Interaction between the Unphosphorylated Receptor with High Affinity for IgE and Lyn Kinase
2001; Elsevier BV; Volume: 276; Issue: 2 Linguagem: Inglês
10.1074/jbc.m003397200
ISSN1083-351X
AutoresBecky M. Vonakis, Hana Haleem‐Smith, Peter S. Benjamin, Henry Metzger,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoChinese hamster ovary fibroblasts previously transfected with the high affinity receptor for IgE (FcεRI) were further transfected with the α subunit of the receptor for interleukin 2 (Tac) or with chimeric constructs in which the cytoplasmic domain of Tac was replaced with the C-terminal cytoplasmic domain of either the β subunit or the γ subunit of FcεRI. Whereas native Tac failed to affect the aggregation-induced phosphorylation of FcεRI, both chimeric constructs substantially inhibited this reaction. Alternatively, the FcεRI-bearing fibroblasts were transfected with two chimeric constructs in which the cytoplasmic domain of Tac was replaced with a modified short form of Lyn kinase. The Lyn in both of the chimeric constructs had been mutated to remove the sites that are normally myristoylated and palmitoylated, respectively; one of the constructs had in addition been altered to be catalytically inactive. The catalytically active construct enhanced, and the inactive construct inhibited, aggregation-induced phosphorylation of the receptors. All of the chimeric constructs were largely distributed outside the detergent resistant microdomains, and whereas aggregation caused them to move to the domains in part, their aggregation was neither necessary nor enhanced their effects. These results and others indicate that the receptor and Lyn interact through protein-protein interactions that neither are dependent upon either the post-translational modification of the kinase with lipid moieties nor result exclusively from their co-localization in specialized membrane domains. Chinese hamster ovary fibroblasts previously transfected with the high affinity receptor for IgE (FcεRI) were further transfected with the α subunit of the receptor for interleukin 2 (Tac) or with chimeric constructs in which the cytoplasmic domain of Tac was replaced with the C-terminal cytoplasmic domain of either the β subunit or the γ subunit of FcεRI. Whereas native Tac failed to affect the aggregation-induced phosphorylation of FcεRI, both chimeric constructs substantially inhibited this reaction. Alternatively, the FcεRI-bearing fibroblasts were transfected with two chimeric constructs in which the cytoplasmic domain of Tac was replaced with a modified short form of Lyn kinase. The Lyn in both of the chimeric constructs had been mutated to remove the sites that are normally myristoylated and palmitoylated, respectively; one of the constructs had in addition been altered to be catalytically inactive. The catalytically active construct enhanced, and the inactive construct inhibited, aggregation-induced phosphorylation of the receptors. All of the chimeric constructs were largely distributed outside the detergent resistant microdomains, and whereas aggregation caused them to move to the domains in part, their aggregation was neither necessary nor enhanced their effects. These results and others indicate that the receptor and Lyn interact through protein-protein interactions that neither are dependent upon either the post-translational modification of the kinase with lipid moieties nor result exclusively from their co-localization in specialized membrane domains. detergent-resistant membrane 2,4-dinitrophenyl Chinese hamster ovary phosphate-buffered saline N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 1,4-piperazinediethanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid bovine serum albumin Cellular responses mediated by the high affinity receptor for IgE (FcεRI) begin with the phosphorylation of the tyrosines in each of the three immunoreceptor tyrosine activation motifs of the receptor (1Cambier J.C. Immunol. Today. 