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Anti-peptide Antibodies Detect Conformational Changes of the Inter-SH2 Domain of ZAP-70 Due to Binding to the ζ Chain and to Intramolecular Interactions

1998; Elsevier BV; Volume: 273; Issue: 15 Linguagem: Inglês

10.1074/jbc.273.15.8916

1083-351X

Laura Grazioli, Valérie Germain, Arthur Weiss, Oreste Acuto,

Endoplasmic Reticulum Stress and Disease

T cell receptor (TCR) triggering induces association of the protein tyrosine kinase ZAP-70, via its two src-homology 2 (SH2) domains, to di-phosphorylatedImmunoreceptor Tyrosine-basedActivation Motifs (2pY-ITAMs) present in the intracellular tail of the TCR-ζ chain. The crystal structure of the SH2 domains complexed with a 2pY-ITAM peptide suggests that the 60-amino acid-long inter-SH2 spacer helps the SH2 domains to interact with each other to create the binding site for the 2pY-ITAM. To investigate whether the inter-SH2 spacer has additional roles in the whole ZAP-70, we raised antibodies against two peptides of this region and probed ZAP-70 structure under various conditions. We show that the reactivity of antibodies directed at both sequences was dramatically augmented toward the tandem SH2 domains alone compared with that of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by sequences C-terminal to the SH2 domains, namely, the linker region and/or the kinase domain. Moreover, antibody binding to the same two determinants was also inhibited when ZAP-70 or the SH2 domains bound to the ζ chain or to a 2pY-ITAM. Together, these two observations suggest a model in which intramolecular contacts keep ZAP-70 in a closed configuration with the two SH2 domains near to each other. T cell receptor (TCR) triggering induces association of the protein tyrosine kinase ZAP-70, via its two src-homology 2 (SH2) domains, to di-phosphorylatedImmunoreceptor Tyrosine-basedActivation Motifs (2pY-ITAMs) present in the intracellular tail of the TCR-ζ chain. The crystal structure of the SH2 domains complexed with a 2pY-ITAM peptide suggests that the 60-amino acid-long inter-SH2 spacer helps the SH2 domains to interact with each other to create the binding site for the 2pY-ITAM. To investigate whether the inter-SH2 spacer has additional roles in the whole ZAP-70, we raised antibodies against two peptides of this region and probed ZAP-70 structure under various conditions. We show that the reactivity of antibodies directed at both sequences was dramatically augmented toward the tandem SH2 domains alone compared with that of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by sequences C-terminal to the SH2 domains, namely, the linker region and/or the kinase domain. Moreover, antibody binding to the same two determinants was also inhibited when ZAP-70 or the SH2 domains bound to the ζ chain or to a 2pY-ITAM. Together, these two observations suggest a model in which intramolecular contacts keep ZAP-70 in a closed configuration with the two SH2 domains near to each other. ZAP-70 is a protein tyrosine kinase (PTK) 1The abbreviations used are: PTK, protein tyrosine kinase; Ab, antibody; mAb, monoclonal antibody; TCR, T-cell antigen receptor; ITAM, immunoreceptor tyrosine-based activation motif; SH2, Src homology domain 2; IA, interdomain A; IB, interdomain B; 2pY-ITAM, di-phosphorylated ITAM; VSV, vesicular stomatitis virus. essential for the initiation of the signaling cascade activated by T cell antigen receptor triggering (1Qian D. Weiss A. Curr. Opin. Cell Biol. 1997; 8: 205-212Crossref Scopus (286) Google Scholar). Overall, ZAP-70 displays two structurally and functionally distinct moieties, an N-terminal one composed of two SH2 domains and a C-terminal kinase domain tethered by an ∼80-amino acid-long linker (2Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (889) Google Scholar) (also referred to as Interdomain B, (IB); Ref.3Hatada M.H. Lu X. Laird E.R. Green J. Morgenstern J.P. Lou M. Marr C.S. Phillips T.B. Ram M.K. Theriault K. Zoller M.J. Karas J.L. Nature. 