albumin; fura-2/AM, fura-2 pentaacetoxymethyl ester; U-73122, 1-(6-[(17β-3-methoxestra-1,3,5(10)trine-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione; U-73343, 1-(6-[(17β-3-methoxestra-1,3,5(10)trine-17-yl)amino]-hexyl)-2,5-pyrrolidine-dione; 2-APB, 2-aminoethoxydiphenyl borate; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; ER, endoplasmic reticulum; siRNA, short interfering RNA; GFP, green fluorescent protein; InsP3, inositol 1,4,5-trisphosphate; HGF, human gingival fibroblast; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). is a crucially important, proinflammatory cytokine associated strongly with the destruction of extracellular matrices in several highly prevalent diseases, including rheumatoid arthritis, periodontitis, and cancer (1Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar, 2O'Neill L.A. Dinarello C.A. Immunol. Today. 2000; 21: 206-209Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 3van den Berg W.B. Z. Rheumatol. 1999; 58: 136-141Crossref PubMed Scopus (102) Google Scholar, 4Honig J. Rordorf-Adam C. Siegmund C. Wiedemann W. Erard F. J. Periodontal Res. 1989; 24: 362-367Crossref PubMed Scopus (172) Google Scholar). IL-1 mediates the degradation of extracellular matrix proteins by enhancing the synthesis and secretion of proteases that are expressed by connective tissue cells including 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), particularly those signals that lead to the activation of 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). Previous studies (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) of IL-1 signaling pathways in cultured fibroblasts have shown that the IL-1-induced activation of the mitogen-activated protein kinase ERK requires the formation of focal adhesions, suggesting that focal adhesion-associated proteins such as integrins and focal adhesion kinase (FAK) (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 play critical, modulatory roles in the generation and regulation of IL-1-induced signals. Although IL-1 type 1 (signaling) receptors are confined 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, 11Qwarnstrom E.E. Page R.C. Gillis S. Dower S.K. J. Biol. Chem. 1988; 263: 8261-8269Abstract Full Text PDF PubMed Google Scholar, 12Luo L. Cruz T. McCulloch C. Biochem. J. 1997; 324: 653-658Crossref PubMed Scopus (49) Google Scholar) and thus may be responsible for the focal adhesion restriction of IL-1 signaling, it is likely that other focal adhesion proteins with intrinsic capacities for post-translational modification also contribute to this phenomenon. Many signaling systems in cells involve protein phosphorylation, which in turn is controlled by the net relative activities of protein kinases and protein phosphatases. Notably, although tyrosine phosphatases may play a role in the termination of various receptor tyrosine kinase-induced signals, they often play positive roles in signaling as well. For example, several receptor tyrosine kinase-signaling pathways that lead to ERK activation utilize protein tyrosine phosphatases such as SHP-2 to help sustain ERK activity (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 from integrins or integrin-associated proteins. SHP-2 also has an impact on IL-1-induced ERK activation, and this activation is tightly linked to integrin clustering in focal adhesions (11Qwarnstrom E.E. Page R.C. Gillis S. Dower S.K. J. Biol. Chem. 1988; 263: 8261-8269Abstract Full Text PDF PubMed Google Scholar). Previous work (14Oh E.S. Gu H. Saxton T.M. Timms J.F. Hausdorff S. Frevert E.U. Kahn B.B. Pawson T. Neel B.G. Thomas S.M. Mol. Cell. Biol. 1999; 19: 3205-3215Crossref PubMed Scopus (193) Google Scholar) has established that SHP-2 is required for integrin-mediated ERK activation, possibly by Src family-related kinase phosphorylation of SHPS-1 and the 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). Furthermore, SHP-2 regulates cell spreading on fibronectin and focal adhesion formation (15Yu 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). However, the