FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2072835510> ?p ?o ?g. }

• W2072835510 endingPage "1944" @default.
• W2072835510 startingPage "1938" @default.
• W2072835510 abstract "SHP-1 is a cytosolic tyrosine phosphatase implicated in down-regulation of B cell antigen receptor signaling. SHP-1 effects on the antigen receptor reflect its capacity to dephosphorylate this receptor as well as several inhibitory comodulators. In view of our observation that antigen receptor-induced CD19 tyrosine phosphorylation is constitutively increased in B cells from SHP-l-deficient motheaten mice, we investigated the possibility that CD19, a positive modulator of antigen receptor signaling, represents another substrate for SHP-1. However, analysis of CD19 coimmunoprecipitable tyrosine phosphatase activity in CD19 immunoprecipitates from SHP-1-deficient and wild-type B cells revealed that SHP-1 accounts for only a minor portion of CD19-associated tyrosine phosphatase activity. As CD19 tyrosine phosphorylation is modulated by the Lyn protein-tyrosine kinase, Lyn activity was evaluated in wild-type and motheaten B cells. The results revealed both Lyn as well as CD19-associated Lyn kinase activity to be constitutively and inducibly increased in SHP-1-deficient compared with wild-type B cells. The data also demonstrated SHP-1 to be associated with Lyn in stimulated but not in resting B cells and indicated this interaction to be mediated via Lyn binding to the SHP-1 N-terminal SH2 domain. These findings, together with cyanogen bromide cleavage data revealing that SHP-1 dephosphorylates the Lyn autophosphorylation site, identify Lyn deactivation/dephosphorylation as a likely mechanism whereby SHP-1 exerts its influence on CD19 tyrosine phosphorylation and, by extension, its inhibitory effect on B cell antigen receptor signaling. SHP-1 is a cytosolic tyrosine phosphatase implicated in down-regulation of B cell antigen receptor signaling. SHP-1 effects on the antigen receptor reflect its capacity to dephosphorylate this receptor as well as several inhibitory comodulators. In view of our observation that antigen receptor-induced CD19 tyrosine phosphorylation is constitutively increased in B cells from SHP-l-deficient motheaten mice, we investigated the possibility that CD19, a positive modulator of antigen receptor signaling, represents another substrate for SHP-1. However, analysis of CD19 coimmunoprecipitable tyrosine phosphatase activity in CD19 immunoprecipitates from SHP-1-deficient and wild-type B cells revealed that SHP-1 accounts for only a minor portion of CD19-associated tyrosine phosphatase activity. As CD19 tyrosine phosphorylation is modulated by the Lyn protein-tyrosine kinase, Lyn activity was evaluated in wild-type and motheaten B cells. The results revealed both Lyn as well as CD19-associated Lyn kinase activity to be constitutively and inducibly increased in SHP-1-deficient compared with wild-type B cells. The data also demonstrated SHP-1 to be associated with Lyn in stimulated but not in resting B cells and indicated this interaction to be mediated via Lyn binding to the SHP-1 N-terminal SH2 domain. These findings, together with cyanogen bromide cleavage data revealing that SHP-1 dephosphorylates the Lyn autophosphorylation site, identify Lyn deactivation/dephosphorylation as a likely mechanism whereby SHP-1 exerts its influence on CD19 tyrosine phosphorylation and, by extension, its inhibitory effect on B cell antigen receptor signaling. B cell antigen receptor B cell receptor for IgG Fc region glutathione S-transferase motheaten viable motheaten polyacrylamide gel electrophoresis phosphatidylinositol 3-kinase protein-tyrosine kinase protein-tyrosine phosphatase Src homology domain N-tris-[hydroxymethyl]methylglycine 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid phosphate-buffered saline immunoreceptor tyrosine-based inhibitory motif B cell responses to antigen stimulation are transduced intracellularly via the B cell antigen receptor (BCR),1 a multimeric receptor complex that comprises membrane immunoglobulin and the immunoglobulin α and β chains (1Justement L.B. Curr. Top. Microbiol. Immunol. 2000; 245: 1-51PubMed Google Scholar, 2Reth M. Wienands J. Annu. Rev. Immunol. 