FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2007181639> ?p ?o ?g. }

• W2007181639 endingPage "16421" @default.
• W2007181639 startingPage "16415" @default.
• W2007181639 abstract "Ligation of the FcγR on natural killer (NK) cells results in the tyrosine phosphorylation of multiple substrates critical for intracellular signaling and activation of NK cell effector functions. However, it remains unclear which nonreceptor protein-tyrosine kinases (PTK) participate in this process. In this report we demonstrate that FcγR ligation induced the tyrosine phosphorylation and increased the catalytic activities of both syk family PTKs, ZAP-70, and syk. The phosphorylation of ZAP-70 and syk was enhanced markedly by overexpression of wild-type lck but not by a kinase-inactive mutant, suggesting that early FcγR-initiated activation of lck results in the subsequent regulation of syk family PTKs. The regulatory interplay between src and syk family PTKs was emphasized further by the observation that lck overexpression enhanced the association of ZAP-70 with the ζ chain of the FcγR complex. Additional analyses indicated that lck induced the subsequent tyrosine phosphorylation of phospholipase C (PLC)-γ2. Interestingly, the regulatory effects of lck on ZAP-70, syk, and PLC-γ2 could not be replaced by overexpression of either fyn or src, demonstrating a selective role for lck in effectively coupling FcγR stimulation to critical downstream signaling events. Taken together, our results suggest not only that FcγR stimulation on NK cells is coupled to the intracellular activation of both ZAP-70 and syk, but that the src family member, lck, can selectively regulate this tyrosine kinase cascade. Ligation of the FcγR on natural killer (NK) cells results in the tyrosine phosphorylation of multiple substrates critical for intracellular signaling and activation of NK cell effector functions. However, it remains unclear which nonreceptor protein-tyrosine kinases (PTK) participate in this process. In this report we demonstrate that FcγR ligation induced the tyrosine phosphorylation and increased the catalytic activities of both syk family PTKs, ZAP-70, and syk. The phosphorylation of ZAP-70 and syk was enhanced markedly by overexpression of wild-type lck but not by a kinase-inactive mutant, suggesting that early FcγR-initiated activation of lck results in the subsequent regulation of syk family PTKs. The regulatory interplay between src and syk family PTKs was emphasized further by the observation that lck overexpression enhanced the association of ZAP-70 with the ζ chain of the FcγR complex. Additional analyses indicated that lck induced the subsequent tyrosine phosphorylation of phospholipase C (PLC)-γ2. Interestingly, the regulatory effects of lck on ZAP-70, syk, and PLC-γ2 could not be replaced by overexpression of either fyn or src, demonstrating a selective role for lck in effectively coupling FcγR stimulation to critical downstream signaling events. Taken together, our results suggest not only that FcγR stimulation on NK cells is coupled to the intracellular activation of both ZAP-70 and syk, but that the src family member, lck, can selectively regulate this tyrosine kinase cascade. Exposure to foreign antigens elicits an antibody response from the immune system. The subsequent interaction of Fc receptor-bearing effector cells with antibodies complexed to soluble or cell-bound antigens initiates a variety of effector functions. Natural killer (NK)1( 1The abbreviations used are: NKnatural killerFcγRlow affinity IgG Fc receptors (FcγR type III)PLCphospholipase CPTKprotein- tyrosine kinaseTCRT cell antigen receptorBCRB cell antigen receptormAbmonoclonal antibodyPAGEpolyacrylamide gel electrophoresisTween 20polyoxyethylene sorbitan monolaurate.) cells represent a distinct subpopulation of lymphocytes which express the IgG Fc receptor type IIIA, hereafter referred to as FcγR(1Trinchieri G. Adv. Immunol. 1989; 47: 187-376Crossref PubMed Scopus (2717) Google Scholar). The FcγR on human NK cells is a multimeric receptor complex consisting of the ligand-binding α subunit (i.e. CD16), which associates noncovalently with dimers of ζ and γ chains(2Ravetch J.V. Kinet J.