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The Unique Domain as the Site on Lyn Kinase for Its Constitutive Association with the High Affinity Receptor for IgE

1997; Elsevier BV; Volume: 272; Issue: 38 Linguagem: Inglês

10.1074/jbc.272.38.24072

1083-351X

Becky M. Vonakis, Huaxian Chen, Hana Haleem‐Smith, Henry Metzger,

Mast cells and histamine

Aggregation of the high affinity receptor for IgE (FcεRI) leads to the phosphorylation of tyrosines on the β and γ chains of the receptor by the Src family kinase Lyn. We have studied the interaction between Lyn and the FcεRI in vivo using a transfection-based approach. FcεRI were stably transfected into Chinese hamster ovary cells. The small amount of endogenous Src family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of FcεRI but not after addition of dimers of IgE. Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced. In contrast, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase, and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn. Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the β chain but not with the receptor's other cytoplasmic domains. Aggregation of the high affinity receptor for IgE (FcεRI) leads to the phosphorylation of tyrosines on the β and γ chains of the receptor by the Src family kinase Lyn. We have studied the interaction between Lyn and the FcεRI in vivo using a transfection-based approach. FcεRI were stably transfected into Chinese hamster ovary cells. The small amount of endogenous Src family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of FcεRI but not after addition of dimers of IgE. Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced. In contrast, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase, and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn. Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the β chain but not with the receptor's other cytoplasmic domains. The family of proteins known as the "multichain immune recognition receptors" includes the antigen receptors on B and T-lymphocytes and Fc receptors including the receptor with high affinity for IgE (FcεRI) 1The abbreviations used are: FcεRI, high affinity receptor for IgE; bp, base pair(s); BSA, bovine serum albumin; CHO, Chinese hamster ovary; DNP, dinitrophenyl; ITAM, immuno-recognition tyrosine-based activation motif; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Pipes, 1,4-piperazinediethanesulfonic acid; Tricine,N-tris(hydroxymethyl)methylglycine. (1Keegan A.D. Paul W.E. Immunol. Today. 1992; 13: 63-68Abstract Full Text PDF PubMed Scopus (173) Google Scholar). Highly homologous in structure, all these receptors utilize, at least in part, a common mechanism to initiate cellular responses; multivalent interactions with antigen lead to aggregation of the receptors and is followed by enhanced phosphorylation of tyrosines (in the "ITAM" motifs within the cytoplasmic domains) of the receptor itself by a receptor-associated Src family kinase (2Cambier J.C. J. Immunol. 1995; 155: 3281-3285PubMed Google Scholar). For FcεRI, we recently presented direct evidence for a "transphosphorylation" mechanism that accounts for the earliest events (3Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (175) Google Scholar, 4Yamashita T. Mao S.-Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar). The data showed that a small fraction of receptors are constitutively associated with the Src family kinase Lyn (4Yamashita T. Mao S.-Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar, 5Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (417) Google Scholar) and that the enhanced phosphorylation that follows aggregation of the receptors is likely to result simply from the apposition of the kinase with its substrate. We have also shown that when the kinase available to the receptor is limited, shuttling of the enzyme between individual aggregates can regulate the intensity of the signal (6Torigoe C. Goldstein B. Wofsy C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1372-1377Crossref PubMed Scopus (46) Google Scholar). 