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• W1968664666 abstract "As an attempt to analyze the roles of C-terminal Src kinase (Csk) in the high affinity IgE receptor (FcεRI)-mediated signaling, we overexpressed Csk, a membrane-targeted form of Csk (mCsk), and a kinase-defective, membrane-targeted form of Csk (mCsk(−)) in rat basophil leukemia (RBL) 2H3 cells. Specific activity of Lyn at the basal state was decreased in Csk-expressing cells, and further decreased in mCsk-expressing cells. In mCsk(−)-expressing cells, basal specific activity of Lyn was increased, thereby indicating that mCsk(−) functioned as a dominant negative molecule. The onset of FcεRI-mediated Lyn activation was delayed in Csk-expressing cells, and further delayed in mCsk-expressing cells. In mCsk(−)-expressing cells, Lyn activation was rapid and quite long lasting. These findings indicate (i) Csk negatively regulates rapid FcεRI/Lyn coupling, and (ii) Csk activity is potentially required for its termination. The onsets of the series of events including tyrosyl phosphorylation of Syk, mitogen-activated protein (MAP) kinase activation, elevation of intracellular calcium concentration ([Ca2+] i), and histamine release were all stepwisely delayed in Csk-expressing cells and in mCsk-expressing cells. The durations of Syk phosphorylation and MAP kinase activation also closely correlated with those of Lyn activation, but [Ca2+] i elevation and histamine release followed different temporal patterns: the delayed responses in Csk-expressing cells and in mCsk-expressing cells led to sustained [Ca2+] i oscillation and histamine release, while the prompt responses in parent cells and mCsk(−)-expressing cells rapidly subsided. These findings provide further evidence that the initiations of the FcεRI-mediated signals are upstreamly regulated by Src family protein tyrosine kinases and revealed that their terminations are regulated by Lyn-dependent (Syk and MAP kinase) and -independent ([Ca2+] i elevation and histamine release) mechanisms. As an attempt to analyze the roles of C-terminal Src kinase (Csk) in the high affinity IgE receptor (FcεRI)-mediated signaling, we overexpressed Csk, a membrane-targeted form of Csk (mCsk), and a kinase-defective, membrane-targeted form of Csk (mCsk(−)) in rat basophil leukemia (RBL) 2H3 cells. Specific activity of Lyn at the basal state was decreased in Csk-expressing cells, and further decreased in mCsk-expressing cells. In mCsk(−)-expressing cells, basal specific activity of Lyn was increased, thereby indicating that mCsk(−) functioned as a dominant negative molecule. The onset of FcεRI-mediated Lyn activation was delayed in Csk-expressing cells, and further delayed in mCsk-expressing cells. In mCsk(−)-expressing cells, Lyn activation was rapid and quite long lasting. These findings indicate (i) Csk negatively regulates rapid FcεRI/Lyn coupling, and (ii) Csk activity is potentially required for its termination. The onsets of the series of events including tyrosyl phosphorylation of Syk, mitogen-activated protein (MAP) kinase activation, elevation of intracellular calcium concentration ([Ca2+] i), and histamine release were all stepwisely delayed in Csk-expressing cells and in mCsk-expressing cells. The durations of Syk phosphorylation and MAP kinase activation also closely correlated with those of Lyn activation, but [Ca2+] i elevation and histamine release followed different temporal patterns: the delayed responses in Csk-expressing cells and in mCsk-expressing cells led to sustained [Ca2+] i oscillation and histamine release, while the prompt responses in parent cells and mCsk(−)-expressing cells rapidly subsided. These findings provide further evidence that the initiations of the FcεRI-mediated signals are upstreamly regulated by Src family protein tyrosine kinases and revealed that their terminations are regulated by Lyn-dependent (Syk and MAP kinase) and -independent ([Ca2+] i elevation and histamine release) mechanisms. The high affinity IgE receptor (FcεRI) 1The abbreviations used are: FcεRI, the high affinity IgE receptor; ITAM, immunoreceptor