1995; 16: 110Abstract Full Text PDF PubMed Scopus (256) Google Scholar). This phosphorylation is stimulated by aggregation of the receptors and in the cells studied in greatest detail, the RBL-2H3 line (2Barsumian E.L. Isersky C. Petrino M.G. Siraganian R.P. Eur. J. Immunol. 1981; 11: 317-323Crossref PubMed Scopus (481) Google Scholar), is effected by the Src family kinase Lyn (3Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (413) Google Scholar). Two molecular models have been proposed to explain the role of aggregation in stimulating the phosphorylation of the receptors. The transphosphorylation model we have used to study this system is based on the following experimental findings (4Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (172) Google Scholar, 5Yamashita T. Mao S.-Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (157) Google Scholar): (a) Lyn kinase is constitutively and weakly associated with a small fraction of the receptors; b) in vivo and in vitro, the constitutively associated kinase is able to phosphorylate the receptors but only when they are aggregated; and (c) the initial phosphorylation of the receptors, but not their aggregation per se, promotes their association with additional, more firmly bound, Lyn kinase. These findings suggest that aggregation stimulates the phosphorylation of the receptors by stabilizing the juxtaposition of the kinase with its substrate, thereby shifting the balance between phosphorylation and dephosphorylation (6Metzger H. Chen H. Goldstein B. Haleem-Smith H. Inman J.K. Peirce M.J. Torigoe C. Vonakis B.M. Wofsy C. Immunol Lett. 1999; 68: 53-57Crossref PubMed Scopus (8) Google Scholar). A second translocation model is based on the following observations: (a) aggregates of receptors, particularly larger aggregates, become localized, at least transiently, in discrete detergent-resistant membranes (DRM)1 (7Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (269) Google Scholar, 8Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 9Stauffer T.P. Meyer T. J. Cell Biol. 1997; 139: 1447-1454Crossref PubMed Scopus (172) Google Scholar); (b) such microdomains occupy only a small fraction of the total plasma membrane but contain a substantial fraction of the total Lyn kinase (7Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (269) Google Scholar); (c) upon aggregation, those receptors that become phosphorylated are largely found in the DRM; and (d) at least in some experiments (see “Discussion”), cholesterol-depleting agents, which interfere with the formation of such microdomains, inhibit the phosphorylation of the receptors (10Sheets E.D. Holowka D. Baird B. J. Cell Biol. 1999; 145: 877-887Crossref PubMed Scopus (282) Google Scholar). It has been proposed that these findings indicate that the primary effect of aggregation is to localize the receptors to regions of the membrane enriched in Lyn kinase (11Baird B. Sheets E.D. Holowka D. Biophys. Chem. 1999; 82: 109-119Crossref PubMed Scopus (74) Google Scholar). Neither of the two models predicates any aggregation-induced increase in the specific activity of the kinase per se, consistent with the limited experimental data on this question (5Yamashita T. Mao S.-Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (157) Google Scholar). We undertook the present studies for two reasons. First, to clarify whether the translocation of the aggregated receptors to DRM was a necessary and sufficient or simply an accompanying aspect of the interaction between Lyn and the receptor. Second, to establish a system with which we could analyze in as physiological a milieu as possible the effect of genetically introduced changes in the structure of the receptor or Lyn on their interaction. We used genetic constructs of portions of the receptor similar to those that had already been applied to this system by others (12Romeo C. Seed B. Cell. 1991; 64: 1037-1046Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 13Letourneur F. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8905-8909Crossref PubMed Scopus (239) Google Scholar, 14Eiseman E. Bolen J.B. J. Biol. Chem. 1992; 267: 21027-21032Abstract Full Text PDF PubMed Google Scholar, 15Jouvin 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, 16Wilson 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 (62) Google Scholar). Specifically, we used cDNA constructs in which the ecto- and transmembrane domains of a subunit of interleukin-2 receptors (CD25α, Tac) were fused to the cytoplasmic domains of the β or the γ chains of FcεRI, alternatively. The constructs were stably transfected into Chinese hamster ovary (CHO) fibroblasts that we had previously transfected with FcεRI (17Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The latter transfectants contain minimal amounts of endogenous Lyn kinase and, therefore, are particularly sensitive to manipulations that either enhance or inhibit the interaction of active kinase with the receptor (17Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 18Wofsy C. Vonakis B.M. Metzger H. Goldstein B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8615-8620Crossref PubMed Scopus (31) Google Scholar). We performed complementary experiments by constructing chimeric constructs consisting of the ecto- and transmembrane domains of Tac to a construct of Lyn that had been mutated to remove the sites of myristoylation and palmitoylation. We then examined the ability of such constructs to influence the phosphorylation of the aggregated receptors, much as we had previously studied normally anchored Lyn (17Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar,18Wofsy C. Vonakis B.M. Metzger H. Goldstein B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8615-8620Crossref PubMed Scopus (31) Google Scholar). Avidin and polyclonal anti-Tac coupled to biotin were purchased from R & D Systems (Minneapolis, MN). Extravidin coupled to horseradish peroxidase was obtained from Sigma. Monoclonal anti-phosphotyrosine antibody (anti-PY) coupled to biotin (4G10), rabbit polyclonal anti-Lyn kinase, and Src family kinase substrate peptide (19Cheng H.C. Litwin C.M. Hwang D.M. Wang J.H. J. Biol. Chem. 1991; 266: 17919-17925Abstract Full Text PDF PubMed Google Scholar) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Dr. T. Waldmann (NCI, National Institutes of Health) provided a hybridoma producing monoclonal anti-Tac (clone HD245). The hybridoma cells were gradually weaned off serum in successive tissue cultures. The antibody was purified from the serum-free supernatants using a kit containing immobilized protein A (Pierce) and labeled with carrier-free [125I]iodine using Iodogen tubes also from Pierce. (Iodination of the anti-Tac with chloramine T resulted in the complete loss of its bindability to Tac-expressing cells.) Monoclonal anti-Tac coupled to biotin (clone B1.49.9) was from Cappel (Durham, NC). Goat anti-mouse IgE, mouse monoclonal anti-DNP IgE (iodinated as appropriate), and covalently cross-linked oligomers of IgE were prepared as described previously (17Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Polyclonal anti-caveolin was from Santa Cruz (Santa Cruz, CA). SpinZyme® phosphocellulose filters were from Pierce. TableI shows selected sequences of the constructs we used for our studies. Dr. J. Oliver (University of New Mexico) generously provided the cDNAs for constructs consisting of the ecto- and membrane spanning domains of Tac fused to the C-terminal 42 residues of the β or of the γ subunit of FcεRI (TT-β or TT-γ) in pcDL-SRa296 (16Wilson 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 (62) Google Scholar). They were digested with BamHI and EcoRI and subcloned into pZeo-2SV (minus orientation of the multiple cloning site), which had been digested with the same enzymes. The cDNA for wild-type Tac (TT-T), which contains the Tac cytoplasmic domain of 13 residues (20Barclay A.N. Birkeland M.L. Brown M.H. Beyers A.D. Somoza C. Williams A.F. The Leukocyte Antigen Facts Book. Academic Press, Ltd., London1993Google Scholar) in pGM, was obtained from W. Leonard (NHLBI, National Institutes of Health) and similarly subloned into pZeo-2SV. We also prepared a construct in which a stop codon was