1995; 377: 32-38Crossref PubMed Scopus (296) Google Scholar). So far, the only known function of the region comprising the two SH2 domains (hereafter indicated as (SH2)2) is to provide a means to recruit ZAP-70 to the plasma membrane by those TCRs engaged with the ligand (4Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar, 5Iwashima M. Irving B.A. van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). This is achieved through the coordinated anchorage of the SH2 domains to di-phosphorylated tyrosine-containing motifs (D/E)XXYXX(I/L)X6–8YXX(I/L) called ITAMs (for Immunoreceptor Tyrosine-basedActivation Motifs) present within the cytoplasmic tails of TCR subunits ζ and ε (6Wange R.L. Kong A.-N.T. Samelson L.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 267: 11685-11688Google Scholar, 7Straus D.B. Weiss A. J. Exp. Med. 1993; 178: 1523-1530Crossref PubMed Scopus (119) Google Scholar, 8Timson Gauen L.K. Zhu Y. Letourneur F. Hu Q. Bolen J. Matis L.A. Klausner R.D. Shaw A.S. Mol. Cell. Biol. 1994; 14: 3729-3741Crossref PubMed Scopus (129) Google Scholar). Thereafter, ZAP-70 undergoes tyrosine phosphorylation culminating in the up-regulation of its catalytic activity which in turn is required for phosphorylating cellular substrates (9Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 10Watts J.D. Affolter M. Krebs D.L. Wange R.L. Samelson L.E. Aebersold R. J. Biol. Chem. 1994; 269: 29520-29529Abstract Full Text PDF PubMed Google Scholar, 11Chan A.C. Dalton M. Johnson R. Kong G.-H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Crossref PubMed Scopus (325) Google Scholar, 12Mège D. Di Bartolo V. Germain V. Tuosto L. Michel F. Acuto O. J. Biol. Chem. 1996; 271: 32644-32652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The x-ray crystal structure of the (SH2)2 of human ZAP-70 complexed with a di-phosphorylated ITAM (2pY-ITAM) peptide (3Hatada M.H. Lu X. Laird E.R. Green J. Morgenstern J.P. Lou M. Marr C.S. Phillips T.B. Ram M.K. Theriault K. Zoller M.J. Karas J.L. Nature. 1995; 377: 32-38Crossref PubMed Scopus (296) Google Scholar) has revealed an unsuspected structural complementarity and immediate contiguity of the two SH2 domains needed to create a high affinity binding site for the 2pY-ITAM. Thus, while the C-terminal SH2 possesses a binding pocket for the first pY of the ITAM, the corresponding pocket for the second pY in the N-terminal SH2 is contributed, in part, by residues of the C-terminal SH2. Moreover, the 60 amino acids forming the inter-SH2 spacer (hereafter referred to as Interdomain A (IA)) (3Hatada M.H. Lu X. Laird E.R. Green J. Morgenstern J.P. Lou M. Marr C.S. Phillips T.B. Ram M.K. Theriault K. Zoller M.J. Karas J.L. Nature. 1995; 377: 32-38Crossref PubMed Scopus (296) Google Scholar) bulges out the SH2 domains and for the most part is structured as a coiled-coil of two antiparallel α-helices which assists in the formation of an interface between the two SH2 domains. It has been speculated that the IA may mediate additional intra- or inter-molecular interactions required for regulating ZAP-70 (3Hatada M.H. Lu X. Laird E.R. Green J. Morgenstern J.P. Lou M. Marr C.S. Phillips T.B. Ram M.K. Theriault K. Zoller M.J. Karas J.L. Nature. 1995; 377: 32-38Crossref PubMed Scopus (296) Google Scholar). This idea stems from several considerations. First, coiled-coils are often found to intervene in protein-protein interactions (13Cohen C. Parry D.A.D. Science. 1994; 263: 488-489Crossref PubMed Scopus (150) Google Scholar, 14Kohn W.D. Mant C.T. Hodges R.S. J. Biol. Chem. 1997; 272: 2583-2586Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Second, there is at least one highly suggestive example involving the spacer connecting the two SH2 domains of the p85 subunit of the phosphatidylinositol 3′-kinase (PI 3-kinase). This region, predicted to be a coiled-coil, mediates the interaction with the catalytic p110 subunit (15Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 1993: 5560-5566Crossref Scopus (87) Google Scholar, 16Dhand R. Hara K. Hiles I. Bax B. Gout I. Panayotou G. Fry M.L. Yonezawa K. Kasuga M. Waterfield M.D. EMBO J. 1994; 13: 511-521Crossref PubMed Google Scholar), and occupancy of the two SH2 domains influences the enzymatic activity (17Carpenter C.L. Auger K.R. Chanudhuri M. Yoakim M. Schaffhausen B. Shoelson S. Cantley L.C. J. Biol. Chem. 1993; 268: 9478-9483Abstract Full Text PDF PubMed Google Scholar,18Rordorf-Nikolic T. Van Horn D.J. Chen D. White M.F. Backer J.M. J. Biol. Chem. 1995; 270: 3662-3666Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Moreover, binding of singly or doubly phosphorylated peptides to the tandem SH2-containing SH-PTP-2 activates the phosphatase activity (19Puskey S. Wandless T.J. Walsh C.T. Shoelsen S.E. J. Biol. Chem. 1995; 270: 2897-2900Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 20Eck M.J. Atwell S.K. Shoelson S.E. Harrison S.C. Nature. 