-P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1286) Google Scholar). Although none of the components of the receptor complex possesses intrinsic kinase activity, stimulation of the FcγR rapidly activates a protein-tyrosine kinase (PTK) signaling pathway that results in the tyrosine phosphorylation of substrates critical for cellular activation, including the ζ chain and phospholipase C (PLC)-γ isoforms(3O'Shea J.J. Weissman A.M. Kennedy I.C.S. Ortaldo J.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 350-354Crossref PubMed Scopus (102) Google Scholar, 4Vivier E. Morin P. O'Brien C. Druker B. Schlossman S.F. Anderson P. J. Immunol. 1991; 146: 206-210PubMed Google Scholar, 5Azzoni L. Kamoun M. Salcedo T.W. Kanakaraj P. Perussia B. J. Exp. Med. 1992; 176: 1745-1750Crossref PubMed Scopus (104) Google Scholar, 6Liao F. Shin H.S. Rhee S.G. J. Immunol. 1993; 150: 2668-2674PubMed Google Scholar, 7Ting A.T. Karnitz L.M. Schoon R.A. Abraham R.T. Leibson P.J. J. Exp. Med. 1992; 176: 1751-1755Crossref PubMed Scopus (125) Google Scholar). This FcγR-initiated PTK signaling pathway appears to be requisite for the activation of NK cell-mediated cytotoxicity and lymphokine production(8O'Shea J.J. McVicar D.W. Kuhns D.B. Ortaldo J.R. J. Immunol. 1992; 148: 2497-2502PubMed Google Scholar, 9Einspahr K.J. Abraham R.T. Binstadt B.A. Uehara Y. Leibson P.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6279-6283Crossref PubMed Scopus (91) Google Scholar, 10Cone J.C. Lu Y. Trevillyan J.M. Bjorndahl J.M. Phillips C.A. Eur. J. Immunol. 1993; 23: 2488-2497Crossref PubMed Scopus (47) Google Scholar). natural killer low affinity IgG Fc receptors (FcγR type III) phospholipase C protein- tyrosine kinase T cell antigen receptor B cell antigen receptor monoclonal antibody polyacrylamide gel electrophoresis polyoxyethylene sorbitan monolaurate. Elegant studies by several groups have demonstrated that ligation of chimeric receptors containing the intracellular portions of either the ζ or γ chains is sufficient to activate intracellular PTKs as well as mediate downstream cytolytic function and lymphokine production (11Irving B.A. Weiss A. Cell. 1991; 64: 891-901Abstract Full Text PDF PubMed Scopus (633) Google Scholar, 12Romeo C. Seed B. Cell. 1991; 64: 1037-1046Abstract Full Text PDF PubMed Scopus (293) Google Scholar, 13Letourneur F. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8905-8909Crossref PubMed Scopus (245) Google Scholar, 14Wirthmueller U. Kurosaki T. Murakami M.S. Ravetch J.V. J. Exp. Med. 1992; 175: 1381-1390Crossref PubMed Scopus (148) Google Scholar). Additional studies have identified a conserved motif in the ζ and γ chains, with the consensus amino acid sequence YXXL-X(6-8)-YXXL, which is sufficient to mediate intracellular signaling(15Romeo C. Amiot M. Seed B. Cell. 1992; 68: 889-897Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 16Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (256) Google Scholar). src family members are candidates for the FcγR-associated PTKs. Recent reports have shown that lck, a member of the src family, is detectable in anti-FcγR immunoprecipitates and that ligation of FcγR increases the in vitro catalytic activity of lck(10Cone J.C. Lu Y. Trevillyan J.M. Bjorndahl J.M. Phillips C.A. Eur. J. Immunol. 1993; 23: 2488-2497Crossref PubMed Scopus (47) Google Scholar, 17Pignata C. Prasad K.V.S. Robertson M.J. Levine H. Rudd C.E. Ritz J. J. Immunol. 1993; 151: 6794-6800PubMed Google Scholar, 18Salcedo T.W. Kurosaki T. Kanakaraj P. Ravetch J.V. Perussia B. J. Exp. Med. 1993; 177: 1475-1480Crossref PubMed Scopus (110) Google Scholar). Nonetheless, the precise regulatory function of lck during FcγR-initiated signaling remains unclear. The syk family PTKs represent an additional class of cytoplasmic PTKs that have been implicated in lymphoid cell signal transduction. T cell antigen receptor (TCR) ligation has been shown to induce the tyrosine phosphorylation of ZAP-70 and its association with the ζ chains of activated TCR complexes(19Chan A.C. Irving B.A. Fraser J.D. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9166-9170Crossref PubMed Scopus (338) Google Scholar, 20Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (889) Google Scholar). Furthermore, a deficiency in ZAP-70 expression severely impairs TCR-mediated signaling(21Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (484) Google Scholar, 22Chan A.C. Kadleck T.A. Elder M.E. Filipovich A.H. Kuo W.