1997; 15: 453-479Crossref PubMed Scopus (366) Google Scholar). The signals transmitted consequent to antigen engagement drive B lymphocyte activation via a complex signaling network, which biochemically links the receptor complex to cellular responses such as to proliferation, differentiation, and antibody secretion. Transmission of BCR signals via this intracellular circuitry is further regulated by the integration of accessory signals from BCR comodulators (3Campbell K.S. Curr. Opin. Immunol. 1999; 11: 256-264Crossref PubMed Scopus (183) Google Scholar) and is highly dependent on reversible protein-tyrosine phosphorylation mediated by the balanced activities of protein-tyrosine kinases (PTKs) and phosphatases (PTPs) (4Tedder T.F. Semin. Immunol. 1998; 10: 259-265Crossref PubMed Scopus (44) Google Scholar, 5Siminovitch K.A. Neel B.J. Semin. Immunol. 1998; 10: 329-347Crossref PubMed Scopus (61) Google Scholar). The initial events of BCR signal relay are characterized by the activation of several PTKs, including Lyn, Fyn, Blk, Syk, and Btk, and the subsequent recruitment of secondary signaling molecules, including phosphatidylinositol 3-kinase (PI3K), Shc, BLNK/SLP-65, Vav, SOS1, and phospholipase Cγ (6De Franco A.L. Curr. Opin. Immunol. 1997; 9: 296-308Crossref PubMed Scopus (281) Google Scholar, 7Gold M.R. Aebersold R.A. J. Immunol. 1994; 152: 42-50PubMed Google Scholar, 8Lankester A.C. van Schijndel G.M. Rood P.M. Verhoven A.J. van Lier R.A. Eur. J. Immunol. 1994; 24: 2818-2825Crossref PubMed Scopus (50) Google Scholar, 9Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 10Wienands J. Schweikert J. Wollscheid B. Jumaa H. Nielson P.J. Reth M. J. Exp. Med. 1998; 188: 791-795Crossref PubMed Scopus (231) Google Scholar, 11Saxton T.M. van Oostveen J. Bowtell D. Aebersold R. Gold M.R. J. Immunol. 1994; 153: 623-636PubMed Google Scholar, 12Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (244) Google Scholar). These initial interactions induce Ras activation, phosphoinositide turnover, increases in intracellular free calcium, and other intermediary events, which ultimately transduce the BCR-evoked signal to the nucleus and consequent proliferation, apoptosis, maturation, or other physiological responses. The mechanisms whereby BCR ligation can induce such a wide diversity of biological outcomes are not well understood but are likely to involve modulation of the BCR signaling pathway by a spectrum of transmembrane and cytosolic signaling effectors that qualitatively and/or quantitatively alter the relay and downstream interpretation of BCR signal (13Smith K.G.C. Fearon D.T. Curr. Top. Microbiol. Immunol. 2000; 245: 195-212PubMed Google Scholar). Among the myriad of proteins implicated in the regulation of BCR signaling, the cytosolic protein-tyrosine phosphatase (PTP) SHP-1 is distinguished by its predominant role as an inhibitor of BCR-driven activation events (5Siminovitch K.A. Neel B.J. Semin. Immunol. 1998; 10: 329-347Crossref PubMed Scopus (61) Google Scholar). The inhibitory effect of SHP-1 on BCR signaling was initially revealed by the demonstration that BCR-evoked proliferation of mature B cells and clonal deletion of self-reactive B cell precursors are aberrantly increased in the context of SHP-1 deficiency (14Pani G. Kozlowski M. Cambier J.C. Mills G.B. Siminovitch K.A. J. Exp. Med. 1995; 181: 2077-2084Crossref PubMed Scopus (240) Google Scholar, 15Cyster J.G. Goodnow C.C. Immunity. 1995; 2: 13-24Abstract Full Text PDF PubMed Scopus (349) Google Scholar). These latter studies involved analysis of B cells from motheaten (me/me) and viable motheaten(me v /me v )mice, animals in which expression of no SHP-1 or a catalytically inactive form of SHP-1 protein, respectively, is associated with increased levels of serum immunoglobulins, high autoantibody titer, and a marked expansion of CD5+ B-1 cells in the periphery (16Kozlowski M. Mlinaric-Rascan I. Feng G.-S. Shen R. Pawson T. Siminovitch K.A. J. Exp. Med. 1993; 178: 2157-2163Crossref PubMed Scopus (210) Google Scholar,17Shultz L.D. Sidman C.L. Annu. Rev. Immunol. 