tyrosine-based activation motif; PTK, protein tyrosine kinase; Csk, C-terminal Src kinase; mCsk, membrane-targeted Csk; mCsk(−), membrane-targeted, kinase-defective Csk; RBL, rat basophil leukemia; MAP kinase, mitogen-activated protein kinase; TCR and BCR, T and B cell receptor, respectively; SH, Src homology; anti-DNP IgE, anti-dinitrophenyl IgE; DNP-BSA, 2,4-dinitrophenylated bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; WT, wild-type. belongs to a family of multi-subunit receptors for antigen, which include T cell receptor (TCR), B cell receptor (BCR), Fcγ, and Fcα receptors (1Metzger H. Immunol. Rev. 1992; 125: 37-48Crossref PubMed Scopus (225) Google Scholar, 2Ravetch J.V. Kinet J.P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1286) Google Scholar, 3Beaven M.A. Metzger H. Immunol. Today. 1993; 14: 222-226Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 4Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar). FcεRI is composed of one α subunit possessing IgE binding site and one β and two γ subunits functioning as signal transducers (3Beaven M.A. Metzger H. Immunol. Today. 1993; 14: 222-226Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 5Eiseman E. Bolen J.B. J. Biol. Chem. 1992; 267: 21027-21032Abstract Full Text PDF PubMed Google Scholar, 6Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar, 7Letourneur F. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8905-8909Crossref PubMed Scopus (245) Google Scholar, 8Blank U. Ra C. Miller L. White K. Metzger H. Kinet J.P. Nature. 1989; 337: 187-189Crossref PubMed Scopus (397) Google Scholar). The cytoplasmic tails of the β and the γ subunits possess characteristic tyrosine-based amino acid sequence motifs (immunoreceptor tyrosine-based activation motif (ITAM)) (4Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar, 9Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1168) Google Scholar), which are also found in the signal transducing subunits of TCR and BCR (4Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar). Tyrosine residues in the ITAM sequences are rapidly phosphorylated after the triggering of the antigen receptors (10Benhamou M. Siraganian R.P. Immunol. Today. 1992; 13: 195-197Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 11Jouvin M.H. Numerof R.P. Kinet J.P. Semin. Immunol. 1995; 7: 29-35Crossref PubMed Scopus (46) Google Scholar, 12Paolini R. Jouvin M.H. Kinet J.P. Nature. 1991; 353: 855-858Crossref PubMed Scopus (222) Google Scholar, 13Weiss A. Cell. 1993; 73: 209-212Abstract Full Text PDF PubMed Scopus (476) Google Scholar), and the phosphorylated ITAM sequences serve as sites for recruiting signaling molecules mainly through their interaction with Src homology (SH) 2 domains (5Eiseman E. Bolen J.B. J. Biol. Chem. 1992; 267: 21027-21032Abstract Full Text PDF PubMed Google Scholar, 6Jouvin M.H. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar, 14Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar, 15Scharenberg A.M. Lin S. Cuenod B. Yamamura H. Kinet J.P. EMBO J. 1995; 14: 3385-3394Crossref PubMed Scopus (143) Google Scholar, 16Shiue L. Green J. Green O.M. Karas J.L. Morgenstern J.P. Ram M.K. Taylor M.K. Zoller M.J. Zydowsky L.D. Bolen J.B. Brugge J.S. Mol. Cell. Biol. 1995; 15: 272-281Crossref PubMed Google Scholar). Several lines of evidence have implicated Src family PTKs in the primary phosphorylation of the ITAM tyrosine (4Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar, 13Weiss A. Cell. 1993; 73: 209-212Abstract Full Text PDF PubMed Scopus (476) Google Scholar,17Kurosaki T. Takata M. Yamanashi Y. Inazu T. Taniguchi T. Yamamoto T. Yamamura H. J. Exp. Med. 1994; 179: 1725-1729Crossref PubMed Scopus (254) Google Scholar, 18Turner J.M. Brodsky M.H. Irving B.A. Levin S.D. Perlmutter R.M. Littman D.R. Cell. 1990; 60: 755-765Abstract Full Text PDF PubMed Scopus (492) Google Scholar, 19Yamanashi Y. Kakiuchi T. Mizuguchi J. Yamamoto T. Toyoshima K. Science. 