1994; 368: 764-769Crossref PubMed Scopus (240) Google Scholar). Finally, it has been reported that the catalytic activity of p72syk, a PTK homologue of ZAP-70, may be increased by binding to a 2pY-ITAM (21Rowley R.B. Burkhardt A.L. Chao H.-G. Matsueda G.R. Bolen J.B. J. Biol. Chem. 1995; 270: 11590-11594Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 22Shiue L. Zoller M.J. Brugge J.S. J. Biol. Chem. 1995; 270: 10498-10502Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). The latter examples are suggestive of allosteric regulation mediated by the SH2-containing region of the protein. To explore what could be the structural role of the IA in the entire ZAP-70, anti-peptide antibodies directed at this region were generated. The use of these antibodies revealed that binding of ZAP-70 or its isolated (SH2)2 to a 2pY-ITAM or the TCR-ζ chain influences the conformation of the IA. Additional experiments also indicated that conformational constraints are imposed on the IA by the regions of the protein downstream of the (SH2)2. These two observations, in combination, suggest a structural model for the entire ZAP-70. Anti-human ZAP-70 4.06 and 2.06 polyclonal antisera were produced by immunizing rabbits (a total of four, two for each peptide) with the synthetic peptides described in Table I coupled via their C- and N-terminal cysteines (cysteine 117 of ZAP-70), respectively, to maleimide-activated keyhole lympet hemocyanin as recommended (Pierce). A similar procedure was used to generate an anti-ζ antiserum (named zeta-N), which is directed at the first 11 amino acid residues of the human ζ chain. The anti-kinase domain polyclonal antiserum 21.11 has been previously described (23Duplay P. Thome M. Herve F. Acuto O. J. Exp. Med. 1994; 179: 1163-1172Crossref PubMed Scopus (156) Google Scholar). The ZAP-4 antibody (24Huby D.J. Carlile G.W. Ley S.C. J. Biol. Chem. 1995; 270: 30241-30244Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) was kindly provided by S. Ley (National Institute of Medical Research, Mill Hill, London. UK). The anti-phosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology, Inc., (Lake Placid, NY). The anti-human TCR Vβ8 (101.5.2) mAb was provided by E. L. Reinherz (Dana Farber Cancer Institute, Boston, MA). Anti-VSV-G epitope antiserum (kindly provided by M. Arpin, Institut Curie, Paris, France) reacts against an 11-amino acid determinant derived from a vesicular stomatitis virus glycoprotein (VSV-G) (25Kreis T.E. EMBO J. 1986; 5: 931-941Crossref PubMed Scopus (283) Google Scholar). WT15.8, a Jurkat cell line expressing a ZAP-70 containing a VSV-Tag at the C terminus (12Mège D. Di Bartolo V. Germain V. Tuosto L. Michel F. Acuto O. J. Biol. Chem. 1996; 271: 32644-32652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, l-glutamine, penicillin, and streptomycin (Life Technologies Inc., France). The generation of the cDNA construct that encodes the amino acids 1–276 of ZAP-70, comprising the SH2(N+C) plus 22 residues of the IB, and the expression vector used (pBJ1) have been previously described (26Qian B.D. Mollenauer M.N. Weiss A. J. Exp. Med. 1996; 183: 611-620Crossref PubMed Scopus (93) Google Scholar). The prokaryotic vector expressing the (SH2)2 of ZAP-70 as a glutathione S-transferase fusion protein (a gift from L. Samelson, National Institutes of Health, Bethesda, MD) and the purification procedure of the protein have been reported (4Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar). The (SH2)2 molecule was cleaved from the glutathioneS-transferase with factor Xa on a glutathione-Sepharose column during the purification