1987; 5: 367-403Crossref PubMed Scopus (139) Google Scholar). At present, the biochemical basis whereby SHP-1 exerts its inhibitory effects on BCR-evoked responses is not entirely defined. This PTP has, however, been shown to interact with the BCR complex in resting B cells and likely acts in this context to maintain the receptor in a tyrosine-dephosphorylated state (14Pani G. Kozlowski M. Cambier J.C. Mills G.B. Siminovitch K.A. J. Exp. Med. 1995; 181: 2077-2084Crossref PubMed Scopus (240) Google Scholar). Following BCR ligation, SHP-1 no longer associates with the BCR, but instead interacts with a number of BCR-inducible tyrosine-phosphoryated transmembrane coreceptors (18Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (480) Google Scholar, 19Blery M. Kubagawa H. Chen C.C. Vely F. Cooper M.D. Vivier E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2446-2451Crossref PubMed Scopus (176) Google Scholar, 20Wu Y. Nadler M.J. Brennan L.A. Gish G.D. Timms J.F. Fusaki N. Jongstra-Bilen J. Tada N. Pawson T. Wither J. Neel B.G. Hozumi N. Curr. Biol. 1998; 8: 1009-1017Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). These coreceptors, which include CD22, PIR-B, and CD72, have all been implicated in the down-regulation of BCR signaling (21O'Keefe T.L. Williams G.T. Davies S.L. Neuberger M.S. Science. 1996; 274: 798-801Crossref PubMed Scopus (465) Google Scholar, 22Maeda A. Kurosaki M. Ono M. Takai T. Kurosaki T. J. Exp. Med. 1998; 187: 1355-1360Crossref PubMed Scopus (171) Google Scholar, 23Nomura T. Han H. Howard M.C. Yagita H. Yakura H. Honjo T. Tsubata T. Int. Immunol. 1996; 8: 867-875Crossref PubMed Scopus (56) Google Scholar) and have been shown to interact with the SHP-1 SH2 domains via phosphorylated tyrosine residues embedded within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (18Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (480) Google Scholar, 19Blery M. Kubagawa H. Chen C.C. Vely F. Cooper M.D. Vivier E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2446-2451Crossref PubMed Scopus (176) Google Scholar, 20Wu Y. Nadler M.J. Brennan L.A. Gish G.D. Timms J.F. Fusaki N. Jongstra-Bilen J. Tada N. Pawson T. Wither J. Neel B.G. Hozumi N. Curr. Biol. 1998; 8: 1009-1017Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The inhibitory effects of these receptors depend on their binding to SHP-1 and appear to be realized via SHP-1-mediated dephosphorylation of tyrosine residues within the receptor cytosolic domains and/or other intracellular signaling effectors recruited to these receptors following BCR engagement. In contrast to the ITIM-containing coreceptor molecules, a number of B cell transmembrane coreceptors modulate BCR signaling so as to amplify the signal and promote its downstream propagation. Among these positive modulatory receptors, the B lineage-specific CD19 molecule appears to play a central role in enhancing BCR coupling to a spectrum of cellular behaviors. CD19, which is expressed as a component of a multimeric complex on the B cell surface (24Bradbury L. Kansas G.S. Levy S. Evans R.L. Tedder T.F. J. Immunol. 1992; 149: 2841-2850PubMed Google Scholar), becomes rapidly tyrosine-phosphorylated following BCR engagement (25Uckun F.M. Burkhardt A.L. Jarvis L. Jun X. Stealey B. Dibirdik I. Myers D.E. Tuel-Ahlgren L. Bolen J.B. J. Biol. Chem. 1993; 268: 21172-21184Abstract Full Text PDF PubMed Google Scholar) and consequently interacts with SH2 domain-containing signaling effectors, such as Lyn, Fyn, Syk, Vav, and PI3K, which play integral roles in BCR signal delivery (25Uckun F.M. Burkhardt A.L. Jarvis L. Jun X. Stealey B. Dibirdik I. Myers D.E. Tuel-Ahlgren L. Bolen J.B. J. Biol. Chem. 1993; 268: 21172-21184Abstract Full Text PDF PubMed Google Scholar, 26van Noesel C.J.M. Lankester A.C. van Schijndel G.M.V. van Lier R.A.W. Int. Immunol. 1993; 5: 699-705Crossref PubMed Scopus (79) Google Scholar, 27Tuveson D.A. Carter R.H. Soltoff S.P. Fearon D.T. Science. 1993; 260: 986-989Crossref PubMed Scopus (281) Google Scholar, 28Weng W.K. Jarvis L. Le Bien T.W. J. Biol. Chem. 1994; 269: 32514-32521Abstract Full Text PDF PubMed Google Scholar). Recent data suggest that CD19 effects on BCR signaling reflect its capacity to not only interact with Src-family PTKs but also to amplify the activities of these enzymes (29Fujimoto M. Poe J.C. Jansen P.J. Sato S.S. Tedder T.F. J. Immunol. 