-L. Iwashima M. Parslow T.G. Weiss A. Science. 1994; 264: 1599-1601Crossref PubMed Scopus (436) Google Scholar). Likewise, stimulation of the B cell antigen receptor (BCR) results in the tyrosine phosphorylation of the receptor-associated syk(23Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1991; 266: 14846-14849Abstract Full Text PDF PubMed Google Scholar, 24Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1992; 267: 8613-8619Abstract Full Text PDF PubMed Google Scholar). In contrast to T and B cells, the function of syk family PTKs in NK cell activation is less clear. Ligation of the FcγR on NK cells can induce the association of a 70-kDa phosphotyrosyl protein with the receptor complex(25Vivier E. da Silva A.J. Ackerly M. Levine H. Rudd C.E. Anderson P. Eur. J. Immunol. 1993; 23: 1872-1876Crossref PubMed Scopus (73) Google Scholar), but the precise roles of ZAP-70 and/or syk during FcγR signal transduction and the nature of their potential interaction with src family PTKs are unknown. We focused our initial examinations of the PTKs involved in FcγR signaling on lck and its role in coupling FcγR stimulation to subsequent tyrosine phosphorylation events. Using the vaccinia virus expression system, we overexpressed wild-type lck in cloned human NK cells. Overexpression of wild-type active lck, but not a kinase-deficient mutant of this PTK, markedly enhanced the FcγR-induced tyrosine phosphorylation of ZAP-70, syk, and PLC-γ2. Furthermore, only lck effectively coupled the FcγR to downstream PTKs, as neither fyn nor src could substitute for lck. Taken together, our data strongly implicate a role for the src family PTK, lck, in the FcγR-initiated regulation of ZAP-70, syk, and PLC-γ. Human CD16+ NK cell lines were isolated and passaged as described previously(26Ting A.T. Einspahr K.J. Abraham R.T. Leibson P.J. J. Immunol. 1991; 147: 3122-3127PubMed Google Scholar). The cell surface phenotype of these NK cell lines was monitored by flow cytometry. All NK cell lines used in these studies were >90% CD16+. In addition, all CD16+ NK cells expressed the following additional phenotypic markers: CD56+, CD11b+, CD2+, and HLA-DR+. All chemicals and drugs, unless otherwise noted, were obtained from Sigma. Fluorescein- and phycoerythrin-conjugated monoclonal antibodies (mAb) were obtained from Becton-Dickinson Monoclonal Center (Mountain View, CA). The anti-FcγR mAb (3G8) has been described previously(27Perussia B. Trinchieri G. J. Immunol. 1984; 132: 1410-1415PubMed Google Scholar). Immunoprecipitating ZAP-70 and syk rabbit antisera and immunoblotting anti-phosphotyrosine mAb 4G10 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Immunoblotting ZAP-70 and syk antisera were raised in rabbits injected with keyhole limpet hemocyanin-conjugated synthetic peptides (ZAP-70 residues 326-341; 28 C-terminal amino acids of porcine p72syk)(20Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (889) Google Scholar, 28Taniguchi T. Kobayashi T. Kondo J. Takahashi K. Nakamura H. Suzuki J. Nagai K. Yamada T. Nakamura S. Yamamura H. J. Biol. Chem. 1991; 266: 15790-15796Abstract Full Text PDF PubMed Google Scholar). The generation and characterization of the lck and PLC-γ2 rabbit antisera have been described previously(7Ting A.T. Karnitz L.M. Schoon R.A. Abraham R.T. Leibson P.J. J. Exp. Med. 1992; 176: 1751-1755Crossref PubMed Scopus (125) Google Scholar, 29Karnitz L. Sutor S.L. Torigoe T. Reed J.C. Bell M.P. McKean D.J. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1992; 12: 4521-4530Crossref PubMed Scopus (184) Google Scholar). The ζ antiserum was generously provided by Augusta Ochoa (National Cancer Institute). Recombinant vaccinia viruses encoding wild-type or mutant src family PTK were generated essentially as described(30Earl P.L. Moss B. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocol in Molecular Biology. John Wiley and Sons, New York1991: 16.15.1-16.17.16Google Scholar). Blunt ended cDNA fragments were obtained from the following sources. A a StuI fragment of c-lck was excised from the plasmid NT18 (31Marth J.D. Peet R. Krebs E.G. Perlmutter R.M. Cell. 1985; 43: 393-404Abstract Full Text PDF PubMed Scopus (363) Google Scholar) and a EcoRV-SmaI fragment of c-fyn was excised from the plasmid pMTFR(32Karnitz L.M. Sutor S.L. Abraham R.T. J. Exp. Med. 1994; 179: 1799-1808Crossref PubMed Scopus (73) Google Scholar), both of which were generously provided by Roger Perlmutter (University of Washington, Seattle). A StuI fragment of lck with a lysine to arginine mutation at position 273 was excised from a plasmid generously provided by Bart Sefton (Salk Institute, San Diego). A HindIII fragment of c-src was obtained from the plasmid pM5H (33Wilson L.K. Kuttrell D.K. Parsons J.T. Parsons S.J. Mol. Cell. Biol. 1989; 9: 1536-1544Crossref PubMed Scopus (95) Google Scholar) kindly provided by Sarah Parsons (University of Virginia, Charlottesville). These blunt ended cDNAs were inserted into the SmaI cloning site of the vector pSC11 (34Chakrabarti S. Brechling K. Moss B. Mol. Cell. Biol. 1985; 5: 3403-3409Crossref PubMed Scopus (623) Google Scholar) and introduced into WR strain vaccinia by homologous recombination. The recombinant vaccinia viruses were characterized by infection of CV-1 cells and subsequent detection by immunoblotting with antisera specific for the different src family PTKs. In vitro autophosphorylation assays were also performed on immunoprecipitated src family PTKs to confirm their catalytic activities. Viruses were propagated in HeLa cell cultures and released by lysing infected HeLa cells with a probe sonicator. The lysate was then layered over a cushion of 36% sucrose solution in 10 mM Tris-HCl, pH 9.0, and centrifuged in a Beckman SW 28 rotor (13,500 rpm, 2 h) to purify the virus. Viruses were subsequently titered on confluent BSC-1 monolayers using the method described previously(35Leibson P.J. Hunter-Laszlo M. Douvas G.S. Hayward A.R. J. Clin. Immunol. 1986; 6: 216-224Crossref PubMed Scopus (29) Google Scholar). NK cells (2 × 106 cells/ml in serum-free RPMI) were infected for 1 h at 37°C at a multiplicity of infection of 20. Cells were then incubated for an additional 3-5 h at 1 × 106 cells/ml in RPMI with 10% bovine calf serum. Infected NK cells were washed twice and resuspended in RPMI with 0.5% bovine serum albumin for stimulation. 100-μl aliquots of NK cells (2 × 106 cells/sample) were incubated at 4°C for 3 min with 10 μl of anti-FcγR mAb (3G8) (final concentration, 100 μg of goat F(ab′)2 fragment anti-mouse IgG (Organon Teknika Corp., West Chester, PA) was then added to the cell suspension, mixed, and briefly pelleted. After incubating at 37°C for the indicated time, pelleted cells were lysed with buffer containing 10 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 500 μM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 10 μg/ml leupeptin, and 1% Triton X-100, pH 7.4. Insoluble material was removed by centrifugation at 15,000 × g for 10 min, and detergent-soluble proteins resolved by 8.5% SDS-polyacrylamide gel electrophoresis (PAGE). Resolved proteins were electrophoretically transferred to Immobilon-P membranes (Millipore, Bedford, MA), and immunoblotting with the anti-phosphotyrosine mAb 4G10 was performed as described previously (7Ting A.T. Karnitz L.M. Schoon R.A. Abraham R.T. Leibson P.J. J. Exp. Med. 1992; 176: 1751-1755Crossref PubMed Scopus (125) Google Scholar). Immunodetection with rabbit antisera specific for ZAP-70, PLC-γ2, and lck were performed as described previously(7Ting A.T. Karnitz L.M. Schoon R.A. Abraham R.T. Leibson P.J. J. Exp. Med. 1992; 176: 1751-1755Crossref PubMed Scopus (125) Google Scholar). Briefly, membranes containing resolved proteins were blocked overnight in Tris-buffered saline containing 2% milk and 0.2% polyoxyethylene sorbitan monolaurate (Tween 20) and incubated for 1 h with the antiserum diluted in Tris-buffered saline containing 2% bovine serum albumin, 0.2% Tween 20, and 0.05% NaN3. After three washes with 0.2% Tween 20 in Tris-buffered saline, immunoreactive proteins were detected with protein A-horseradish peroxidase and the ECL detection system from Amersham Corp. 200-μl aliquots of NK cells (2 × 107/sample) were incubated at 4°C for 3 min with 10 μl of anti-FcγR mAb (3G8) (final concentration, 10 μg/ml). The cells were pelleted gently (700 × g, 30 s, 4°C) and resuspended in 200 μg of goat