1991; 251: 192-194Crossref PubMed Scopus (338) Google Scholar). In the case of FcεRI, Lyn, Src, and Yes (members of Src family PTKs) are rapidly activated after the receptor triggering (20Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (417) Google Scholar), Lyn possibly associates with the the β subunit of FcεRI (14Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar, 20Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (417) Google Scholar,21Alber G. Miller L. Jelsema C.L. Varin-Blank N. Metzger H. J. Biol. Chem. 1991; 266: 22613-22620Abstract Full Text PDF PubMed Google Scholar), and deletion of the cytoplasmic region of the β subunit abrogates FcεRI signalings in murine mast cells (21Alber G. Miller L. Jelsema C.L. Varin-Blank N. Metzger H. J. Biol. Chem. 1991; 266: 22613-22620Abstract Full Text PDF PubMed Google Scholar). Most notably, recent heterologous expression studies have demonstrated that Lyn expression is required for the efficient phosphorylation of the β and the γ subunits as well as phosphorylation and activation of Syk (15Scharenberg A.M. Lin S. Cuenod B. Yamamura H. Kinet J.P. EMBO J. 1995; 14: 3385-3394Crossref PubMed Scopus (143) Google Scholar,22Lin S. Cicala C. Scharenberg A.M. Kinet J.P. Cell. 1996; 85: 985-995Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar), another cytoplasmic PTK. The kinase activity of Src family PTKs has been shown to be regulated by the phosphorylation level of the C-terminal tyrosine conserved among the family members (23Cooper J.A. Howell B. Cell. 1993; 73: 1051-1054Abstract Full Text PDF PubMed Scopus (495) Google Scholar, 24Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2187) Google Scholar). It is postulated that the phosphorylated tyrosine associates with the intramolecular SH2 domain (23Cooper J.A. Howell B. Cell. 1993; 73: 1051-1054Abstract Full Text PDF PubMed Scopus (495) Google Scholar) and that the “closed” conformation (25Kaplan K.B. Swedlow J.R. Morgan D.O. Varmus H.E. Genes Dev. 1995; 9: 1505-1517Crossref PubMed Scopus (295) Google Scholar) prevents the catalytic domain from interacting with external kinase substrates. Disruption of the intramolecular interaction, presumably through the dephosphorylation of the C-terminal tyrosine or through the replacement of the phosphorylated C-terminal tyrosine with high affinity protein ligands (23Cooper J.A. Howell B. Cell. 1993; 73: 1051-1054Abstract Full Text PDF PubMed Scopus (495) Google Scholar), results in augmented kinase activity (“open” conformation). In hematopoietic cells, C-terminal Src kinase (Csk) and CD45 tyrosine phosphatase are implicated in the phosphorylation and the dephosphorylation of the regulatory tyrosine, respectively (4Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar, 23Cooper J.A. Howell B. Cell. 1993; 73: 1051-1054Abstract Full Text PDF PubMed Scopus (495) Google Scholar). Csk, first purified as a PTK that specifically phosphorylates the regulatory tyrosine (26Okada M. Nada S. Yamanashi Y. Yamamoto T. Nakagawa H. J. Biol. Chem. 1991; 266: 24249-24252Abstract Full Text PDF PubMed Google Scholar), has structural similarities with Src family PTKs but is devoid of the C-terminal regulatory tyrosine and the N-terminal myristylation signal required for membrane localization (26Okada M. Nada S. Yamanashi Y. Yamamoto T. Nakagawa H. J. Biol. Chem. 1991; 266: 24249-24252Abstract Full Text PDF PubMed Google Scholar, 27Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (511) Google Scholar, 28Jove R. Hanafusa H. Annu. Rev. Cell Biol. 1987; 3: 31-56Crossref PubMed Scopus (299) Google Scholar). Creation of Csk-deficient mice, which could not survive embryonic stages, has revealed that Src family PTKs are commonly activated in Csknull cells, thereby indicating that Csk is one of major negative regulators of the basal activity of Src family PTKs (29Nada S. Yagi T. Takeda H. Tokunaga T. Nakagawa H. Ikawa Y. Okada M. Aizawa S. Cell. 1993; 73: 1125-1135Abstract Full Text PDF PubMed Scopus (364) Google Scholar, 30Imamoto A. Soriano P. Cell. 1993; 73: 1117-1124Abstract Full Text PDF PubMed Scopus (347) Google Scholar). Negative impacts of Csk on receptor-mediated dynamic signaling have also been noted in T lymphocytes: overexpression of Csk, and mutant forms of Csk possessing membrane-attachment signals