2C. Wofsy, C. Torigoe, U. M. Kent, H. Metzger, and B. Goldstein, submitted for publication. The experiments described in this paper mainly explored the sites of interaction between Lyn kinase and FcεRI. For the most part, the prior studies of others explored the interaction between Lyn and isolated portions of the receptor (7Jouvin M.-H.E. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar, 8Lin S. Cicala C. Scharenberg A.M. Kinet J.P. Cell. 1996; 85: 985-995Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 9Wilson 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, 10Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar). The yeast two-hybrid system (11Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-292Abstract Full Text PDF PubMed Scopus (526) Google Scholar) used in some of our studies is an analogous approach. We also employed transfection techniques, which allowed us to examine the kinase-receptor interactions in a more physiological setting. The latter experiments also allowed us to test the effect of varying the level of Lyn on the responsiveness of the receptors to discrete stimuli, and thereby to test certain quantitative predictions made by the current model. The yeast strains (CG1945 and Y187) and cloning vectors (pAS2-1 and pACT) were obtained fromCLONTECH (Palo Alto, CA); the expression vectors pBlueBac, pCDM8, and pZeo, as well as a baculovirus MAXBAC expression kit from Invitrogen (Carlsbad, CA); polyacrylamide gels used for electrophoresis (PAGE) from NOVEX (San Diego, CA); the antibiotics (G418, zeocin) from Life Technologies, Inc. and Invitrogen, respectively; and plasmid DNA purification kits from Qiagen (Santa Clarita, CA). Monoclonal anti-phosphotyrosine (anti-Tyr(P)) antibodies were obtained conjugated to horseradish peroxidase from Transduction Laboratories (PY-20) or Upstate Biotechnology, Inc. (4G10). Polyclonal antibodies to human Src family kinases Lyn and Fyn were purchased from Upstate Biotechnology, Inc.; antibodies to c-Src and c-Yes were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-DNP IgE (12Liu F.T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. Klinman N.R. Katz D.H. J. Immunol. 1980; 124: 2728-2737PubMed Google Scholar) and rat IgE (of unknown specificity) (13Bazin H. Querinjean P. Beckers A. Heremans J.F. Dessy F. Immunology. 1974; 26: 713-723PubMed Google Scholar) were purified as described previously (14Holowka D. Metzger H. Mol. Immunol. 1982; 19: 219-227Crossref PubMed Scopus (79) Google Scholar, 15Kulczycki Jr., A. Metzger H. J. Exp. Med. 1974; 140: 1676-1695Crossref PubMed Scopus (223) Google Scholar) and labeled with carrier-free 125I using chloramine T (16McConahey P.J. Dixon F.J. Int. Arch. Allergy Appl. Immunol. 1966; 29: 185-189Crossref PubMed Scopus (1397) Google Scholar). Goat anti-mouse IgE was purchased from ICN (Costa Mesa, CA); rabbit anti-rat IgE was purified as described (17Taurog J.D. Mendoza G.R. Hook W.A. Siraganian R.P. Metzger H. J. Immunol. 1977; 119: 1757-1761PubMed Google Scholar). Covalently cross-linked IgE oligomers were prepared and analyzed as described (6Torigoe C. Goldstein B. Wofsy C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1372-1377Crossref PubMed Scopus (46) Google Scholar). Rat basophilic leukemia (RBL-2H3) cells were maintained as described previously (18Barsumian E.L. Isersky C. Petrino M.G. Siraganian R.P. Eur. J. Immunol. 1981; 11: 317-323Crossref PubMed Scopus (485) Google Scholar). Chinese hamster ovary cells (CHO) were grown in stationary flasks at 37 °C in a humidified atmosphere containing 5% CO2 in Iscove's modified Eagle's minimum essential medium, 10% fetal calf serum, 25 mm HEPES, and the appropriate antibiotics to maintain expression of the transfected genes. Spodoptera frugiperda(Sf9) insect cells were maintained in spinner culture at 27 °C as described previously (191555Summers, M. D., and Smith, G. E. (1988) Tex. Agric. Expt. Stn. Bull. , 1555.Google Scholar). The nucleotide sequence of each expression construct was confirmed by automated DNA sequencing using a dye terminator kit obtained from Applied Biosystems (Foster City, CA). A 5′-stretch cDNA library was prepared from mRNA isolated from RBL cells. Two separate priming reactions with either oligo(dT) or random primers were performed to generate the first strand. The reactions were pooled prior to second strand synthesis. The cDNA library was then prepared in the expression vector pCDM8 (20Gubler U. Hoffman B.J. Gene ( Amst. ). 