F(ab′)2 fragment anti-mouse IgG. After incubating at 37°C for the indicated time, reactions were terminated with 1 ml of ice-cold lysis buffer containing 20 mM Tris-HCl, 40 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 0.1% bovine serum albumin, 500 μM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 10 μg/ml leupeptin, and 1% Triton X-100, pH 7.4. After 10 min at 4°C, the samples were centrifuged (15,000 × g, 10 min) to remove nuclear and cellular debris. Postnuclear supernatants were immunoprecipitated for 1-2 h at 4°C with rabbit antisera bound to protein A-Sepharose beads. The immunoprecipitates were washed three times and bound proteins eluted with 50 μl of SDS-sample buffer and resolved by SDS-PAGE. Anti-phosphotyrosine immunoblotting was performed as described above. Anti-ζ and anti-syk immune complex kinase reactions were performed using a modification of the method described previously(36Wange R.L. Isakov N. Burke Jr., T.R. Otaka A. Roller P.P. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 944-948Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Briefly, cell stimulation was terminated in a Brij 96 lysis buffer (1% Brij, 25 mM Tris, pH 7.6, 150 mM NaCl, 1 mM Na3VO4, 5 mM EDTA, and 10 μg/ml aprotinin and leupeptin). Immunoprecipitates were washed two times, and the beads were incubated at 25°C for 5 min in 20 mM Tris, pH 7.6, 10 mM MnCl2, 1 μM ATP, 0.25 μg of cfb3 (cytoplasmic fragment of band 3; generously provided by Lawrence E. Samelson, NIH), 10 μCi of [γ-32P]ATP. The radiolabeled 43-kDa cfb3 was quantitated after scanning the membrane using the Radioanalytic Imaging System (model 4000, AMBIS, Inc., San Diego). Studies on NK cells have been hampered by the lack of a suitable methodology to genetically manipulate these cells. We report here the use of the vaccinia virus expression systems to overexpress efficiently src family PTKs in this cell type. Vaccinia viruses encoding wild-type src, fyn, lck, and a kinase-inactive lckR273 mutant (lysine to arginine mutation at position 273) were generated by homologous recombination. The absence of kinase activity in the lckR273 mutant was verified by infection of CV-1 cells with the different viruses followed by a lck-specific immune complex autophosphorylation assay (data not shown). After a 4-h infection of cloned NK cells with recombinant viruses encoding either wild-type or kinase-inactive lck, high levels of lck expression were detected by immunoblotting with a lck-specific antiserum (Fig. 1). Likewise, infection of NK cells with recombinant viruses encoding src or fyn resulted in the efficient overexpression of catalytically active PTKs (data not shown). Immunoblot analyses in multiple experiments revealed that infection of NK cells with the lck- and fyn-encoding viruses resulted in approximately 2-5-fold increases in both lck and fyn protein relative to endogenous levels. Furthermore, infection with either the lck-encoding or fyn-encoding viruses resulted in 2-3-fold increases in the total catalytic activity of anti-lck + anti-fyn immunoprecipitates for their shared substrate, enolase (data not shown). Whereas there was no detectable src in uninfected NK cells, infection with the src-encoding virus resulted in high levels of src expression (data not shown). The secretory and cytotoxic functions of NK cells remained intact during the 4-6-h infection with the vaccinia viruses. lck can physically associate with the FcγR complex and exhibits enhanced in vitro catalytic activity after FcγR ligation in NK cells(10Cone J.C. Lu Y. Trevillyan J.M. Bjorndahl J.M. Phillips C.A. Eur. J. Immunol. 1993; 23: 2488-2497Crossref PubMed Scopus (47) Google Scholar, 17Pignata C. Prasad K.V.S. Robertson M.J. Levine H. Rudd C.E. Ritz J. J. Immunol. 