more effectively, suppress TCR-mediated protein tyrosine phosphorylation and lymphokine production (31Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (237) Google Scholar, 32Cloutier J.F. Chow L.M. Veillette A. Mol. Cell. Biol. 1995; 15: 5937-5944Crossref PubMed Scopus (57) Google Scholar). As an attempt to explore the roles of Csk in FcεRI signaling, and to further assess the functional significance of Src family PTKs in the context of mast cells, we variously modulated Csk activity in rat basophil leukemia (RBL) 2H3 cell line, a widely used model system for analyzing FcεRI functions (14Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar, 15Scharenberg A.M. Lin S. Cuenod B. Yamamura H. Kinet J.P. EMBO J. 1995; 14: 3385-3394Crossref PubMed Scopus (143) Google Scholar, 20Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (417) Google Scholar, 42Zhang J. Berenstein E.H. Evans R.L. Siraganian R.P. J. Exp. Med. 1996; 184: 71-79Crossref PubMed Scopus (240) Google Scholar). We generated RBL2H3 sublines overexpressing Csk, a membrane-targeted form of Csk (mCsk) (31Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (237) Google Scholar, 32Cloutier J.F. Chow L.M. Veillette A. Mol. Cell. Biol. 1995; 15: 5937-5944Crossref PubMed Scopus (57) Google Scholar), and a kinase-defective form of mCsk (mCsk(−)) that functioned as a dominant negative molecule. By using these cell lines, we show herein that (i) Csk is involved in the initiation and the termination of FcεRI-mediated Lyn activation, (ii) the initiations of the series of events leading to granule release followed Lyn activation, and (iii) the termination of the signals are regulated by both Lyn-dependent and -independent mechanisms. All the culture media and Geneticin® were purchased from Life Technologies Oriental (Osaka, Japan). Fetal calf serum was from Equitech-Bio (Ingram, TX). Protein G-Sepharose beads were from Pharmacia-LKB (Uppsala, Sweden). Enolase and mouse monoclonal anti-dinitrophenyl IgE (anti-DNP IgE) were from Sigma. 2,4-dinitrophenylated bovine serum albumin (DNP-BSA, 30 ± 5 mol of DNP/1 mol of BSA) was from LSL (Tokyo, Japan). [γ-32P]ATP was from Amersham Corp. (Buckinghamshire, United Kingdom). All other chemical reagents were of analytical grade. Polyclonal antibody against amino acids 44–63 of human p56lyn and that against amino acids 257–352 of human Syk were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phosphotyrosine monoclonal antibody, 4G10 was from ICN Immunobiologicals (Lisle, IL). Anti-Erk 1/Erk 2 polyclonal antibody (33Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar, 34Tobe K. Kadowaki T. Tamemoto H. Ueki K. Hara K. Koshio O. Momomura K. Gotoh Y. Nishida E. Akanuma Y. Yazaki Y. Kasuga M. J. Biol. Chem. 1991; 266: 24793-24803Abstract Full Text PDF PubMed Google Scholar) and anti-Csk polyclonal antibody (27Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (511) Google Scholar, 29Nada S. Yagi T. Takeda H. Tokunaga T. Nakagawa H. Ikawa Y. Okada M. Aizawa S. Cell. 1993; 73: 1125-1135Abstract Full Text PDF PubMed Scopus (364) Google Scholar) were prepared as described. cDNAs of rat Csk and a kinase-defective Csk (Csk(−)), in which Lys-222 is replaced with Arg, were prepared as described (27Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (511) Google Scholar). Csk- and Csk(−) chimeras possessing N-terminal 16 amino acids of rat c-Src (c-Src tag: (M)GSNKSKPKDASQRRR), harboring a myristylation signal (28Jove R. Hanafusa H. Annu. Rev. Cell Biol. 1987; 3: 31-56Crossref PubMed Scopus (299) Google Scholar, 31Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (237) Google Scholar) were prepared by polymerase chain reaction-based techniques. Two N-terminal overhanging primers (primers 1 and 2) and a C-terminal primer (primer 3) were designed. These were: primer 1, 5′-AGC-AAG-CCC-AAG-GAC-GCC-AGC-CAG-CGG-CGC-CGC-TCG-GCT-ATA-CAG-GCC-TCC-TGG-3′, coding for the C-terminal 11 amino acids of the c-Src tag and the N-terminal 7 amino acids of rat Csk without first ATG; primer 2, 5′-CT-CGG-TAC-CCG-TCG-ACC-ACC-ATG-GGC-AGC-AAC-AAG-AGC-AAG-CCC-AAG-GAC-GCC-AGC-3′, possessing 