mechanisms and the protein-protein interactions that are required for integrins and SHP-2 to mediate the IL-1 activation of ERK are not defined. Recent studies (16Zhang 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) have demonstrated that SHP-2 regulates the activity of Src family kinases, and ultimately the activation of ERK, by controlling the recruitment of C-terminal Src kinase. The activation of ERK through the Src family kinase system is thought to involve the phosphorylation of PLCγ1, a step in the signaling pathway required for the release of calcium from internal stores and the subsequent activation of the Ras-Raf-MEK-ERK (where MEK is mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) cascade. Ras activation may in turn rely on its anchorage to endoplasmic reticulum membranes (17Chiu V.K. Bivona T. Hach A. Sajous J.B. Silletti J. Wiener H. Johnson II, R.L. Cox A.D. Philips M.R. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (510) Google Scholar) as well as its recruitment of SHP-2 to plasma and endoplasmic reticulum membranes (16Zhang 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). However, the mechanisms whereby the various participants in this signaling pathway are marshaled to specific sites within the cell are not known. As noted earlier, IL-1-induced ERK activation is focal adhesion-restricted (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), requires the release of Ca2+ from the endoplasmic reticulum (18Wang Q. Downey G.P. Choi C. Kapus A. McCulloch C.A. FASEB J. 2003; 17: 1898-1900Crossref PubMed Google Scholar), and may depend on the sequestration of signaling molecules such as Ras to endoplasmic reticulum membranes (17Chiu V.K. Bivona T. Hach A. Sajous J.B. Silletti J. Wiener H. Johnson II, R.L. Cox A.D. Philips M.R. Nat. Cell Biol. 2002; 4: 343-350Crossref PubMed Scopus (510) Google Scholar). Accordingly, we considered that focal adhesions and the endoplasmic reticulum may provide spatially discrete staging sites (19Cohen P. Nat. Cell Biol. 2002; 4: 127-130Crossref PubMed Scopus (746) Google Scholar) that enable the SHP-2-dependent activation of ERK by IL-1. Materials—Bovine fibronectin, poly-l-lysine, BSA, sodium orthovanadate, mouse monoclonal antibodies to vinculin, and goat anti-rabbit antibodies coupled to fluorescein isothiocyanate were obtained from Sigma. Rabbit polyclonal antibodies to ERK1/2 and mouse monoclonal anti-phospho-ERK1/2 were purchased from New England Biolabs (Beverly, MA). Phospho-SHP-2 (Tyr-542), PLCγ2, PLCγ1, and phospho-PLCγ1 (Tyr-783) antibodies were purchased from Cell Signaling (Beverly, MA). Rabbit polyclonal antibodies to SHP-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-calnexin was obtained from BD Biosciences. Horseradish peroxidase-conjugated goat anti-mouse (H+L) and goat anti-rabbit (H+L) were purchased from Cedarlane Laboratories (Hornby, Ontario, Canada). The ECL chemiluminescent kit was purchased from Amersham Biosciences. Acidified bovine type I collagen (Vitrogen) was purchased from Cohesion Technologies, Inc. (Palo Alto, CA). Recombinant human IL-1β was obtained from R&D Systems (Minneapolis, MN). Fura-2/AM, mag-fura-2/AM, and fura-C18 were obtained from Molecular Probes, Inc. (Eugene, OR). 2-Aminoethoxydiphenyl borate (2-APB), xestospongin C, PLC inhibitor 1-(6-[(17β-3-methoxestra-1,3,5(10)trine-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) and its inactive congener 1-(6-[(17β-3-methoxestra-1,3,5(10)trine-17-yl)amino]hexyl)-2,5-pyrrolidine-dione (U-73343), and swinholide A were obtained from Calbiochem. Cell Culture and Bead Preparations—Human gingival fibroblasts were grown in minimal essential medium. Murine embryonic fibroblasts (SHP-2 (Δ46-110) (a kind gift of Dr. G. S. Feng, Burnham Institute, La Jolla, CA) and the same cells transfected with wild-type SHP-2) were grown in high glucose Dulbecco's modified Eagle's medium. All media contained 10% fetal bovine serum and antibiotics (0.17% penicillin V, 0.1% gentamicin sulfate, and 0.01% amphotericin). Cells