1993; 151: 6794-6800PubMed Google Scholar, 18Salcedo T.W. Kurosaki T. Kanakaraj P. Ravetch J.V. Perussia B. J. Exp. Med. 1993; 177: 1475-1480Crossref PubMed Scopus (110) Google Scholar). Furthermore, we demonstrated in NK clones that FcγR ligation induced a transient 2-3-fold increase in lck-specific in vitro kinase activity, whereas no change in fyn-specific activity was detected (data not shown). We next examined the effects of lck overexpression on FcγR-mediated signal transduction. As shown in Fig. 2, overexpression of lck in unstimulated NK cells led to the increased tyrosine phosphorylation of several intracellular proteins (fifth lane). Interestingly, the immunoreactive proteins migrating at molecular masses of approximately 150, 120, 116, 85, and 75 kDa displayed electrophoretic mobilities identical to those induced by stimulation of uninfected NK cells with anti-FcγR mAb (second and fifth lanes). FcγR ligation of lck-overexpressing NK cells led to further increases in the tyrosine phosphorylation levels of these substrates (sixth lane). Moreover, infection of NK cells with either the control wild-type WR strain vaccinia (third and fourth lanes) or the recombinant virus expressing the kinase-inactive lck (seventh and eighth lanes) did not alter the FcγR-induced tyrosine phosphorylation events. We next tested whether other members of the src family PTKs can induce the same effects as lck. In contrast to the dramatic effects observed with lck, overexpression of fyn had only minimal effects on the tyrosine phosphorylation levels of both resting and FcγR-stimulated NK cells (Fig. 3, seventh and eighth lanes). Furthermore, overexpression of src had no detectable effects on the FcγR-induced tyrosine phosphorylation of proteins (Fig. 3, ninth and tenth lanes). In Fig. 3, both fyn (seventh and eighth lanes) and src (ninth and tenth lanes) are detectable as phosphotyrosyl proteins migrating at molecular mass of 60 kDa. These observations are consistent with a specific role for lck in FcγR-initiated tyrosine phosphorylation events.Figure 3:Effects of src family PTK overexpression on tyrosine phosphorylation in NK cells. NK cells (2 × 106/sample) were either uninfected or infected with control WR, lck-, fyn-, or src-encoding vaccinia virus. FcγR stimulation and phosphotyrosine detection of cellular lysates were performed as described in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next investigated whether members of the syk family PTK are involved in FcγR signaling. NK cells were stimulated with cross-linked anti-FcγR mAb, and then either ZAP-70 or syk was immunoprecipitated with its respective antiserum. Precipitated proteins were resolved by SDS-PAGE, transferred to Immobilon-P membranes, and blotted with the anti-phosphotyrosine mAb, 4G10. Stimulation of FcγR rapidly elevated the phosphotyrosine levels of both ZAP-70 (Fig. 4A) and syk (Fig. 4B). Both phosphorylation events exhibited similar kinetics, with phosphorylation peaking at 1 min and returning to basal level by 30 min. Although the 21-23-kDa tyrosine-phosphorylated isoforms of ζ associated with ZAP-70 after FcR ligation or after pervanadate-induced(37Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar, 38O'Shea J.J. McVicar D.W. Bailey T.L. Burns C. Smyth M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10306-10310Crossref PubMed Scopus (144) Google Scholar) stimulation (Fig. 5), similar associations with syk were not detected (data not shown). These results are consistent with an earlier report of the FcγR complex in activated NK cells associating with a 70-kDa phosphotyrosyl protein that displayed a peptide map similar to that of ZAP-70(25Vivier E. da Silva A.J. Ackerly M. Levine H. Rudd C.E. Anderson P. Eur. J. Immunol. 1993; 23: 1872-1876Crossref PubMed Scopus (73) Google Scholar).Figure 5:Kinetics of FcγR-induced association of phospho-ζ with ZAP-70. NK cells (2 × 107/sample) were stimulated for the indicated time (min) with either cross-linked anti-FcγR mAb 3G8 or pervanadate (PV). ZAP-70 immunoprecipitates were resolved by SDS-PAGE (12.5% gel), transferred to Immobilon-P membrane, and probed with the anti-phosphotyrosine mAb 4G10.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We extended this analysis to determine whether the FcγR-induced modifications of ZAP-70 and syk were associated with any changes in their catalytic activity. Recent reports have demonstrated that a peptide fragment of the human erythrocyte band 3 (i.e. cfb3) is an in vitro substrate for both ZAP-70 and syk(36Wange R.L. Isakov N. Burke Jr., T.R. Otaka A. Roller P.P. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 944-948Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 39Harrison M.L. Isaacson C.C. Burg D.L. Geahlen R.L. Low P.S. J. Biol. Chem. 1994; 269: 955-959Abstract Full Text PDF PubMed Google Scholar). Using this exogenous substrate in an in vitro kinase assay, we demonstrated that FcR ligation induced rapid and kinetically similar increases in the catalytic activities of ZAP-70 and syk, with maximal increases by 1 min and returns toward base line by 30 min (Fig. 6). The pervanadate-induced(37Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar, 38O'Shea J.J. McVicar D.W. Bailey T.L. Burns C. Smyth M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10306-10310Crossref PubMed Scopus (144) Google Scholar) tyrosine-phosphorylated forms of ZAP-70 and syk also had increased in vitro catalytic activity (Fig. 6). The data presented thus far implicate both src and syk family PTKs in FcγR signaling. However, the precise regulatory role of lck in the FcγR-initiated PTK pathway, including its potential interaction with syk family PTKs, is not known. To evaluate these questions, NK cells were first infected with either control WR vaccinia virus or the recombinant lck-encoding vaccinia virus and then stimulated with cross-linked anti-FcγR mAb (Fig. 7). Similar to our previous observation with uninfected NK cells (Fig. 4), cross-linking of the FcγR on control WR-infected cells induces the tyrosine phosphorylation of ZAP-70 (first and second lanes) and syk (fifth and eighth lanes). Significantly, overexpression of lck markedly enhanced the FcγR-induced tyrosine phosphorylation of both ZAP-70 (third and fourth lanes) and syk (seventh and eighth lanes). When the FcγR-induced tyrosine phosphorylation levels of ZAP-70 and syk in lck-overexpressing cells were compared with their respective counterparts in control cells, the enhancement was consistently greater for ZAP-70 (approximately 3-fold) than for syk (approximately 1.5-fold) (Fig. 7). This quantitative difference in the augmentation of the phosphorylation of ZAP-70 and syk by lck was observed reproducibly in three separate experiments. These effects caused by lck overexpression required an active kinase as they were not seen with the kinase-inactive mutant (data not shown). Furthermore, neither fyn nor src overexpression enhanced the FcγR-induced tyrosine phosphorylation of ZAP-70 and syk (Fig. 8). Taken together, these results suggest that lck can selectively regulate ZAP-70 and syk during FcγR signaling. In addition, the quantitative difference in lck's effect on the two related members of the syk family may reflect a difference in their requirement for src family PTKs.Figure 8:Effects of src family PTK overexpression on ZAP-70 and syk tyrosine phosphorylation. NK cells (2 × 107/sample) infected with either the control WR, lck-, fyn-, or src-encoding vaccinia virus were stimulated (+), or not (-), for 1 min with cross-linked anti-FcγR mAb 3G8. Phosphotyrosine detection of ZAP-70 (upper panel) or syk (lower panel) immunoprecipitates was performed as described in Fig. 7.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next investigated whether lck-mediated events regulate the association of ZAP-70 with the FcγR complex. NK cells overexpressing lck were stimulated with cross-linked anti-FcγR mAb, lysed in a buffer containing 1% Triton X-100, and the ζ chain immunoprecipitated. Precipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting. Detection with the anti-phosphotyrosine mAb revealed a phosphotyrosyl-containing protein migrating at approximat" @default.
• W2007181639 created "2016-06-24" @default.
• W2007181639 creator A5010517097 @default.
• W2007181639 creator A5011636458 @default.
• W2007181639 creator A5019205026 @default.
• W2007181639 creator A5048801471 @default.
• W2007181639 creator A5060264036 @default.
• W2007181639 creator A5073271296 @default.
• W2007181639 date "1995-07-01" @default.
• W2007181639 modified "2023-09-26" @default.
• W2007181639 title "Interaction between lck and syk Family Tyrosine Kinases in Fcγ Receptor-initiated Activation of Natural Killer Cells" @default.
• W2007181639 cites W136542906 @default.
• W2007181639 cites W1481629090 @default.
• W2007181639 cites W1525901333 @default.
• W2007181639 cites W1551296854 @default.
• W2007181639 cites W1551636999 @default.
• W2007181639 cites W1556867369 @default.
• W2007181639 cites W1562110281 @default.