1999; 162: 7088-7094PubMed Google Scholar). As is consistent with the positive role for CD19 in regulation of BCR signaling, mice, which overexpress CD19 consequent to the expression of a CD19 transgene, manifest augmented B cell proliferative responses to BCR cross-linking and show markedly increased serum immunoglobulin levels (30Engel P. Zhou L.-J. Ord D.C. Sato S. Koller B. Tedder T. Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (482) Google Scholar). These animals also display a dramatic increase in the numbers of B-1 lineage cells and a proportionate decrease in the numbers of conventional B cells within the periphery (31Sato S. Ono N. Steeber D.A. Pisetsky D.S. Tedder T.F. J. Immunol. 1996; 157: 4371-4378PubMed Google Scholar). These observations, therefore, reveal the phenotype engendered by CD19 overexpression to be very similar to the B cell phenotype conferred by SHP-1 deficiency, a finding that suggests that the influence of these respective proteins on BCR signaling thresholds reflects the modulation of a common signaling element or cascade. This hypothesis is further supported by our previous data revealing BCR-evoked CD19 phosphorylation to be markedly reduced in cells lacking both the CD45 and SHP-1 PTPs (32Pani G. Siminovitch K.A. Paige C.J. J. Exp. Med. 1997; 186: 581-588Crossref PubMed Scopus (52) Google Scholar) and thus identifying CD19 as a possible target of SHP-1-mediated dephosphorylation. In the current study, we have directly investigated the role for SHP-1 in modulating the tyrosine phosphorylation of CD19. The results of these studies confirm that BCR-induced tyrosine phosphorylation of CD19 is enhanced in SHP-1-deficient mice but also suggest that the contribution of SHP-1 to the direct dephosphorylation of CD19 is small. Because of these observations, as well as data revealing CD19 to be associated with the Lyn protein-tyrosine kinase following BCR engagement (25Uckun F.M. Burkhardt A.L. Jarvis L. Jun X. Stealey B. Dibirdik I. Myers D.E. Tuel-Ahlgren L. Bolen J.B. J. Biol. Chem. 1993; 268: 21172-21184Abstract Full Text PDF PubMed Google Scholar, 26van Noesel C.J.M. Lankester A.C. van Schijndel G.M.V. van Lier R.A.W. Int. Immunol. 1993; 5: 699-705Crossref PubMed Scopus (79) Google Scholar) and identifying a central role for Lyn in modifying CD19 effects on B cell survival (33Uckun F.M. Evans W.E. Waddick K.G. Tuel-Ahlgren L. Chelstrom L.M. Burkhardt A.C. Bolen J.B. Myers D.E. Science. 1995; 267: 886-891Crossref PubMed Scopus (259) Google Scholar, 34Myers D.E. Jun X. Waddick K.G. Forsyth C. Chelstrom L.M. Gunther R.L. Tumer N.E. Bolen J. Uckun F.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9575-9579Crossref PubMed Scopus (38) Google Scholar), we next investigated the possibility that SHP-1 influence on CD19 tyrosine phosphorylation is mediated via the regulation of Lyn activity. The results of this analysis indicate both BCR-induced tyrosine phosphorylation and activation of the Lyn protein-tyrosine kinase to be markedly augmented in me/me andme v /me v compared with wild-type B cells. In addition, Lyn inducibly associates with the SHP-1 N-terminal SH2 domain and is dephosphorylated at its autophosphorylation site (Tyr-397) by incubation with SHP-1. These data identify Lyn as a substrate for SHP-1-mediated dephosphorylation/deactivation and suggest that SHP-1 inhibitory effects on Lyn activity contribute to the down-regulation of CD19 tyrosine phosphorylation and may thereby provide an important mechanism for disrupting CD19 interactions with downstream effectors involved in the relay and amplification of BCR-initiated activation signal. Antibodies used for these studies included the following: PE-conjugated B220 antibody from PharMingen (Mississauga, Ontario), rabbit polyclonal anti-Lyn antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), monoclonal anti-phosphotyrosine antibody 4G10 from Upstate Biotechnology Inc. (Lake Placid, NY), and goat F(ab′)2 anti-mouse IgM antibody from Jackson ImmunoResearch (West Grove, PA). Rat anti-mouse CD19 antibody was produced by the 1D3 hybridoma (provided by Dr. D. Fearon, University of Cambridge School of Medicine, Cambridge, UK) (35Krop I. de Fougerolles A.R. Hardy R.R. Allison M. Schlissel M.S. Fearon D.T. Eur. J. Immunol. 