1983; 25: 263-269Crossref PubMed Scopus (3077) Google Scholar). Probes were prepared by restriction digestion of human Lyn A-pSVL. Probes representing the N terminus (amino acid residues 1–298) and the C terminus (residues 163–512) were purified. The library was plated, and colony lifts were hybridized with either probe. Positive colonies went through secondary and tertiary screening. The nucleotide sequences of two clones, designated N14 (2052 bp) and C18 (2316 bp), were determined by primer walking and DNA sequencing of both strands. The Wisconsin package from the Genetics Computer Group, Inc. was used to assemble and analyze the nucleotide sequences of the isolated clones. N14 contained an open reading frame of Lyn A, beginning with ATG from bp 80 to bp 1616, while clone C18 encoded Lyn B beginning with ATG between bp 236 and bp 1709. The sequence of Lyn A in the coding region was identical to a previously published sequence (21Minoguchi K. Kihara H. Nishikata H. Hamawy M.M. Siraganian R.P. Mol. Immunol. 1994; 31: 519-529Crossref PubMed Scopus (24) Google Scholar); the sequence of rat Lyn B lacks an "insert" of 21 amino acids found in the A form of the kinase at a position identical to that previously shown for human and murine Lyn (22Yamanashi Y. Fukushige S. Semba K. Sukegawa J. Miyajima N. Matsubara K. Yamamoto T. Toyoshima K. Mol. Cell Biol. 1987; 7: 237-243Crossref PubMed Scopus (164) Google Scholar, 23Yi T.L. Bolen J.B. Ihle J.N. Mol. Cell Biol. 1991; 11: 2391-2398Crossref PubMed Scopus (126) Google Scholar) but is otherwise identical to Lyn A. Therefore, it differs somewhat from the previously published sequence for rat Lyn B (24Rider L.G. Raben N. Miller L. Jelsema C. Gene ( Amst. ). 1994; 138: 219-222Crossref PubMed Scopus (7) Google Scholar). 3The open reading frame of our Lyn B cDNA is identical to the Lyn A except for the missing 21-residue "insert." The differences in the sequence originally reported (24Rider L.G. Raben N. Miller L. Jelsema C. Gene ( Amst. ). 1994; 138: 219-222Crossref PubMed Scopus (7) Google Scholar) probably resulted from sequencing errors or errors introduced during amplification with PCR. Two further clones, a partial Lyn A cDNA and a full-length Lyn B cDNA isolated from an RBL library, confirmed our sequence. CHO cells were transiently transfected with the Lyn-pCDM8 plasmids by electroporation, harvested 48–72 h later, and a lysate of the whole cells was prepared using SDS. After separation by PAGE and transfer, Western blotting with anti-human Lyn confirmed that the expressed proteins had the expected the size for Lyn A (56 kDa) and Lyn B (53 kDa) (data not shown). To generate DNA binding domain fusion proteins, the N-terminal (1–58) and C-terminal (201–243) cytoplasmic domains of the rat FcεRIβ were amplified by PCR from the full-length cDNA and cloned into theEcoRI/BamHI sites of pAS2-1. The cytoplasmic domain of rat FcεRIγ (residues 27–68), as a PCR fragment, was cloned into NcoI/BamHI site of pAS2-1, to generate pAS2-1-γC. To create activation domain fusion proteins, the full-length Lyn A and Lyn B and various deletion mutants were amplified by PCR and cloned into BamHI/XhoI sites of pACT (Fig. 1). Plasmid constructs were introduced into yeast cells by lithium acetate, following the protocol provided byCLONTECH. Transformants were plated on synthetic medium containing 5 mm 3-amino-1,2,4-trizole and lacking leucine, tryptophan, and histidine (SD-3) to detect the His phenotype, or synthetic medium lacking leucine and tryptophan (SD-2), to measure transformation efficiency. The β-galactosidase activity of transformants was measured in a filter assay with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside as substrate