inserted after that coding for leucine residue 238 so that no cytoplasmic domain would be expressed (TT-0).Table ICharacteristics of Tac and chimeric constructsConstructC terminus of CD25αN-terminus of CD25α, of FcɛRI β or γ, or of LynJunctional sequenceBaseAmino acidBaseAmino acidTT-T714237715238..LLSGLTWQRR..TT-β714237660202..LLSGLRIGQE..TT-γ72023915027..SGLTWRLKIQ..TT-Lyn714237124..LLSGLEIKSK....TT-LynKR714237124..LLSGLEIKSK....Columns 2 and 3 show the number for the 3′ base in the 3′ codon of the open reading frame and for the corresponding C-terminal amino acid residue from the transmembrane domain of the Tac protein (CD25α). Columns 4 and 5 show the number for the 5′ base in the 5′ codon of the open reading frame and for the corresponding N-terminal amino acid residue from the cytoplasmic domains of the Tac protein, the β subunit (C-terminal), the γ subunit, and the Lyn protein, respectively. The junctional sequence is given in the last column with the joined residues shown in bold type. In the construct Tac-LynKR, arginine substitutes for lysine at residue 279. Open table in a new tab Columns 2 and 3 show the number for the 3′ base in the 3′ codon of the open reading frame and for the corresponding C-terminal amino acid residue from the transmembrane domain of the Tac protein (CD25α). Columns 4 and 5 show the number for the 5′ base in the 5′ codon of the open reading frame and for the corresponding N-terminal amino acid residue from the cytoplasmic domains of the Tac protein, the β subunit (C-terminal), the γ subunit, and the Lyn protein, respectively. The junctional sequence is given in the last column with the joined residues shown in bold type. In the construct Tac-LynKR, arginine substitutes for lysine at residue 279. For the Lyn-based chimeric constructs, we generated fusion proteins by polymerase chain reaction. cDNA coding for Lyn kinase mutated at its N terminus was prepared as follows: sense and antisense primers for the desired mutations of the intact wild-type short form of Lyn (5′-CTCGAGATTAAATCAAAAAGGAAAGACAATC-3′ and 5′-CTCGAGCCACTATGGCTGCTGCTGATACTGCCCTTCCGTGGCACTG-3′) were synthesized and used in polymerase chain reaction with the wild-type Lyn as template. The sense primer encodes a XhoI site at its N terminus that excludes the N-terminal methionine and glycine. The primer also substitutes the codon for cysteine with one for glutamic acid. A construct of Tac coding for the ecto- and transmembrane domain was generated by polymerase chain reaction with an XhoI site at its C terminus. The two cDNAs were digested with XhoI and then ligated to generate the fusion chimera (TT-Lyn) and subsequently subcloned into the expression vector pZeo. TT-LynKR was generated similarly using the Lyn cDNA that encodes the catalytically inactive Lyn generated previously (17Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Analysis of each construct was determined using the Big Dye® kit obtained from Applied Biosystems (Foster City, CA) and shown to be as planned. Plasmid expression constructs (10 μg) were electroporated (0.4 cm gap, 200 volts, 500 millifarad) into 1 × 107 CHO-B12 cells, a cell line stably expressing 170,000 FcεRI under G418 selection (Ref. 17Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholarand Table II). After 72 h, the transfected cells were placed under selection with 250 μg/ml zeocin. Clones were picked after 5–7 days, expanded, and screened for expression of FcεRI and the Tac construct. A second set of transfectants for TT-γ and all of the TT-Lyn transfectants were prepared using LipofectAMINE (Life Technologies, Inc.). In a 6-well plate, 3 × 105 CHO-B12 cells were plated and grown overnight to 60–70% confluence. 