5′ KpnI and XhoI sites and coding for N-terminal 12 amino acids of the c-Src Tag; and primer 3, 5′-CTG-AAG-CTG-CTA-CAG-ACA-ATA-GG-3′, corresponding to the antisense sequence (nucleotides 623–601) of the coding region of rat Csk. Csk cDNA was first amplified using primers 1 and 3, and the fragment was further amplified using primers 2 and 3. The resultant fragment was digested with KpnI (in the primer 2) and with ApaI (nucleotide 586 in the coding region of Csk). The digested fragment was ligated at the ApaI site with the C-terminal segment of Csk, or Csk(−), thus forming a c-Src-Csk chimera (mCsk) and mCsk(−). Clones without misincorporation of nucleotides were chosen, and the entire coding regions of the cDNAs excised with XhoI were inserted into a mammalian expression vector pCXN2 (35Miyazaki J. Takaki S. Araki K. Tashiro F. Tominaga A. Takatsu K. Yamamura K. Gene (Amst.). 1989; 79: 269-277Crossref PubMed Scopus (370) Google Scholar) at SalI site. RBL2H3 cells, routinely maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 100 μg/ml streptomycin, and 100 units/ml penicillin, were freshly subcloned by limiting dilution and used for transfection. RBL2H3 cells (106 cells in a 6-cm dish) were transfected by lipofection with 30 μl of Transfectum® (Promega) and 10 μg of the expression vectors. Geneticin-resistant clones were selected in the medium containing 1.2 μg/ml Geneticin, and clones overexpressing Csk and Csk mutants were screened by immunoblotting with anti-Csk polyclonal antibody. Multiple independent clones overexpressing Csk (Csk 1 to 9), mCsk (mCsk 1 to 7), and mCsk(−) (mCsk(−) 1 to 4) were obtained. Of these, we mainly used Csk 5 and 9, mCsk 1 and 3, and mCsk(−) 1 and 3, with similar expression levels of these molecules. The data described below were reproducible in the two sets of clones for each cDNA. 106cells in 6-cm dishes were cultured for 36 h and then starved for serum for another 24 h. Cells were sensitized with anti-DNP IgE (a final concentration of 1 μg/ml) for 1 h in serum-free DMEM containing 0.1% fatty acid-free BSA, washed with phosphate-buffered saline, and incubated in serum-free DMEM at 37 °C under humidified 5% CO2. Cells were stimulated with the indicated concentration of DNP-BSA for varying periods. Medium was aspirated, and cells were quickly frozen by floating the dishes on liquid nitrogen. Cells were lysed on ice with 400 μl/dish lysis buffer (buffer A: 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 20 mm β-glycerophosphate, 1 mm sodium orthovanadate, 1.0% Nonidet P-40, 0.1% deoxycholate, and 0.2 mm phenylmethylsulfonyl fluoride) and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was used as cell lysate. Cell lysate was incubated with first antibodies, and the immunecomplex was collected with protein G-Sepharose beads. Beads were washed twice with buffer A. The immunoprecipitated proteins were subjected to immunoblotting or to kinase reaction. In the immunecomplex kinase assay of Lyn, beads were washed once with a kinase buffer (25 mmTris-HCl, pH 8.0, 2 mm MnCl2, and 10 mm MgCl2) and resuspended in the same buffer. Kinase reaction was conducted in 25 μl of the kinase buffer containing 10 μm ATP (1 μCi of [γ-32P]ATP in 25 μl) and 1 μg of acid-treated enolase for 2 min at 25 °C. Under the reaction conditions,32P incorporation into enolase was linear with time for 5 min. Cell lysates or the immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Proteins were probed with first antibodies and then reacted with horseradish peroxidase- or alkaline phosphatase-conjugated second antibodies. The signals were detected by enhanced chemiluminescence (ECL Western blotting system, Amersham Corp.) or by the color development with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, as described (33Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar). Cells were cultured on glass coverslips for 24 h, starved for serum for another 24 h, and simultaneously sensitized with anti-DNP IgE (a final concentration of 1 μg/ml) and loaded with 5 μm Fura-2/AM in serum-free DMEM containing 0.1% fatty acid-free BSA at 37 °C for 1 h. Then, cells