as recommended (Amersham Pharmacia Biotech). Gel-filtration analysis revealed the absence of aggregated protein.Table IAnti-ZAP-70 antisera used in this studyNameImmunizing peptide1-2001Numbering of the human ZAP-70 sequence is according to Ref. 2.Protein region1-bThe limits of the ZAP-70 regions are according to Ref. 2.4.06106–117Interdomain A2.06117–130Interdomain AZAP-4271–290Interdomain B21.11483–499Kinase domain1-2001 Numbering of the human ZAP-70 sequence is according to Ref. 2Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (889) Google Scholar.1-b The limits of the ZAP-70 regions are according to Ref. 2Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (889) Google Scholar. Open table in a new tab Peptides corresponding to human ZAP-70 sequences 106–117 and 117–130 and to amino acids 1 through 11 of the human TCR ζ chain (an additional cysteine was added to the C terminus of this peptide for coupling to the carrier) purified by high performance liquid chromatography were purchased from Neosystems (Strasbourg, France). Peptides corresponding to the first ITAM of the human TCR ζ chain (ζ1, residues 48–66) plus a 4-amino acid linker at the N terminus (final sequence SGSGNQLYNELNLGRREEYDVLD) were synthesized as mono- (on Tyr62) and di-phosphorylated (on Tyr51 and Tyr62) forms (by F. Baleaux, Dept. of Organic Chemistry, Institut Pasteur, Paris). Peptides were purified by reverse-phase high performance liquid chromatography and in part biotinylated at the N terminus by Biotin sulfo-NHS (Pierce). The purity and the molecular weight of the peptides were confirmed by ion electro-spray ionization mass spectroscopy. For activation, cells were stimulated with anti-TCR mAb 101.5.2 at 1:200 dilution of ascites for 2 min at 37 °C. Unstimulated or TCR-activated cells were solubilized at 108 cells/ml for 15 min in ice-cold lysis buffer containing 1% Nonidet P-40, 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm MgCl2, and 1 mm EGTA in the presence of inhibitors of proteases and phosphatases (10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm Pefabloc-sc, 50 mm NaF, 10 mmNa4P2O7, and 1 mmNaVO4). Precleared postnuclear lysates were incubated with or without the synthetic ITAM peptides at the indicated concentration for 90 min at 4 °C. After incubation, lysates were subjected to immunoprecipitation for 1–2 h with 1 to 5 μl of intact polyclonal antisera preadsorbed to protein A-Sepharose as described (12Mège D. Di Bartolo V. Germain V. Tuosto L. Michel F. Acuto O. J. Biol. Chem. 1996; 271: 32644-32652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In some experiments streptavidin-agarose beads (Amersham Pharmacia Biotech) were utilized to precipitate ITAM-bound ZAP-70. Similar procedures were employed when purified recombinant ZAP-70 (SH2)2, typically ∼50 ng in 100 μl of lysis buffer containing 0.1 mg/ml of bovine serum albumin, were reacted with anti-IA antisera and ITAM peptides. After separation on SDS-polyacrylamide gel electrophoresis under reducing conditions and blotting, proteins were detected by enhanced chemiluminescence when the anti-phosphotyrosine antibody 4G10 was used or by 125I-labeled protein A (Amersham Pharmacia Biotech) in all the other experiments as described previously (12Mège D. Di Bartolo V. Germain V. Tuosto L. Michel F. Acuto O. J. Biol. Chem. 1996; 271: 32644-32652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Molecular weight markers, Mark12 MW standards were purchased from Novex. Quantitation of 125I-labeled proteins was performed using ImageQuant software after scanning in a PhosphorImager (Molecular Dynamics). Transfection of Jurkat cells was performed by electroporating (at 260 V, 960 microfarads) 107 Jurkat cells in 0.5 ml of RPMI 1640 medium supplemented with 20% fetal calf serum in a Gene Pulse cuvette (Bio-Rad) (12Mège D. Di Bartolo V. Germain V. Tuosto L. Michel F. Acuto O. J. Biol. Chem. 1996; 271: 32644-32652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) with various amounts (5–30 μg) of pBJ1 vector expressing the (SH2)2 domains of ZAP-70. Forty h later, transfected cells were either left unstimulated or stimulated with anti-TCR mAb for 2 min at 37 °C, lysed, and subjected to immunoprecipitation with the indicated antisera. Two antisera (named 4.06 and 2.06) were raised against synthetic peptides corresponding to the amino acid sequences 106–117 and 117–130 of ZAP-70, respectively (TableI). The peptide segment 106–117 begins shortly after the end of the N-terminal SH2 domain, and together, the two peptides cover ∼40% of the IA (2Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (889) Google Scholar, 3Hatada M.H. Lu X. Laird E.R. Green J. Morgenstern J.P. Lou M. Marr C.S. Phillips T.B. Ram M.K. Theriault K. Zoller M.J. Karas J.L. Nature. 