1 μg of plasmid DNA was diluted into 100 μl of Opti-MEM serum-free medium (Life Technologies, Inc.) and mixed with a second solution containing 6 μl of LipofectAMINE in 100 μl of Opti-MEM serum-free medium. The mixture was incubated at room temperature for 30 min., diluted with 0.8 ml of Opti-MEM, and overlaid onto the cells. After 5 h at 37 °C, 1 ml of CHO-B12 growth medium containing 20% (2×) fetal bovine serum was added to the cells. The transfectants were then selected with zeocin, cloned and expanded as for transfectants generated by electroporation. Duplicate wells of each clone to be tested were grown to confluence in 24-well plates (≈2.5 × 105 cell/well). The cells in one set of wells were incubated with 1 μg/ml of [125I]IgE in 0.1 ml for 1 h at 37 °C, washed five times with phosphate-buffered saline (PBS), and then solubilized with boiling hot 1% SDS, in a 62.5 mm Tris, 0.5× PBS buffer. The lysates were then γ-counted. For assessing expression of the Tac constructs, cells were incubated with 0.5 μg/ml of [125I]anti-Tac in 0.5 ml, at 4 °C for 30 min, washed four times with ice-cold Iscove's medium, and then solubilized and counted as for the cells labeled with IgE. To maintain the integrity of cell surface Tac, cells were harvested using 3 mm EDTA in PBS rather than trypsin for passaging and stimulation. In addition, all CHO transfectants were cryopreserved in growth media supplemented with 5% (CH3)2SO, and a new vial of cells was routinely thawed every 2 months to assure a consistent ratio of Tac to FcεRI.Table IIExpression of Tac and FcɛRI on transfected CHO-B12 cellsTransfected cDNAClone2-aClones with numbers ending in E were transfected by electroporation; those with numbers ending in L were transfected by LipofectAMINE.FcɛRI (×10−5)Tac (×10−5)Tac:FcɛRITT-β1A3E1.2 ± 0.102.7 ± 0.502.21A4E1.0 ± 0.012.1 ± 0.202.12A3E1.0 ± 0.070.9 ± 0.100.92C9E1.3 ± 0.010.4 ± 0.080.3TT-γA1L0.8 ± 0.102.0 ± 0.402.5A3E1.4 ± 0.200.3 ± 0.040.2B1E1.3 ± 0.090.9 ± 0.100.7C1L0.9 ± 0.102.2 ± 0.102.4D6E1.6 ± 0.104.0 ± 0.702.5TT-LynD6L1.5 ± 0.160.43 ± 0.040.29A6L1.2 ± 0.100.5 ± 0.100.43B1L0.78 ± 0.010.5 ± 0.070.64A5L1.5 ± 0.170.6 ± 0.060.40C6L1.4 ± 0.120.64 ± 0.080.44C4L0.95 ± 0.121.4 ± 0.181.5342C2E1.7 ± 0.160.02 ± 0.0140.01TT-LynKR33A4E1.2 ± 0.140.22 ± 0.030.1831A4E1.3 ± 0.150.62 ± 0.090.4834A3E1.5 ± 0.262.18 ± 0.481.44TT-T21A4E1.2 ± 0.110.68 ± 0.080.5824B2E0.96 ± 0.070.16 ± 0.050.17TT-051B2E0.800.160.2052C4E0.930.330.3652C2E1.050.440.4251C2E0.934.404.73pZeo4E1.5 ± 0.08NA2-bNA, Not applicable.NA5E1.2 ± 0.04NANAnoneB121.7 ± 0.19NANAA CHO cell line previously transfected with rat FcɛRI by electroporation and selected with G418 (CHO-B12 (last row)), was further transfected by electroporation or LipofectAMINE either with vector containing the zeocin antibiotic resistance marker only (pZeo) or in addition with Tac chimeras (TT-β, TT-γ, TT-Lyn, or TT-LynKR) or the wild-type or truncated Tac (TT-T and TT-0), and selected with zeocin. FcɛRI were quantitated using [125I]IgE in duplicate incubations two to seven times for each transfectant except those transfected with TT-0. Tac was quantitated using [125I]anti-Tac from clone HD245 in duplicate incubations two to seven times for each transfectant except those transfected with TT-0. We assumed each molecule of anti-Tac bound two molecules of Tac (22Junghans R.P. Immunol. Today. 1999; 20: 401-406Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The values shown are the means ± S.E.2-a Clones with numbers ending in E were transfected by electroporation; those with numbers ending in L were transfected by LipofectAMINE.2-b NA, Not applicable. Open table in a new tab A CHO cell line previously transfected with rat FcɛRI by electroporation and selected with G418 (CHO-B12 (last row)), was further transfected by electroporation or LipofectAMINE either with vector containing the zeocin antibiotic resistance marker only (pZeo) or in addition with Tac chimeras (TT-β, TT-γ, TT-Lyn, or TT-LynKR) or the wild-type or truncated Tac (TT-T and TT-0), and selected with zeocin. FcɛRI were quantitated using [125I]IgE in duplicate incubations two to seven times for each transfectant except those transfected with TT-0. Tac was quantitated using [125I]anti-Tac from clone HD245 in duplicate incubations two to seven times for each transfectant except those transfected with TT-0. We assumed each molecule of anti-Tac bound two molecules of Tac (22Junghans R.P. Immunol. Today. 