were washed twice with HEPES-Tyrode buffer (25 mm HEPES-NaOH, pH 7.4, 140 mm NaCl, 2.7 mm KCl, 1.8 mm CaCl2, 12 mm NaHCO3, 5.6 mmd-glucose, 0.49 mm MgCl2, 0.37 mm NaH2PO4) containing 0.1% fatty acid-free BSA and incubated in the same buffer. Cells were stimulated with DNP-BSA (a final concentration of 100 ng/ml) at ambient temperature (20–25 °C). Fluorometric cell images were recorded with ICCD camera/image analysis system (ARGUS-50, Hamamatsu Photonics, Hamamatsu, Japan), ratio images (340/380 nm) were sequentially collected, and the ratio images were represented as a color gradient according to a Ca2+ calibration curve (36Mori M. Aihara M. Kume K. Hamanoue M. Kohsaka S. Shimizu T. J. Neurosci. 1996; 16: 3590-3600Crossref PubMed Google Scholar). 106 cells in 6-cm dishes were cultured for 36 h, starved for serum for 24 h, and sensitized with anti-DNP IgE (a final concentration of 1 μg/ml) for 1 h in serum-free DMEM containing 0.1% fatty acid-free BSA. Cells were washed with phosphate-buffered saline twice, incubated in HEPES-Tyrode buffer containing 0.1% fatty acid-free BSA, and stimulated with various concentrations of DNP-BSA. Aliquots of the supernatant were sequentially collected, and histamine contents were measured with an automated histamine analyzer as described (37Morita Y. Asakawa M. Hirai K. Takaishi T. Miyamoto T. Int. Arch. Allergy Appl. Immunol. 1989; 88: 332-336Crossref PubMed Scopus (4) Google Scholar). Previous studies have shown that TCR signaling is inhibited by Csk and almost completely blocked by Csk chimeras possessing myristylation or isoprenylation signals (31Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (237) Google Scholar, 32Cloutier J.F. Chow L.M. Veillette A. Mol. Cell. Biol. 1995; 15: 5937-5944Crossref PubMed Scopus (57) Google Scholar). The augmented functions caused by the membrane targeting are presumed to be due to close positioning of these Csk mutants to Src family PTKs (31Chow L.M. Fournel M. Davidson D. Veillette A. Nature. 1993; 365: 156-160Crossref PubMed Scopus (237) Google Scholar,32Cloutier J.F. Chow L.M. Veillette A. Mol. Cell. Biol. 1995; 15: 5937-5944Crossref PubMed Scopus (57) Google Scholar). We, therefore, prepared a membrane-targeted Csk in which the N-terminal myristylation signal of rat c-Src (28Jove R. Hanafusa H. Annu. Rev. Cell Biol. 1987; 3: 31-56Crossref PubMed Scopus (299) Google Scholar) was fused to the entire coding sequence of rat Csk (mCsk). A kinase defective mCsk (mCsk(−)) was also created. Wild-type Csk, mCsk, and mCsk(−) cDNAs were stably expressed in a freshly subcloned line of RBL2H3. An immunoblot with anti-Csk antibody of two independent clones expressing these Csk-based molecules is shown in Fig.1, lane Csk:blot. Because of the additional 14 amino acids, mCsk and mCsk(−) exhibited a slightly retarded electrophoretic mobility compared with wild-type Csk. To explore the effects of the overexpression of these Csk-based molecules on the basal activity of Lyn, protein levels of Lyn were first compared. Cells at quiescence were solubilized with the buffer containing 1% Nonidet P-40 and 0.1% deoxycholate, and the lysates were subjected to immunoblotting with anti-Lyn antibody. As shown in Fig. 1 (lane Lyn:blot), the amount of Lyn was reproducibly decreased in mCsk(−) cells as compared with that in parent cells. Lyn was immunoprecipitated from each volume of the cell lysate containing an equal amount of Lyn, and the relative specific activity of Lyn toward enolase was compared. As shown in Fig. 1 (lane Lyn:spec), the specific activity of Lyn was decreased in Csk cells and further decreased in mCsk cells while it was increased in mCsk(−) cells. Decreased abundance of Src family PTKs as seen in mCsk(−) cells has also been noted in Csknull cells (29Nada S. Yagi T. Takeda H. Tokunaga T. Nakagawa H. Ikawa Y. Okada M. Aizawa S. Cell. 1993; 73: 1125-1135Abstract Full Text PDF PubMed Scopus (364) Google Scholar, 38Gross J.A. Appleby M.W. Chien S. Nada S. Bartelmez S.H. Okada M. Aizawa S. Perlmutter R.M. J. Exp. Med. 1995; 181: 463-473Crossref PubMed Scopus (34) Google Scholar), where the