1995; 377: 32-38Crossref PubMed Scopus (296) Google Scholar). To investigate whether the two antisera recognized native ZAP-70 and to compare their reactivity with that of other anti-ZAP-70 antisera directed at different regions of the molecule, immunoprecipitation experiments were carried out from lysates of Jurkat cells unstimulated or stimulated with an anti-TCR mAb (Fig. 1). Similarly to 21.11 and ZAP-4 anti-peptide antisera, specific for sequences contained within the kinase domain and the IB, respectively (Table I), 4.06 and 2.06 immunoprecipitated tyrosine-phosphorylated ZAP-70 (Fig. 1,pY-ZAP-70 in lanes 1–8). In this as well as in other experiments, 2.06 was found to be weaker than 4.06 (see also below). However, in striking contrast with the antisera 21.11 and ZAP-4, in TCR-activated Jurkat cells, 4.06 and 2.06 did not show co-immunoprecipitation of phosphorylated ζ chain (cf. pY-ζ in lanes 2 and 4 with lanes 6 and 8). The ζ chain was positively identified by an anti-ζ antiserum (zeta-N, lane 9) which, as expected, in activated Jurkat cells co-immunoprecipitated with ZAP-70. The lack of detection of the ζ chain with both anti-IA antisera was not due to a lower capacity to immunoprecipitate ZAP-70. Indeed, with the anti-IA antisera, ζ was not visible even when the signal of ZAP-70 was similar to that obtained with 21.11 and ZAP-4 (cf. lane 2 with lane 6 or lane 4 with lane 5). In addition, the ζ chain remained undetectable after longer exposure times (not shown). Phosphorylated ZAP-70, not associated to the ζ chain but observable with anti-IA antisera is likely to represent a fraction of the molecules which detached from ζ spontaneously or as a consequence of anti-IA Ab binding. These results suggested that the epitopes recognized by the anti-IA antisera are masked or structurally modified when ZAP-70 is complexed with the ζ chain. The lack of recognition of ZAP-70·ζ complexes by anti-IA Abs could be due to an alteration of the corresponding epitopes consequent to the binding of the SH2 domains to the ITAMs. To directly test this hypothesis, we assessed whether a synthetic peptide corresponding to a 2pY-ITAM inhibited recognition of ZAP-70 by anti-IA Abs. For these experiments, we used a Jurkat cell line named WT15.8, stably expressing ZAP-70 tagged at its C terminus with a VSV sequence (12Mège D. Di Bartolo V. Germain V. Tuosto L. Michel F. Acuto O. J. Biol. Chem. 1996; 271: 32644-32652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The use of tagged ZAP-70 was preferred since the anti-tag antiserum was found to be the strongest, thus allowing sensitive detection of ZAP-70. Cell lysates from unstimulated WT15.8 were incubated with increasing concentrations of mono- (pY) or di- (2pY) phosphorylated ITAM peptides containing a biotin molecule at the N terminus and then reacted with steptavidin-agarose or with the anti-ZAP-70 antisera. In agreement with previous reports (5Iwashima M. Irving B.A. van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar), only the 2pY-ITAM was able to bind ZAP-70, as shown by precipitation with streptavidin-agarose (Fig. 2, lanes 12 and 13). Moreover, the 2pY-ITAM, but not the pY-ITAM, inhibited the immunoprecipitation of ZAP-70 with the 4.07 antibody in a dose-dependent manner (Fig. 2, cf. lanes 4, 5 and 6 with lanes 1, 2, and 3). Quantitation of the ZAP-70 band allowed calculation to an ∼90% inhibition of ZAP-70 immunoprecipitation in the presence of 10 μm of 2pY-ITAM. Similar levels of inhibition were obtained when untagged ZAP-70 was immunoprecipitated from Jurkat cells or