or in a liquid assay with ano-nitrophenyl-β-d-galactoside substrate according to the CLONTECH protocol. The 2.3-kilobase pair Lyn B XhoI-digested insert was isolated from pCDM8 and subcloned into the XhoI site of pZeo. The unique domain construct was generated by PCR amplification using internal sense and antisense primers for the unique domain: (5′-CGGGCGGCTCGATGGGATGTATTAAATCAAAAAGGAAAG-3′, and 5′-CGGCGGCTCGAGCTAGTCCCCTTGCTCCTCTGGATC-3′, respectively). The final PCR product was digested with XhoI and cloned back into the pZeo XhoI site. The catalytically inactive Lyn B(K279R)-pZeo construct was prepared using the Altered Sites in vitro mutagenesis system from Promega (Madison, WI) as follows. A 2.3-kilobase pair XbaI fragment of Lyn B from Lyn-pCDM8 was ligated into the XbaI site of pAlter-1. Mutagenesis followed the manufacturer's protocol using the ampicillin repair primer provided in the kit and a Lyn single mutation antisense oligonucleotide: (5′-GCCAGGCTTGAGGGTCCTTACAGCCACTTTTGTGC-3′) to convert TTC (Lys) → TCC (Arg). The mutant Lyn B was digested with XhoI and BstBI and ligated back into pZeo. Using Lipofectin reagent-mediated transfection (Life Technologies, Inc.), pSVL constructs of the α, β, and γ subunits of rat FcεRI along with pSV2neo had been previously introduced into CHO cells, and a clone expressing a high number of receptors was frozen. 4C. Pucillo, unpublished studies. After thawing, expression of receptors decreased rapidly with time in culture, so we recloned the culture by incubating it with fluorescein-conjugated IgE and sorting on a fluorescence activated cell sorter. The 1% of cells expressing the highest number of receptors were resorted on 96-well plates at 0.5 cell/well. Fifty surviving clones were screened for expression of receptors, by growing the cells to confluence and sensitizing them with 125I-labeled mouse IgE. The washed adherent cells were solubilized with boiling SDS sample buffer and the IgE in the extract quantitated by γ-counting. Of five high expressing clones, one (CHO-B12) proved highly stable and was used for all subsequent studies. CHO-B12 cells were cryopreserved by freezing in 5% dimethyl sulfoxide, 95% growth medium; higher concentrations of dimethyl sulfoxide caused a rapid decline in FcεRI. The cells were electroporated (0.4-cm gap cuvettes, 200 V, 500 microfarads) in the presence of one of several rat Lyn-pZeo constructs or empty pZeo vector, which had been linearized by digestion with Eco57I. To select resistant clones, the medium was supplemented with 250 μg/ml zeocin 72 h post-transfection. The human Lyn B cDNA (1.5 kilobase pairs) was excised from pSVL by XbaI digestion and ligated into the homologous NheI site of pBlueBac. Sf9 cells were co-transfected with wild type AcMNPV DNA and the Lyn construct to generate recombinant Lyn baculoviruses. Adherent Sf9 cells were infected with plaque-purified baculovirus at a multiplicity of infection of 0.4 and, after 48 h, lysed in 0.1% Nonidet P-40 buffer containing protease and phosphatase inhibitors. Western blotting with anti-Tyr(P) indicated that the Lyn B protein was phosphorylated on tyrosine as it was produced in the insect cells (data not shown). CHO cells to be stimulated with antigen were sensitized overnight with 125I-labeled mouse anti-DNP mouse IgE, washed three times in buffer A (150 mmNaCl, 5 mm KCl, 25 mm Pipes, pH 7.2) plus 0.1% (w/v) gelatin and 5.4 mm dextrose, and resuspended at 1 × 107 cells/ml. DNP6-BSA was added as a 5-fold stock solution to 5 × 106 cells at 37 °C for the times indicated. CHO cells stimulated with IgE oligomers were incubated with the indicated concentrations at 37 °C for the times indicated. After stimulation, the receptors were solubilized in 0.05% Triton X-100 (3Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (175) Google Scholar). For immunoprecipitation, anti-mouse or anti-rat IgE antibody was prebound to 30 μl of protein A-Sepharose beads overnight in borate-buffered saline, pH 8, containing 0.1% gelatin. The beads were recovered by centrifugation and combined with the lysates ("precleared" with 100 μl of protein A-Sepharose beads overnight) for 2 h. After recentrifugation the immunoprecipitates were washed four times as described previously (3Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (175) Google Scholar), and the bound proteins released by boiling in SDS sample buffer for 5 min. Immunoprecipitated receptors were separated by electrophoresis in SDS on 10% polyacrylamide gels equilibrated with Tricine and the phosphorylated proteins detected with an anti-Tyr(P) antibody and an enhanced chemiluminescent detection system (ECL, Amersham) (25Alber G. Kent U.M. Metzger H. J. Immunol. 1992; 149: 2428-2436PubMed Google Scholar). Autophotographs of Western blots were quantitated by computerized densitometry (Molecular Dynamics, Sunnyvale, CA). Three steps were taken to ensure equal numbers of receptors were being compared in those studies in which cells co-transfected with inactive forms of Lyn were compared with cells that had not been co-transfected. First, the cells were incubated with IgE that had been labeled with125I and equal numbers of counts were loaded per lane. Second, one lane on each gel was loaded with the same amount of phosphorylated human Lyn B to correct for differences in transfer, antibody staining, washing, etc. Third, the primary anti-Tyr(P) blots were stripped and reprobed with an antibody (JRK) to the β chain of the receptor (26Rivera J. Kinet J.-P. Kim J. Pucillo C. Metzger H. Mol. Immunol. 1988; 25: 647-661Crossref PubMed Scopus (79) Google Scholar), and densitometric analysis was repeated. The densitometric values from the primary anti-Tyr(P) blots were then corrected for any differences in anti-Tyr(P) staining or loading of receptors. In separate experiments, the linearity of antibody staining (anti-Tyr(P), anti-β) was verified by loading increasing amounts of an appropriate protein extract and quantitating the band intensity. To quantitate the relative amounts of Lyn, whole cell lysates containing either 7 × 104 or 1.6 × 105 cell eq were prepared with SDS for each transfectant. Depending on which molecules had been transfected, the samples were separated on 8% (Lyn B, RK Lyn), 10% (CHO-B12, pZeo), or 4–20% (unique Lyn A) Tris-glycine gels and blotted with an anti-Lyn antibody and an HRP-conjugated anti-rabbit secondary antibody. One (central) lane on each gel was loaded with a fixed amount of human Lyn B (above). The densitometric readings for the bands corresponding to Lyn were normalized relative to the human Lyn B standard. CHO cells were suspended at 5 × 106 cells/ml and incubated with 5 μg/ml125I-labeled IgE for 1 h at 37 °C. Nonspecific binding was evaluated by preincubating the cells with a 10-fold excess of unlabeled IgE for 30 min at 37 °C. Cells were separated from unbound IgE by pelleting through phthalate oil (15Kulczycki Jr., A. Metzger H. J. Exp. Med. 1974; 140: 1676-1695Crossref PubMed Scopus (223) Google Scholar, 27Matthyssens G.E. Hurwitz E. Givol D. Sela M. Mol. Cell. Biochem. 1975; 7: 119-126Crossref PubMed Scopus (13) Google Scholar). CHO cells were sonicated, and the 140,000 × g supernatant (cytosolic fraction) and pellet (membrane fraction) were prepared from the post-nuclear supernatant as described previously (28Pribluda V.S. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11446-11450Crossref PubMed Scopus (33) Google Scholar). Membrane proteins were solubilized in 0.5% Triton X-100, for 30 min at 4 °C. Each subcellular fraction was treated with an equal volume of boiling 2 × SDS sample buffer for 5 min prior to gel electrophoresis. Coupled in vitrotranscription-translation reactions were conducted with [35S]Cys according to the manufacturer's recommendation (T3 TnT® coupled reticulocyte lysate system, Promega). Initial identification of potentially interacting domains was conducted by co-transforming constructs containing the cytoplasmic domains of the FcεRI fused to the binding domain of the Gal4 transcription factor with constructs containing Lyn or various mutated forms of Lyn fused to the activation domain of Gal4 (Fig.1). The nucleotide sequence coding for the N- and C-terminal cytoplasmic domains of the β subunit of the rat IgE receptor, βNand βC, were subcloned into pAS2 to generate Gal4 DNA binding domain fusion proteins. Unfortunately, both fusion proteins autonomously activated the reporter genes. This is presumably due to the acidic hemagglutinin epitope located between the Gal4 DNA binding domain and the inserted proteins (29Bartel P.L. Chien C.