C-terminal regulatory tyrosine in Src PTKs are underphosphorylated. Although precise mechanisms of the altered Lyn levels in mCsk(−) cells remain to be elucidated, it could be ascribed to the shortened half-lives of Src family PTKs whose C-terminal tyrosine is dephosphorylated (39Hata A. Sabe H. Kurosaki T. Takata M. Hanafusa H. Mol. Cell. Biol. 1994; 14: 7306-7313Crossref PubMed Scopus (79) Google Scholar, 40Iba H. Cross F.R. Garber E.A. Hanafusa H. Mol. Cell. Biol. 1985; 5: 1058-1066Crossref PubMed Scopus (76) Google Scholar). The decreased protein level, along with the increased specific activity of Lyn in mCsk(−) cells, strongly indicated that mCsk(−) interfered with intrinsic Csk activity as a dominant negative molecule. As an initial experiment, these cell lines were sensitized with a saturating concentration of anti-DNP IgE (1 μg/ml) (41Wilson B.S. Kapp N. Lee R.J. Pfeiffer J.R. Martinez A.M. Platt Y. Letourneur F. Oliver J.M. J. Biol. Chem. 1995; 270: 4013-4022Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), stimulated with various concentrations of DNP-BSA (1–1,000 ng/ml) for 30 min, and released histamine was measured. Contrary to the expectation that Csk and mCsk might inhibit histamine release, all the clones responded well to these concentrations of antigen after the long incubation periods, with an optimal concentration of 100 ng/ml (not shown, and see Fig. 6 B). These findings suggest that Csk or mCsk does not substantially suppress the overall cell activation. To further assess the influence of these molecules, we followed the temporal patterns of FcεRI-mediated signaling under the optimal stimulation conditions (1 μg/ml anti-DNP IgE, and 100 ng/ml DNP-BSA). FcεRI-mediated changes in the tyrosine phosphorylation of cellular proteins are shown in Fig. 2 A. Under the quiescent conditions, two sets of doublets with molecular masses of around 55 kDa (identified as two isoforms of Lyn (not shown)) and 180 kDa (not identified in this study) were already tyrosyl phosphorylated in all the clones, and tyrosyl phosphorylation of other proteins was minimal. After the triggering of FcεRI, multiple proteins were tyrosyl phosphorylated in all the clones, but their temporal patterns were different. Among the tyrosyl-phosphorylated proteins, those around 70 kDa (indicated by arrows) gave strongest signals in all the clones. In the wild-type cells, the signal became rapidly detectable within 30 s, peaked at around 5 min, and then declined. The time span of the signal was delayed in Csk-expressing cells (peaked at around 10–20 min) and was further delayed in mCSK-expressing cells (peaked at around 30–60 min) (Fig.2 A). In mCsk(−)-expressing cells, it was as rapidly detectable as in the wild-type cells, and, intriguingly, its duration was markedly prolonged (Fig. 2 A). The time-dependent changes in the intensity of the 70-kDa phosphoproteins were densitometrically determined in the two sets of clones (WT, Csk 5 and 9, mCsk 1 and 3, and mCsk(−) 1 and 3), and the average was depicted in the graph (Fig. 2 B). The stepwisely delayed responses in Csk- and mCsk-expressing cells and the rapid response in mCsk(−)-expressing cells clearly indicated that Csk negatively regulates the initiation of FcεRI-mediated protein tyrosine phosphorylation in a manner dependent on its catalytic activity. In addition, the prolonged response in mCsk(−)-expressing cells suggested that the dominant negative Csk delayed the termination of the signal. To examine if the Csk-based molecules altered cell signaling by directly affecting Lyn activation, time-dependent changes in the Lyn activity were followed. Lyn was immunoprecipitated from the cell lysate at each time after FcεRI stimulation, and kinase activity in the immunoprecipitate was assayed using an external substrate, enolase. Protein levels of Lyn recovered in the c" @default.
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• W1968664666 title "Roles of C-terminal Src Kinase in the Initiation and the Termination of the High Affinity IgE Receptor-mediated Signaling" @default.
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