• W2007181639 cites W1963569988 @default.
• W2007181639 cites W1971302204 @default.
• W2007181639 cites W1983960385 @default.
• W2007181639 cites W1990173454 @default.
• W2007181639 cites W2002779187 @default.
• W2007181639 cites W2004458954 @default.
• W2007181639 cites W2007794993 @default.
• W2007181639 cites W2009597900 @default.
• W2007181639 cites W2011067475 @default.
• W2007181639 cites W2012694659 @default.
• W2007181639 cites W2014154395 @default.
• W2007181639 cites W2019300008 @default.
• W2007181639 cites W2021278166 @default.
• W2007181639 cites W2030655955 @default.
• W2007181639 cites W2035562653 @default.
• W2007181639 cites W2042582068 @default.
• W2007181639 cites W2044336492 @default.
• W2007181639 cites W2044907066 @default.
• W2007181639 cites W2049030343 @default.
• W2007181639 cites W2052561585 @default.
• W2007181639 cites W2055444005 @default.
• W2007181639 cites W2058885870 @default.
• W2007181639 cites W2061604045 @default.
• W2007181639 cites W2065978478 @default.
• W2007181639 cites W2068982799 @default.
• W2007181639 cites W2075955998 @default.
• W2007181639 cites W2077237119 @default.
• W2007181639 cites W2078991312 @default.
• W2007181639 cites W2080627154 @default.
• W2007181639 cites W2120576246 @default.
• W2007181639 cites W2123030732 @default.
• W2007181639 cites W2127141367 @default.
• W2007181639 cites W2151151593 @default.
• W2007181639 cites W2166504548 @default.
• W2007181639 cites W4249736906 @default.
• W2007181639 doi "https://doi.org/10.1074/jbc.270.27.16415" @default.
• W2007181639 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7541798" @default.
• W2007181639 hasPublicationYear "1995" @default.
• W2007181639 type Work @default.
• W2007181639 sameAs 2007181639 @default.
• W2007181639 citedByCount "89" @default.
• W2007181639 countsByYear W20071816392012 @default.
• W2007181639 countsByYear W20071816392013 @default.
• W2007181639 countsByYear W20071816392014 @default.
• W2007181639 countsByYear W20071816392016 @default.
• W2007181639 countsByYear W20071816392017 @default.
• W2007181639 countsByYear W20071816392018 @default.
• W2007181639 countsByYear W20071816392022 @default.
• W2007181639 crossrefType "journal-article" @default.
• W2007181639 hasAuthorship W2007181639A5010517097 @default.
• W2007181639 hasAuthorship W2007181639A5011636458 @default.
• W2007181639 hasAuthorship W2007181639A5019205026 @default.
• W2007181639 hasAuthorship W2007181639A5048801471 @default.
• W2007181639 hasAuthorship W2007181639A5060264036 @default.
• W2007181639 hasAuthorship W2007181639A5073271296 @default.
• W2007181639 hasBestOaLocation W20071816391 @default.
• W2007181639 hasConcept C101544691 @default.
• W2007181639 hasConcept C108636557 @default.
• W2007181639 hasConcept C120642132 @default.
• W2007181639 hasConcept C139563560 @default.
• W2007181639 hasConcept C154317977 @default.
• W2007181639 hasConcept C170493617 @default.
• W2007181639 hasConcept C184235292 @default.
• W2007181639 hasConcept C185592680 @default.
• W2007181639 hasConcept C202751555 @default.
• W2007181639 hasConcept C2779931124 @default.
• W2007181639 hasConcept C2909867262 @default.
• W2007181639 hasConcept C2911166042 @default.
• W2007181639 hasConcept C35174551 @default.
• W2007181639 hasConcept C42362537 @default.
• W2007181639 hasConcept C502942594 @default.
• W2007181639 hasConcept C55493867 @default.
• W2007181639 hasConcept C86803240 @default.
• W2007181639 hasConcept C95444343 @default.
• W2007181639 hasConceptScore W2007181639C101544691 @default.
• W2007181639 hasConceptScore W2007181639C108636557 @default.
• W2007181639 hasConceptScore W2007181639C120642132 @default.
• W2007181639 hasConceptScore W2007181639C139563560 @default.
• W2007181639 hasConceptScore W2007181639C154317977 @default.
• W2007181639 hasConceptScore W2007181639C170493617 @default.

• next