-T. Sternglanz R. Fields S. BioTechniques. 1993; 14: 920-924PubMed Google Scholar). However, the fusion protein containing the cytoplasmic domain of the γ subunit was not autonomously active. Therefore, we subcloned nucleotide sequences coding for βN and βC into the newly developed vector pAS-2-1, which is similar to pAS2, but has the acidic hemagglutinin epitope removed. Neither pAS2-1-βC or pAS2-1-βN were autonomously active. The activities of the His and LacZ reporter genes in CG1945 yeast transformants expressing Lyn and βN, βC or γC were tested as described (see "Experimental Procedures"). Both the full-length and unique domain of both Lyn A and Lyn B interacted directly with βC (data not shown). However, the interaction was much weaker than the interaction detected between the p53 and SV40 fusion proteins used as a positive control. Thus, per microgram of DNA, co-transformation with p53 and SV40 resulted in more colonies on His-deficient medium (SD-3) and rapid growth into large colonies. All of the colonies containing p53 and SV40 rapidly turned blue. In contrast, co-transformation with the Lyn and βC constructs resulted in fewer colonies and slower growth on His-deficient medium and only the large colonies turned blue. No interaction was detected between βN or γC with any forms of Lyn in this assay. To quantitate the interaction between Lyn and βC or βN, we measured the β-galactosidase activity of these co-transformants in yeast strain Y187 in a liquid assay. In addition to the full-length Lyn and the construct containing only the unique domain, we tested a series of Lyn mutants based on the results of Pleiman et al. (30Pleiman C.M. Abrams C. Gauen L.T. Bedzyk W. Jongstra J. Shaw A.S. Cambier J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4268-4272Crossref PubMed Scopus (126) Google Scholar) and Timson Gauen et al. (31Timson Gauen L.K. Kong A.N. Samelson L.E. Shaw A.S. Mol. Cell Biol. 1992; 12: 5438-5446Crossref PubMed Scopus (163) Google Scholar). The negative control in this experiment was the 40 amino acid residues (from 27 to 66) of Lyn fused out-of-frame to the Gal4 activation domain (pACT-27–66-OOF). As shown in Fig. 2, the activity of the LacZ reporter gene from co-transformants with the unique domain of either Lyn A or B was as high as that from co-transformants with the full-length form of either Lyn. These values are 3-fold higher than those than from the negative control pACT-27–66-OOF. Consistent with the result from CG1945 strain, the interaction between Lyn and βC is weaker (on the basis of the β-galactosidase activity, only 1% as strong) than that between p53 and SV40. Co-transformants containing Lyn residues 1–10, 1–27, or 27–66 produced only slightly higher amounts of β-galactosidase than the negative control. Again, no interaction between Lyn and βN was detected. A clone of transfected CHO cells that stably expressed ≈170,000 receptors/cell (CHO-B12) (Table I) was further characterized. When immunoblotted with anti-human Lyn antibody, extracts of these cells, like those of the untransfected CHO cells, show a weakly reactive component at ≈58 kDa, i.e. slightly greater than the apparent molecular mass of 53 and 56 kDa observed for rat Lyn (Fig. 3 A). There was no reactivity with a panel of antibodies to human c-Src, Fyn, or c-Yes (data not shown). Cells from the B12 clone were incubated with anti-DNP-specific mouse IgE and after solubilization with detergent, the bound (unaggregated) receptors were immunoprecipitated with goat anti-mouse IgE. Upon Western blotting with anti-Tyr(P), no evidence for phosphorylation was observed (Fig. 3 B, lane 1). When the cells were incubated with multivalent antigen (DNP-BSA) prior to solubilization, phosphorylation of tyrosines on the β and γ subunits of the transfected receptors was observed (lane 2). Disaggregation of the receptors in vivo by addition of hapten (DNP-caproic acid) after the exposure to DNP-BSA led to the complete reversal of the antigen-induced phosphorylation of receptor tyrosines within ≤1 min (data not shown).Table ICHO transfectantsNameCloneInsertFcεRILyn per FcεRILyn (Inact.) per Lyn (End.)