were used between the 5th and 12th passages as described previously (20MacGillivray 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). Murine embryonic fibroblasts (SHP-2 (Δ46-110) and the same cells reconstituted with wild-type murine SHP-2) were generated and grown in high glucose Dulbecco's modified Eagle's medium as described previously (21Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (399) 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), 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, resuspended in PBS, and sonicated for 10 s (output setting 3, 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- or BSA-coated magnetite beads. Focal adhesion complexes were isolated from cells after specific incubation time periods as described previously (22Plopper 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 then scraped into ice-cold cytoskeleton extraction buffer (CSKB) (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, power 15%), and the beads were isolated from the lysate using a magnetic separation stand. The beads were resuspended in fresh ice-cold CSKB, homogenized with a Dounce homogenizer (20 strokes), and re-isolated magnetically. The beads were washed in CSKB, sedimented with a microcentrifuge, resuspended in Laemmli sample buffer, and placed in a boiling water bath for 10 min to allow the collagen-associated complexes to dissociate from the beads. The beads were sedimented, and lysates were collected for analysis. Calcium Measurement—For measuring whole cell [Ca2+]i, cells on coverslips were loaded with 3 μm fura-2/AM for 20 min at 37 °C, and cells were measured by ratio fluorometry as described previously (23Ko K.S. Glogauer M. McCulloch C.A. Ellen R.P. Infect. Immun. 1998; 66: 703-709Crossref PubMed Google Scholar). For estimating the Ca2+ concentration near the plasma membrane (juxtamembrane concentration), cells attached to coverslips were briefly permeabilized with saponin and incubated with 2 μm fura-C18, pentapotassium salt at 25 °C for 20 min followed by washing with PBS (24Ko K.S. Arora P.D. McCulloch C.A. J. Biol. Chem. 2001; 276: 35967-35977Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). For estimating [Ca2+]ER, cells were incubated with mag-fura-2/AM (4 μm) for 150 min at 37 °C in minimum essential medium containing 10% fetal bovine serum (25Golovina V.A. Blaustein M.P. Science. 1997; 275: 1643-1648Crossref PubMed Scopus (423) Google Scholar). 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. 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. After incubation with fura-2/AM, an inspection of the cells by fluorescence microscopy demonstrated no vesicular compartmentalization of fura-2, suggesting that the dye loading method permitted measurement of cytosolic [Ca2+]i. A visual inspection of fura-C18-loaded cells and mag-fura-2-loaded cells showed fluorescent labeling of the plasma membrane and intracellular organelles, respectively. Whole cell and plasma membrane [Ca2+]i measurements were obtained with C·IMAGING Systems Simple PCI software (Compix, Inc., Cranberry, PA), with excitation wavelengths of 340 and 380 nm, respectively, and an emission wavelength of 520 nm. Changes in [Ca2+]i were monitored by the ratios of fura-2 fluorescence at 340 and 380 nm. Immunoblotting—The protein concentrations of cell lysates were determined by the Bradford assay (Bio-Rad). 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 BSA to block nonspecific binding sites. Membranes were incubated overnight with the primary antibodies 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 BSA. Labeled proteins were visualized by chemiluminescence according to the manufacturer's instructions (Amersham Biosciences). Immunofluorescence—Chamber slides (8-well, Labtek) were coated with poly-l-lysine (100 μg/ml in PBS). Cells were plated for 4-6 h prior to incubation with fibronectin-coated latex microbeads for 30 min at 37 °C. Prior to immunostaining, cells were stimulated with IL-1 (20 ng/ml for 20 min), fixed (3.7% paraformaldehyde in