when non-biotinylated 2pY-ITAM was used. Moreover, similar results were reproduced with 2.06 antiserum (data not shown). This effect was restricted to the anti-IA antibodies since no inhibition was detected with the anti-kinase domain 21.11 (lanes 8 and 9) or with anti-Tag antisera (lanes 10 and 11). From these results, we conclude that the binding of the 2pY-ITAM to ZAP-70 is the event that determines the loss of immunoreactivity. One possible explanation for the above results is that the ITAM itself may sterically hinder the epitopes and prevent antibody binding. However, the three-dimensional structure of the (SH2)2·2pY-ITAM complex (3Hatada M.H. Lu X. Laird E.R. Green J. Morgenstern J.P. Lou M. Marr C.S. Phillips T.B. Ram M.K. Theriault K. Zoller M.J. Karas J.L. Nature. 1995; 377: 32-38Crossref PubMed Scopus (296) Google Scholar) shows that both amino acid segments lie on the opposite side of the (SH2)2·2pY-ITAM interface (see also “Discussion”). Moreover, it is difficult to imagine how the two sequences which are part of an extended structure may both be sterically hindered by such a short peptide. Thus, it is extremely unlikely that this mechanism can account for the observed changes in anti-IA Ab reactivity. Another possibility could be that upon ITAM binding, either the kinase domain or the IB or both interact with the IA and induce a conformational change or a masking effect resulting in loss of anti-IA Ab immunoreactivity. Alternatively, this effect may be simply due to a conformational change of the IA induced by 2pY-ITAM binding independently of the rest of the molecule. To discriminate between these two possibilities, experiments similar to those presented in Figs. 1 and 2 were performed using the (SH2)2 moiety of ZAP-70. To this end, Jurkat cells were transiently transfected with a vector expressing the tandem SH2 domains of ZAP-70 (26Qian B.D. Mollenauer M.N. Weiss A. J. Exp. Med. 1996; 183: 611-620Crossref PubMed Scopus (93) Google Scholar) and subjected to immunoprecipitation with the 4.06 antiserum after TCR-mediated activation or after addition of the 2pY-peptide. Fig. 3 A(top panel) shows that, in cells transfected with (SH2)2 activated with anti-TCR mAb, the TCR-ζ chain (with associated ZAP-70) was detected as a series of strongly phosphorylated bands when immunoprecipitated with the anti-ζ antiserum but remained undetected when using the 4.06 antiserum. However, the (SH2)2 was efficiently immunoprecipitated from the same cells with the 4.06, as shown by stripping and reprobing the blot with this antiserum (Fig. 3 A, bottom panel, lanes 1 and 2). Moreover, as previously demonstrated (26Qian B.D. Mollenauer M.N. Weiss A. J. Exp. Med. 1996; 183: 611-620Crossref PubMed Scopus (93) Google Scholar), the (SH2)2 molecule could be detected in part associated with the TCR-ζ chain. Indeed, in the anti-ζ immunoprecipitation, a band corresponding to a small fraction of the transfected (SH2)2was visible and increased in intensity after activation (Fig. 3 A, bottom panel, lanes 3 and 4). Of note is that in these experiments no tyrosine phosphorylation of the (SH2)2 was detected (the corresponding band migrates as an ∼33-kDa molecular species) even after TCR stimulation. Thus, like intact ZAP-70, the (SH2)2·ζ complex also could not be detected by antibodies directed at the 106–117 IA sequence. This was also verified in an in vitro assay since the 2pY-ITAM, but not the pY-ITAM, could almost completely inhibit the immunoprecipitation of the transfected (SH2)2 with the 4.06 antibody (Fig. 3 B). Similar results were obtained when the (SH2)2 was immunoprecipitated with the 2.06 antisera after incubation with the 2pY-ITAM peptide (data not shown). These experiments indicated that the IB and/or the kinase domain were not implicated in the ITAM-induced inhibition of anti-IA antibodies recognition. However, one could not rule out the possibility that in Jurkat cells a putative protein associated with the IA region upon ITAM binding and produced the inhibitory effect by steric hindrance. Fig. 3 C shows that this explanation is unlikely since addition of 2pY-ITAM, but not pY-ITAM (lanes 3 and 2, respectively), to purified