1999; 20: 401-406Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The values shown are the means ± S.E. CHO transfectants were harvested and incubated at 5 × 106cells/ml with 5 μg/ml [125I]IgE for 1 h at 37 °C. Nonspecific binding was assessed by preincubating the cells with 50 μg/ml unlabeled IgE for 30 min. Cells were then centrifuged through a mixture of phthalate oils (21Matthyssens G.E. Hurwitz E. Givol D. Sela M. Mol. Cell. Biochem. 1975; 7: 119-126Crossref PubMed Scopus (13) Google Scholar), and the radioactivity in the pellets was counted. Alternatively, CHO transfectants were incubated at 5 × 106 cells/ml with 0.5 μg/ml [125I]anti-Tac for 30 min at 4 °C. Nonspecific binding was measured in duplicate incubations with 5 μg/ml unlabeled anti-Tac. Cells were then isolated as above, and the pellets were counted. The number of molecules of Tac was calculated from the recovered radioactive counts and the specific activity of the labeled anti-Tac, assuming one molecule of anti-Tac binds two molecules of Tac protein (22Junghans R.P. Immunol. Today. 1999; 20: 401-406Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Stimulation of CHO transfectants with IgE plus antigen or with preformed covalently cross-linked oligomers IgE was conducted as described previously (17Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Where aggregation of Tac was employed, the cells were incubated first with 2 μg/ml monoclonal anti-Tac-biotin (clone B1.49.9) at room temperature and then with 25 μg/ml avidin, for 10 min at 37 °C. FcεRI were aggregated with oligomers of IgE for 30 min at 37 °C when appropriate. Alternatively, Tac constructs were aggregated using a monoclonal anti-Tac followed by a polyclonal anti-mouse IgG. FcεRI were solubilized and immunoprecipitated with anti-IgE, and phosphorylation of tyrosines on their β, and the γ subunits was determined as described previously (18Wofsy C. Vonakis B.M. Metzger H. Goldstein B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8615-8620Crossref PubMed Scopus (31) Google Scholar). Tac constructs were extracted with 1% Nonidet P-40 and immunoprecipitated with monoclonal anti-Tac. Immunoprecipitates were analyzed on 10% Tricine gels followed by immunoblotting first with biotinylated polyclonal anti-Tac or 4G10 and then with horseradish peroxidase-coupled avidin. For monitoring of FcεRI, CHO transfectants were sensitized for 1 h at room temperature with 5 μg/ml IgE, 10% of which had been labeled with 125I. For monitoring of Tac constructs, the CHO cells were labeled with [125I]anti-Tac for 30 min, at 4 °C. After washing the cells three times with buffer A (4Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (172) Google Scholar), plasma membranes were isolated by Dounce homogenization and sedimented in 30% isotonic Percoll (23Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (671) Google Scholar). Successive 1-ml fractions were removed from the top of the gradient, the location of the visible band was noted, and the radioactive counts in each fraction were determined in a γ-counter. The method we used is similar to that described by Rodgers and Rose (24Rodgers W. Rose J.K. J. Cell Biol. 1996; 135: 1515-1523Crossref PubMed Scopus (285) Google Scholar) and by Field et al. (8Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Briefly, cells were detached with 3 mm EDTA in PBS and resuspended in growth medium at 5 × 106/ml. After the desired treatment, 107 cells in 0.5 ml were diluted 2-fold with 0.5 ml of 0.1% Triton X-100. The lysate was kept at 4 °C with gentle agitation for 30 min. It was then mixed with an equal volume of 85% sucrose, transferred to a Beckman 344060 clear centrifuge tube and overlaid with 6 ml of 30% sucrose prepared in a pH 7.5 buffer containing 0.05% Triton X-100, 10 mm Tris, 150 mm NaCl, 5 mm EDTA, 1 mmNa3VO4, and 2 mm sodium iodoacetate supplemented with protease inhibitors leupeptin, pepstatin, aprotinin, each at 1 μg/ml, and 5 mm 4-(2-aminoethyl)-benzensulfonyl fluoride or phenylmethylsulfonyl fluoride (buffer A). The 