1996; 26: 238-242Crossref PubMed Scopus (111) Google Scholar), and a rabbit polyclonal anti-CD19 antibody was derived by immunization with a polylysine-conjugated peptide corresponding to amino acids 504–523 within the CD19 cytosolic domain (SynPep Corp., Dublin, CA). Rabbit polyclonal anti-SHP-1 antibody and monoclonal anti-Thy1.2 antibody from the hybridoma clone J1j.10 (ATCCT1B184) were produced in our laboratory as described previously (14Pani G. Kozlowski M. Cambier J.C. Mills G.B. Siminovitch K.A. J. Exp. Med. 1995; 181: 2077-2084Crossref PubMed Scopus (240) Google Scholar, 16Kozlowski M. Mlinaric-Rascan I. Feng G.-S. Shen R. Pawson T. Siminovitch K.A. J. Exp. Med. 1993; 178: 2157-2163Crossref PubMed Scopus (210) Google Scholar). Low-Tox rabbit complement was purchased from Cedarlane (Hornby, ONT), and chemicals used for immunoprecipitation/immunoblotting were purchased from Sigma Chemical Co. (St. Louis, MO). Single cell suspensions of splenocytes were obtained from 10- to 14-day-old C3HeBFeJ-melme(motheaten), C57BL6-me v /me v (viable motheaten), and congenic wild-type (+/+) mice derived at the Samuel Lunenfeld Research Institute breeding facility by mating C3HeBFeJ-me/+ and +/+ and C57BL/6J-me v /+ and +/+ breeding pairs. Purified populations of splenic B lymphocytes were obtained fromme/me,me v /me v, and wild-type congenic mice by subjecting splenic cell suspensions to erythrocyte lysis in 0.8% ammonium chloride, followed by treatment with anti-Thy1.2 antibody for 30 min on ice and a subsequent 45-min incubation with a 1:15 dilution of rabbit complement (Serotec Ltd., Toronto, Ontario). The cells were then washed and layered over a Percoll gradient (Amersham Pharmacia Biotech, Baie d'Urfé, Province of Quebec) as described previously (14Pani G. Kozlowski M. Cambier J.C. Mills G.B. Siminovitch K.A. J. Exp. Med. 1995; 181: 2077-2084Crossref PubMed Scopus (240) Google Scholar). The resulting cells were >90% mIg and B220 positive as determined by fluorescence-activated cell sorting (Becton Dickinson, Mountainview, CA) analysis. The CD5+ murine B lymphoma line (36Arnold L.W. La Cascio N.J. Lutz P.M. Pennell C.A. Klapper D. Haughton G. J. Immunol. 1983; 131: 2064-2268PubMed Google Scholar) (provided by Dr. A. Kaushik, University of Guelph, Guelph, Ontario) and the WEHI-231 B lymphoma line (purchased from ATCC, Rockville, MD) were cultured at 37 °C in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 5% fetal bovine serum (Sterile System Inc., Logan, UT), 50 μm 2-β mercaptoethanol, and 100 μg/ml penicillin/streptomycin. WEHI-231, CH12, or purified splenic B cells (2–5 × 107) were resuspended in 5 ml of culture medium and stimulated with 40 μg/ml F(ab′)2antibody for varying periods of time. Stimulations were done on ice to retard biochemical reactions when studying the kinetics of CD19 phosphorylation and Lyn kinase activation (37Burg D.L. Furlong M.T. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1994; 269: 28136-28142Abstract Full Text PDF PubMed Google Scholar). For biotinylation, 5–6 × 107 cells/ml were suspended at 107cells/ml in ice-cold PBS and mixed with 0.3 mg/ml sulfo-NHS-Biotin solution (Pierce Chemical Co., Rockford, IL). After 30-min incubation at room temperature, the reaction was quenched by 5-min incubation with 50 mg/ml glycine in PBS. Cells were than washed twice in cold PBS and subjected to stimulation as above. Following stimulation, biotinylated or nonbiotinylated cells were incubated in lysis buffer (50 mm Tris-HCI, pH 8.0, 150 mm NaCl, 50 μm NaF, 2 mm phenylmethylsulfonyl fluoride, 2 mm Na3VO4, 50 mmZnCl2, 50 μm o-phosphate, 2 mm EDTA, 10 μg/ml leupeptin, 10 μg/ml aprotinin) containing either 1% CHAPS (Sigma) or 1% Nonidet P-40. Cell lysates were centrifuged at 14,000 × g for 10 min at 4 °C, and protein concentrations were then determined using the bicinchoninic acid assay (Pierce). Anti-Lyn or anti-IgG (isotype control) antibody was incubated at 4 °C overnight under rocking conditions with protein A-Sepharose 4B (Amersham Pharmacia Biotech) in PBS (about 10 μg of antibody per 10 μl of beads). Beads were then washed two times in 0.1 m sodium borate (pH 8.6) and two times in 0.2 m tri-ethanolamine (pH 8.2). Beads were then resuspended in 0.2 m tri-ethanolamine solution containing 40 mm dimethyl pimelimidate dihydrochloride (Pierce) and incubated for 1 h at room temperature with continual rocking. The antibody-coupled beads were washed two times in 200 