bacterially expressed (SH2)2 of ZAP-70, was sufficient to produce a marked reduction of recognition by 4.06 antibodies. Note that the recombinant protein was able to bind the 2pY-ITAM (lane 5). Together, these data strongly suggest that the loss of anti-IA epitope recognition is essentially due to a conformational change transmitted to the IA region by binding of the ZAP-70 (SH2)2 to a 2pY-ITAM. During the course of the experiments involving the expression of isolated (SH2)2 of ZAP-70 in Jurkat cells, we consistently noted that this molecule was immunoprecipitated by the anti-IA Abs more efficiently than the whole ZAP-70. Experiments were therefore set up to determine the magnitude of this effect and to exclude possible artifacts due to overexpression of the (SH2)2. Thus, Jurkat cells were transiently transfected with different amounts of the expression plasmid containing the (SH2)2 to obtain different (SH2)2/endogenous ZAP-70 ratios. Immunoprecipitations were then carried out with the 4.06 and 2.06 antisera, and the bands corresponding to ZAP-70 and (SH2)2 were quantitated by immunoblotting using 4.06. Before the immunoprecipitation step, an aliquot of the total lysate was used to estimate the relative expression of both molecules. In the experiment shown in Fig. 4 A, endogenous ZAP-70 was expressed at approximately a 10-fold excess compared with the transfected (SH2)2 (see lane 1 and, for quantitation, Experiment I in TableII). If the reactivity of the antisera against the two proteins was the same, then the (SH2)2/ZAP-70 ratio should remain constant in the immunoprecipitate. However, after immunoprecipitation with both 4.06 and 2.06 antisera, a relative increase in the signal of the transfected (SH2)2 over ZAP-70 was clearly evident (Fig. 4 A,lane 2 and 3). Thus, while the amount of (SH2)2 was 10-fold lower than ZAP-70, after immunoprecipitation with 4.06, the signal obtained for the two proteins was nearly the same. This higher reactivity toward the (SH2)2 was even more dramatic for the 2.06 antiserum. This reagent immunoprecipitated ZAP-70 inefficiently compared with 4.06 (cf. lanes 2 and 3) but immunoprecipitated the (SH2)2 as efficiently as 4.06. The magnitude of these modifications in reactivity toward (SH2)2 compared with ZAP-70 can be quantitatively appreciated by confronting the (SH2)2/ZAP-70 signal ratios in the cell lysate and in the immunoprecipitates of 4.06 and 2.06. These ratios are reported in Table II for the experiment shown in Fig. 4 A (Experiment I) and for two additional ones in which higher amounts of (SH2)2 compared with ZAP-70 were expressed. Independently of the initial amounts of (SH2)2and ZAP-70 present, there is an increase of the (SH2)2/ZAP-70 ratio in the immunoprecipitates. It is clear that on average for the epitope recognized by 4.06 Abs there is a gain of reactivity of ∼5-fold, whereas such a change can be estimated to be >100-fold for 2.06 (with this antiserum only, <1% of the total intact ZAP-70 is detected after a single immunoprecipitation). Fig. 4 B also shows, from one of these experiments, a control of the structural intactness of the (SH2)2 versusendogenous ZAP-70. Both proteins were able to bind with similar capacity to the 2pY-ITAM peptide as demonstrated by the fact that their ratio after binding is comparable with the one seen in the total lysate (lane 1). This result excludes that the anti-IA antisera recognize a grossly altered population of (SH2)2 molecules unable to bind to 2pY-ITAM.Table II(SH2)2/ZAP-70 ratios in cell lysates and in anti-IA antisera immunoprecipitatesLysateIP 4.06IP 2.06Experiment I0.10.713.4Experiment II3.012.62-2001Average of three independent immunoprecipitations.ND2-2003ND, not done.Experiment III7.63417292-bAverage of two independent immunoprecipitations.2-2001 Average of three independent immunoprecipitations.2-b Average of two independent immunoprecipitations.2-2003 ND, not done. Open table in a new tab From these experiments, we conclude that the epitopes recognized by both anti-IA are conformationally dependent on (or hindered by?) the C-terminal moiety of the molecule, including the IB and the kinase domain. The most direct interpre