PBS for 10 min at room temperature), blocked, and permeabilized in PBS with 0.2% Triton X-100 and 0.2% BSA for 15 min at room temperature. Antibodies were diluted in PBS with 0.2% Triton X-100 and 0.2% BSA. Immunofluorescence staining was performed with rabbit anti-SHP-2 antibody (1:50), mouse anti-calnexin (1:50), or rabbit anti-PLCγ1 antibody (1:50) for 1 h at room temperature. Slides were washed with PBS, incubated with goat anti-rabbit Texas Red-conjugated antibody or goat anti-mouse fluorescein isothiocyanate-conjugated antibody for 1 h, washed, and sealed with coverslips. The slides were viewed with a Nikon 300 inverted fluorescence microscope equipped with Nomarski optics. Images were captured with a charge-coupled device camera (Imagepoint, Photometrics, Phoenix, AZ). 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 sodium PPi, 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 overnight with 50 μl of agarose-conjugated rabbit polyclonal SHP-2 (5 μl of antibody, 50 μl of agarose beads) at 4 °C on a rotating wheel. The pellet of beads was washed three times with Triton X-100 lysis buffer, resuspended in 2× SDS-PAGE sample buffer, and boiled for 10 min. Equal amounts of the eluted proteins were analyzed by SDS-PAGE followed by immunoblotting using anti-SHP-2, anti-phospho-SHP-2, anti-PLCγ1, and anti-phospho-PLCγ1 antibodies. Short Interfering RNA—Specific inhibition of SHP-2 was conducted with siRNA (sequence AG-CCCAAAAAGAGUUACAUUGCC-DTT, residues 1080-1100 of the SHP-2 sequence). Human gingival fibroblasts grown in 24-well plates were transfected with 100 nm of SHP-2 siRNA or GFP-siRNA (control) using Oligofectamine according to the manufacturer's specifications. Gene silencing was conducted for 72 h. Cells were washed in PBS, lysed with Laemmli buffer, and immunoblotted to assess the efficacy of SHP-2 knockdown. Data Analysis—Means and S.E. were calculated for [Ca2+]i measurements, including the base line [Ca2+]i and the net change of [Ca2+]i above base line. For all continuous variables, means and S.E. were computed, and, when appropriate, comparisons were made between two groups with the unpaired Student's t test or, for multiple samples, with analysis of variance. Post hoc testing was carried out 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. IL-1-induced Ca2+ Influx and ER Release Occur Locally around Fibronectin-coated Beads—IL-1-induced Ca2+ signals are dependent on focal adhesions in cultured cells (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, 18Wang Q. Downey G.P. Choi C. Kapus A. McCulloch C.A. FASEB J. 2003; 17: 1898-1900Crossref PubMed Google Scholar), but the spatial relationships between focal adhesions and sites of IL-1-induced Ca2+ transients have not been described. We first determined whether IL-1-induced Ca2+ signaling is restricted to focal adhesion sites. Focal adhesions were induced by applying fibronectin-coated beads to the dorsal surfaces of cells plated on poly-l-lysine (i.e. with no focal adhesions on their ventral surfaces). Cells plated on poly-l-lysine were loaded with fura-2/AM (to measure cytosolic Ca2+), fura-C18 (to measure juxtamembranous Ca2+), or mag-fura-2/AM (to measure ER Ca2+). Cells were then preincubated with fibronectin- or BSA-coated beads (2-μm diameter) and treated with IL-1. Ratios of [Ca2+]i, fura-C18, and mag-fura-2 were measured by ratio imaging for the whole cell in circumscribed 5-μm zones around the periphery of attached beads and in similarly sized, randomly chosen zones within the projected area of the cell but at least 10 μm from the attached beads (Fig. 1, A-C). IL-1 induced localized increases of intracellular Ca2+ (fura-2/AM), Ca2+ transients subjacent to the cell membrane (fura-C18), and Ca2+ release from the ER (mag-fura-2/AM), but these changes were found only when cells were incubated with fibronectin-coated beads and only in the vicinity of fibronectin beads. No significant changes in Ca2+ were detected at sites remote from the beads, and no evidence for IL-1-induced Ca2+ signaling was