mm ethanolamine (pH 8.2), two times in 0.1m sodium borate (pH 8.0), two times in PBS, and resuspended in PBS supplemented with 0.2% NaN3. Lysates were precleared before immunoprecipitation by incubating 1 mg of cell lysate with protein A-Sepharose beads (Amersham Pharmacia Biotech) at 4 °C for 1 h and for an additional 1 h with 40 μl of rabbit preimmune serum. Lysates were then incubated for 2 h at 4 °C with the appropriate antibody (anti-Lyn, anti-IgG isotype control) and 25 μl of protein A- or protein G-Sepharose beads. Immune complexes were then collected by centrifugation, washed four times in lysis buffer, and boiled for 5 min in reducing SDS-gel sample buffer. Samples were then electrophoresed through SDS-polyacrylamide and transferred to nitrocellulose (Bio-Rad Laboratories, Mississauga, Ontario). After 1-h incubation in 3% gelatin, the filters were incubated for 1 h at room temperature with anti-CD19, anti-Lyn, or anti-phosphotyrosine 4G10 antibodies followed by horseradish peroxidase-labeled secondary antibody (Amersham Pharmacia Biotech, Arlington Heights, ICN) or, for analysis of biotinylated cells, with horseradish peroxidase-avidin (Pierce). Immune complexes were detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech). Stripping and reprobing of the blots were performed according to Amersham Pharmacia Biotech's recommended protocol. For analysis of CD19-associated phosphatase activity, anti-CD19 immunoprecipitates were prepared (as described above) from 1 mg of lysates of unstimulated or anti-IgM antibody-stimulated motheaten and wild-type B cells. Phosphatase assays were also performed on anti-CD19 and anti-IgG (control) immunoprecipitates prepared from lysates of wild-type splenic B cells immunodepleted of SHP-1 by overnight incubation with an excess of anti-SHP-1 antibody followed by addition of 100 μl of protein A-Sepharose. For this experiment, the complete immunodepletion of SHP-1 protein was confirmed by Western immunoblotting analysis (data not shown). The amount of SHP-1 antibody utilized to completely immunodeplete SHP-1 protein from lysates was predetermines by titration and Western immunoblotting analysis (data not shown). Immunoprecipitates were washed twice in phosphatase buffer (10 mm Tris-HCl, 1.0 mm EDTA, 1 mg/ml bovine serum albumin, 0.1% 2-β-mercaptoethanol, 0.01% NaN3, pH 7.34) and then incubated for 12 h at 37 °C overnight in phosphatase buffer containing 2 mm p-nitrophenyl phosphate (Sigma). Under these conditions, SHP-1 activity for the substrate has been shown previously to increase linearly with the amount of SHP-1 used in the reaction (38Kon-Kozlowski M. Pani G. Pawson T. Siminovitch K.A. J. Biol. Chem. 1996; 271: 3856-3862Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 39Pei D. Neel B.G. Walsh C.T. Proc, Natl. Acad. Sci. U. S. A. 1993; 90: 1092-1096Crossref PubMed Scopus (66) Google Scholar). Reactions were terminated by addition of 0.2 n NaOH, and absorbance was measured at 410 nm by spectrophotometry. Lyn kinase activity was evaluated using immunoprecipitates prepared as described above from unstimulated and stimulated splenic B cells. The immunoprecipitates were washed in kinase buffer (20 mm HEPES, pH 7.6, 150 mmNaCl, 5 mm MnCl2, 0.25 mmNa3VO4, 0.5% Nonidet P-40, 0.1 mm2-β-mercaptoethanol) and then incubated for 30 min at 30 °C in 20 μl of kinase buffer containing 10 μCi of [γ-32P]ATP (ICN) with or without 10 μg of GST-Igα/β fusion protein (provided by Dr. Y. Wu, Toronto, Ontario). Samples were resuspended in SDS-gel sample buffer, boiled, and centrifuged at 14,000 × gfor 10 min and resolved over 10% SDS-PAGE gels. The32P-labeled proteins were electrophoretically transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) and then visualized by autoradiography. Lyn quantification was performed by anti-Lyn immunoblotting of the membranes using ECL. GlutathioneS-transferase (GST)-SHP-1 fusion proteins were generated by subcloning the following murine cDNA or polymerase chain reaction-amplified fragments into pGEX2T: the full-length SHP-1 cDNA (GST-SHP-1), a full-length SHP-1 cDNA containing a Cys-453 → Ser mutation (GST-SHP-1 (C453S)), the SHP-1 N-terminal SH2 domain (amino acids 1–95), the SHP-1 C-terminal SH2 domain (amino acids 110–205), and the SHP-1 N- and C-terminal