×10 −5CHO-B121-aA stable CHO FcεRI transfectant (CHO-B12) was generated by electroporation of FcεRI subunits (α, β, γ). Stable double transfectants, likewise generated by electroporation, were prepared by transfecting various Lyn constructs into CHO-B12 cells and selection with zeocin. Control cells, doubly transfected with FcεRI and empty pZeo vectors, were prepared by the same protocol.B12NA1-bNA, not applicable.1.72.51-cThe values shown in this column are strictly relative and were determined as follows. For each transfectant, the normalized densitometric readings of the total Lyn in a fixed number of cell equivalents (see "Experimental Procedures") was divided by the number of FcεRI per cell × 10−3. For example, for the first item in this column the corrected densitometric reading was 430.6. The latter divided by 170 (FcεRI per cell × 10−3) equals 2.5. The quantitation of Lyn was done two to eight times for each transfectant; the enumeration of the FcεRI was done in duplicate.NALyn/B12A6Lyn B1.01.0NALyn/B12A9Lyn B0.79.3NALyn/B12A11Lyn B1.366NALyn/B12D1Lyn B1.334NALyn/B12D7Lyn B1.33.2NALyn/B12D8Lyn B1.30.94NARK Lyn/B12RK17Inact.Lyn B1.97.46.01-dThe amount of endogenous hamster Lyn (Lyn (End.)) and transfected inactive Lyn (Lyn(Inact.)) expressed per cell was determined by Western blotting of SDS lysates with anti-(human) Lyn. The values shown represent the average of two to eight separate determinations.RK Lyn/B12RK21Inact.Lyn B1.84.64.2RK Lyn/B12RK26Inact.Lyn B1.42412Lyn unique/B12C6Unique Lyn A1.25.95.0Lyn unique/B12U7Unique Lyn A1.00.830.42Lyn unique/B12U8Unique Lyn A1.71.90.76pZeo/B12Z1None0.85.2NApZeo/B12Z2None0.86.6NApZeo/B12Z3None1.05.0NApZeo/B12Z4None0.86.8NApZeo/B12Z5None1.52.8NApZeo/B12Z6None1.06.6NA1-a A stable CHO FcεRI transfectant (CHO-B12) was generated by electroporation of FcεRI subunits (α, β, γ). Stable double transfectants, likewise generated by electroporation, were prepared by transfecting various Lyn constructs into CHO-B12 cells and selection with zeocin. Control cells, doubly transfected with FcεRI and empty pZeo vectors, were prepared by the same protocol.1-b NA, not applicable.1-c The values shown in this column are strictly relative and were determined as follows. For each transfectant, the normalized densitometric readings of the total Lyn in a fixed number of cell equivalents (see "Experimental Procedures") was divided by the number of FcεRI per cell × 10−3. For example, for the first item in this column the corrected densitometric reading was 430.6. The latter divided by 170 (FcεRI per cell × 10−3) equals 2.5. The quantitation of Lyn was done two to eight times for each transfectant; the enumeration of the FcεRI was done in duplicate.1-d The amount of endogenous hamster Lyn (Lyn (End.)) and transfected inactive Lyn (Lyn(Inact.)) expressed per cell was determined by Western blotting of SDS lysates with anti-(human) Lyn. The values shown represent the average of two to eight separate determinations. Open table in a new tab RBL cells can be stimulated either by aggregating receptor-bound monomeric IgE with antigen or by incubating the cells with preformed dimers of IgE (Fig. 3 B, lane 8). In contrast, incubation of the CHO-B12 cells with dimeric IgE failed to induce detectable phosphorylation of the receptors (Fig. 3 B,lane 4). These results are consistent with a limiting amount of protein-tyrosine kinase being associated with the receptors in these cells (see "Discussion"). A series of stable transfectants of the CHO-B12 cells with rat Lyn were isolated. The relative ratios of full-length Lyn/receptor of six clones (A6 through D8) are shown