30% layer was overlaid with 3.5 ml of 5% sucrose, again prepared with buffer A. Tubes were centrifuged in a swinging bucket rotor at 38,000 rpm for12–14 h. Successive 1-ml fractions were removed from the top of the gradient. All fractions were counted and then diluted with Triton X-100 to a final concentration of 0.5% prior to immunoprecipitations of FcεRI or 1% Triton X-100 for immunoprecipitation with anti-Tac. Triplicate immunoprecipitates with anti-Lyn, anti-Tac, or control antibodies were incubated in 25 μl of kinase assay buffer (25 mm Pipes, 150 mm NaCl, 5 mm KCl, pH 7.2, 5 mmMnCl2, 2 mm CHAPS, 0.5 mmNa3VO4) containing 2 mm substrate peptide. Reactions were initiated by addition of ATP (100 μm ATP, 10 μCi of [γ-32P]ATP (PerkinElmer Life Sciences)) at 25 °C and vortexed every 10–15 min. The reactions were quenched by the addition of 10 μl of 50% (v/v) acetic acid. The peptide was then isolated using phosphocellulose filters. The filters were washed twice with 0.5 ml of 0.075m phosphoric acid and then counted in a scintillation counter in Filtron-X mixture. CHO cells previously transfected with FcεRI (17Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) were further transfected by electroporation with Tac (TT-T) or a chimeric construct containing the extracellular and transmembrane domains of Tac and the C-terminal cytoplasmic domain of either the β chain (TT-β) or the γ chain of FcεRI (TT-γ). Stable clones that had been selected with zeocin and characterized for their expression of FcεRI and the Tac constructs are listed in Table II. The ratio of TT-β to FcεRI ranged from 0.3 to 2.2, and the that of TT-γ to FcεRI ranged from 0.2 to 2.5. When analyzed by Western blotting, anti-Tac immunoprecipitates of the detergent extracts of such transfectants revealed a diffuse band of 55 kDa (Fig. 1, lanes 1,4, and 6). Although the peptide molecular mass of the constructs is only ≈30 kDa, glycosylation of the ectodomain of Tac is known to retard its mobility. The bands with apparently lower molecular mass (38, 40 kDa) likely represent non- or hypo-glycosylated constructs not expressed on the cell surface. Such species were previously described for chimeric constructs of Tac fused to the cytoplasmic portion of the T cell receptor ζ chain or the γ chain of FcεRI (13Letourneur F. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8905-8909Crossref PubMed Scopus (239) Google Scholar). Fig.2 shows the results of an experiment in which cells from clone 2A3E expressing on their surface approximately 0.9 TT-β per FcεRI (Table II) were first reacted with anti-Tac, or not, to aggregate the Tac constructs. They were then reacted with 500 ng/ml of either monomeric IgE or a mixture of trimeric and tetrameric IgE for 30 min. Cells transfected with receptors only (CHO-B12) were treated similarly. The receptors were immunoprecipitated and Western blotted with anti-phosphotyrosine. The odd numbered lanes show that there is only minimal phosphorylation of the receptors on cells reacted with the monomeric IgE, whereas substantial phosphorylation of the β and γ subunits is apparent in cells reacted with the oligomers. Similar results were obtained in the cells transfected with TT-β, but in this and repeated similar experiments analyzed quantitatively, the phosphorylation was substantially less (−70% in the experiment illustrated). Prior aggregation of the Tac-β constructs neither enhanced nor diminished the inhibition (lane 6 versus lane 8). Additional studies examined the relationship between the ratio of TT-β to FcεRI and the inhibition of phosphorylation. The IgE receptors on cells with TT-β to FcεRI ratios of either 0.3 or 0.9 were aggregated with increasing concentrations of antigen, and the phosphotyrosine on the receptor was compared with identically stimulated control cells (cells transfected solely with the zeocin marker-containing plasmid). A clone expressing a TT-β to FcεRI ratio of 0.3 (clone 2C9E) and stimulated with 80, 150, or 300 ng antigen/ml showed
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