SH2 domains (amino acids 1–221). These expression plasmids were transfected intoEscherichia coli JM101, and the fusion proteins were purified from isopropyl β-d-thiogalactopyranoside-induced bacteria using glutathione-conjugated Sepharose beads (Amersham Pharmacia Biotech). Equimolar amounts of each GST-SHP-1 fusion protein and GST-beads were then incubated at 4 °C for 1 h with 0.9 μg of in vitro 32P-labeled purified Lyn protein. Beads were washed seven times, and the complexes were resuspended in SDS-sample buffer, boiled, analyzed by SDS-PAGE, and transferred to nitrocellulose, and the Lyn protein was visualized by autoradiography. In vitro[γ-32P]ATP-labeled Lyn was immunoprecipitated using anti-Lyn antibody and incubated at 37 °C with equal amounts of either GST-SHP-1 or GST-SHP-1 (C453S) protein in 200 μl of phosphatase buffer (10 mm Tris-HC1, 1.0 mmEDTA, 1 mg/ml bovine serum albumin, 0.1% 2-β-mercaptoethanol, 0.01% NaN3, pH 7.34). The immune complexes were then resolved over SDS-PAGE and transferred to nitrocellulose. The 56-kDa Lyn-containing bands were then excised from the membranes and subjected to CNBr cleavage as described previously (40Hurley T.R. Hyman R. Sefton B.M. Mol. Cell. Biol. 1993; 13: 1651-1656Crossref PubMed Scopus (166) Google Scholar). The excised Lyn protein was incubated with 60 mg/ml CNBr in 70% formic acid for at least 2 h at room temperature. Samples were then washed and dried, and the CNBr-generated peptide fragments were resuspended in tricine SDS sample buffer, resolved by separation on 10–20% gradient Tricine SDS-PAGE (Novex, San Diego, CA), transferred to an Immobilon-P membrane, and visualized by autoradiography. Complete digestion of each sample was confirmed by the absence of higher molecular weight32P-labeled bands, and the CNBr fragments were quantitated by phosphorimaging (Molecular Dynamics). Previous data from our laboratory and others have revealed modulation of inhibitory coreceptors to represent a major mechanism whereby SHP-1 mediates its down-regulatory effects on BCR signaling (5Siminovitch K.A. Neel B.J. Semin. Immunol. 1998; 10: 329-347Crossref PubMed Scopus (61) Google Scholar, 18Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (480) Google Scholar, 19Blery M. Kubagawa H. Chen C.C. Vely F. Cooper M.D. Vivier E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2446-2451Crossref PubMed Scopus (176) Google Scholar, 20Wu Y. Nadler M.J. Brennan L.A. Gish G.D. Timms J.F. Fusaki N. Jongstra-Bilen J. Tada N. Pawson T. Wither J. Neel B.G. Hozumi N. Curr. Biol. 1998; 8: 1009-1017Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). However, data garnered from the analysis of mice deficient for both the CD45 and SHP-1 PTPs (31Sato S. Ono N. Steeber D.A. Pisetsky D.S. Tedder T.F. J. Immunol. 1996; 157: 4371-4378PubMed Google Scholar) raised the possibility that SHP-1 also influences the tyrosine phosphorylation and, by extension, signaling functions of the positive regulatory coreceptor, CD19. To begin addressing this issue, SHP-1-deficient B cells from me/me andme v /me v mice were evaluated with regards to the kinetics of CD19 phosphorylation following BCR ligation. As illustrated in Fig.1, anti-phosphotyrosine immunoblotting analysis of CD19 immunoprecipitates from the SHP-1-deficient cells revealed tyrosine phosphorylation of the 115- to 120-kDa species representing CD19 to be markedly increased both constitutively and inducibly in the me v/me vand to a lesser extent in the me/me cells compared with wild-type cells. The increased phosphorylation of CD19 detected in the motheaten cells cannot be attributed to expansion of the CD5+ B-1 cell population in these mice, because CD5+ CH12 cells exhibited normal levels of CD19 phosphorylation both before and after BCR cross-linking (data not shown). These data support the contention that SHP-1 modulates CD19 tyrosine phosphor" @default.
• W2072835510 created "2016-06-24" @default.
• W2072835510 creator A5027215304 @default.
• W2072835510 creator A5040888166 @default.
• W2072835510 creator A5062765426 @default.
• W2072835510 creator A5064595347 @default.
• W2072835510 creator A5077628272 @default.
• W2072835510 creator A5086505183 @default.
• W2072835510 date "2001-01-01" @default.
• W2072835510 modified "2023-10-16" @default.