found in cells plated on poly-l-lysine and incubated with BSA-coated beads. Therefore, as shown previously (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, 12Luo L. Cruz T. McCulloch C. Biochem. J. 1997; 324: 653-658Crossref PubMed Scopus (49) Google Scholar), integrin-dependent focal adhesion formation is essential for IL-1-induced Ca2+ signals. The novel aspect of the current observations is that Ca2+ fluxes induced by IL-1 originate from and are strictly localized to the vicinity of focal adhesions. Because there was very tight co-localization of mag-fura-2 Ca2+ signals with fibronectin-coated beads, we asked whether the focal adhesions were co-distributed with the endoplasmic reticulum. Fibronectin or BSA-coated latex beads were added to the dorsal surfaces of cells attached previously to poly-l-lysine to induce focal adhesion assembly at sites of cell-bead contacts. Immunostaining for calnexin, an endoplasmic reticulum-specific protein, showed that within 30 min after adding fibronectin or BSA-beads, calnexin accumulated at sites adjacent to the fibronectin beads but not BSA beads (Fig. 1D), indicating that the endoplasmic reticulum is spatially associated with nascent focal adhesions. These experiments confirmed that the localized generation of focal adhesions on rounded cells was sufficient to restore responsiveness to IL-1 without inducing cell spreading (26MacGillivray 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) and showed that IL-1-induced Ca2+ release from the endoplasmic reticulum is spatially associated with nascent focal adhesions. SHP-2 Is Required for IL-1-induced Cytosolic Ca2+ Flux, ER Ca2+ Release, and ERK Activation—A general role for protein tyrosine phosphorylation in the regulation of Ca2+ fluxes has been reported (27Hsu S. Schmid A. Sternfeld L. Anderie I. Solis G. Hofer H.W. Schulz I. Cell. Signal. 2003; 15: 1149-1156Crossref PubMed Scopus (14) Google Scholar, 28Rosado J.A. Porras T. Conde M. Sage S.O. J. Biol. Chem. 2001; 276: 15666-15675Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and we demonstrated earlier that the protein tyrosine phosphatase SHP-2 is enriched in the focal adhesions of fibroblasts and can modulate IL-1-induced ERK activation (20MacGillivray 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). Although several protein tyrosine phosphatases, including protein tyrosine phosphatase-α and low molecular weight protein tyrosine phosphatase, are known to associate with focal adhesions (20MacGillivray 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, 29Petrone A. Sap J. J. Cell Sci. 2000; 113: 2345-2354PubMed Google Scholar, 30Feng G.S. Exp. Cell Res. 1999; 253: 47-54Crossref PubMed Scopus (252) Google Scholar, 31von Wichert G. Jiang G. Kostic A. De Vos K. Sap J. Sheetz M.P. J. Cell Biol. 2003; 161: 143-153Crossref PubMed Scopus (181) Google Scholar), we focused on SHP-2, a non-receptor type of protein tyrosine phosphatase that has been implicated in the regulation of cytokine, growth factor, and integrin signaling (15Yu 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, 20MacGillivray 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). Importantly, SHP-2 itself undergoes phosphorylation on several key tyrosine residues, including Tyr-580 and Tyr-542, which are modifications that are known to alter its catalytic and adaptor functions (20MacGillivray 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). We utilized a cell model system that allowed direct analysis of the role of SHP-2 in IL-1-induced Ca2+ fluxes and ERK activation through focal adhesions. SHP-2-/- (Δ46-110, designated here as SHP-2-/-) murine embryonic fibroblasts cells express a SHP-2 protein with a deletion of 65 amino acids in the N-terminal SH2 domain (21Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (399) Google Scholar), which prohibits receptor-mediated ERK activation in several different signaling systems (32Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-" @default.
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