• W2072835510 title "The SH2 Domain Containing Tyrosine Phosphatase-1 Down-regulates Activation of Lyn and Lyn-induced Tyrosine Phosphorylation of the CD19 Receptor in B Cells" @default.
• W2072835510 cites W133056582 @default.
• W2072835510 cites W1480844331 @default.
• W2072835510 cites W1487500734 @default.
• W2072835510 cites W1488229599 @default.
• W2072835510 cites W1543886931 @default.
• W2072835510 cites W1602442471 @default.
• W2072835510 cites W1617435269 @default.
• W2072835510 cites W164293537 @default.
• W2072835510 cites W1665416055 @default.
• W2072835510 cites W1670289781 @default.
• W2072835510 cites W1884715737 @default.
• W2072835510 cites W1956939804 @default.
• W2072835510 cites W1964203957 @default.
• W2072835510 cites W1969318766 @default.
• W2072835510 cites W1972602948 @default.
• W2072835510 cites W1979769420 @default.
• W2072835510 cites W1980485261 @default.
• W2072835510 cites W1986150526 @default.
• W2072835510 cites W1996486759 @default.
• W2072835510 cites W2003319634 @default.
• W2072835510 cites W2009818164 @default.
• W2072835510 cites W2011211211 @default.
• W2072835510 cites W2012514963 @default.
• W2072835510 cites W2013611981 @default.
• W2072835510 cites W2015042072 @default.
• W2072835510 cites W2018394492 @default.
• W2072835510 cites W2030793757 @default.
• W2072835510 cites W2032667632 @default.
• W2072835510 cites W2034768126 @default.
• W2072835510 cites W2037375596 @default.
• W2072835510 cites W2049072057 @default.
• W2072835510 cites W2051774115 @default.
• W2072835510 cites W2053541596 @default.
• W2072835510 cites W2060548377 @default.
• W2072835510 cites W2061728533 @default.
• W2072835510 cites W2063825367 @default.
• W2072835510 cites W2066138756 @default.
• W2072835510 cites W2072241069 @default.
• W2072835510 cites W2078991312 @default.
• W2072835510 cites W2083673122 @default.
• W2072835510 cites W2086227162 @default.
• W2072835510 cites W2088549138 @default.
• W2072835510 cites W2088551118 @default.
• W2072835510 cites W2090392995 @default.
• W2072835510 cites W2092354039 @default.
• W2072835510 cites W2101073455 @default.
• W2072835510 cites W2112997504 @default.
• W2072835510 cites W2117309266 @default.
• W2072835510 cites W2121754249 @default.
• W2072835510 cites W2123938732 @default.
• W2072835510 cites W2134495747 @default.
• W2072835510 cites W2134505654 @default.
• W2072835510 cites W2134884900 @default.
• W2072835510 cites W2140678474 @default.
• W2072835510 cites W2144954864 @default.
• W2072835510 cites W2152218437 @default.
• W2072835510 cites W2153072554 @default.
• W2072835510 cites W2166771367 @default.
• W2072835510 cites W2172099254 @default.
• W2072835510 doi "https://doi.org/10.1074/jbc.m006820200" @default.
• W2072835510 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11042209" @default.
• W2072835510 hasPublicationYear "2001" @default.
• W2072835510 type Work @default.
• W2072835510 sameAs 2072835510 @default.
• W2072835510 citedByCount "45" @default.
• W2072835510 countsByYear W20728355102012 @default.
• W2072835510 countsByYear W20728355102013 @default.
• W2072835510 countsByYear W20728355102014 @default.
• W2072835510 countsByYear W20728355102017 @default.
• W2072835510 countsByYear W20728355102018 @default.
• W2072835510 countsByYear W20728355102021 @default.
• W2072835510 countsByYear W20728355102022 @default.
• W2072835510 countsByYear W20728355102023 @default.
• W2072835510 crossrefType "journal-article" @default.
• W2072835510 hasAuthorship W2072835510A5027215304 @default.
• W2072835510 hasAuthorship W2072835510A5040888166 @default.
• W2072835510 hasAuthorship W2072835510A5062765426 @default.
• W2072835510 hasAuthorship W2072835510A5064595347 @default.
• W2072835510 hasAuthorship W2072835510A5077628272 @default.
• W2072835510 hasAuthorship W2072835510A5086505183 @default.
• W2072835510 hasBestOaLocation W20728355101 @default.
• W2072835510 hasConcept C108636557 @default.
• W2072835510 hasConcept C11960822 @default.
• W2072835510 hasConcept C1491633281 @default.
• W2072835510 hasConcept C170493617 @default.
• W2072835510 hasConcept C175818844 @default.
• W2072835510 hasConcept C185592680 @default.

• next