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Dominant-Negative Zeta-Associated Protein 70 Inhibits T Cell Antigen Receptor Signaling by Dapeng Qian, Marianne N. Mollenauer, and Arthurweiss

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Dominant-Negative Zeta-Associated Protein 70 Inhibits T Cell Antigen Receptor Signaling by Dapeng Qian, Marianne N. Mollenauer, and Arthurweiss Dominant-negative Zeta-associated Protein 70 Inhibits T Cell Antigen Receptor Signaling By Dapeng Qian, Marianne N. Mollenauer, and ArthurWeiss From the Departments of Microbiotogy and Immunology, and of Medicine, and the Howard Hughes Medical Institute, University of California, San Frandsco, California 94143 Sunlrnary Zeta-associated protein (ZAP)-70 is a cytoplasmic protein tyrosine kinase required for T cell antigen receptor (TCR) signaling and development. Mutations in ZAP-70 result in severe combined immunodeficiency in humans. ZAP-70 interacts with the TCR by binding to ty- rosine-phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) present in the invariant subunits of the TCR complex. Here we report that two ZAP-70 mutants devoid of kinase activity, generated either by a point mutation in the kinase domain to create an inac- tive kinase, or by truncation of the entire kinase domain (SH2[N+C]), functioned as domi- nant-negative mutants to specifically suppress TCR-mediated activation of NFAT, a nuclear factor essential for inducible interleukin 2 gene expression. Biochemical studies with the SH2(N+C) mutant showed that it also blocked early TCR signaling events, such as p95 vav ty- rosine phosphorylation, extracellular signal-regulated kinase 2 activation, and the association of a number of tyrosine-phosphorylated proteins with growth factor receptor-binding protein 2 (GRB2). The inhibitory effects of the SH2(N+C) mutant revealed that it requires an intact phosphotyrosine-binding site in its COOH-terminal SH2 domain. Using a CD8-~ chimeric receptor to analyze the interaction of the SH2(N+C) mutant with ITANIs of TCR-~, we found that this mutant was constitutively bound to the hyperphosphorylated CD8-~ chimera. These results indicate that tyrosine-phosphorylated ITAM is the target for the action of this dominant-negative mutant, suggesting that the assembly of a functional receptor signaling complex on ITAMs is a critical proximal TCR signaling event leading to downstream activation. he TCR is a multimolecular complex consisting of genetic studies have shown that the TCR initiates signal T clonotypic antigen-binding subunits (or and 13 in most transduction by interacting with and activating at least T cells) and invariant signal-transducing subunits (% B, and three cytoplasmic PTKs, Lck, Fyn, and ZAP-70 (1-3). Lck e chains of the CD3 complex, and ~ family proteins) (1). and Fyn, two members of the Src family PTK, have been Engagement of the TCR by antigen bound to MHC mol- suggested to initially tyrosine phosphorylate the immu- ecules or by anti-TCR mAbs triggers a signal transduction noreceptor tyrosine-based activation motifs (ITAMs) con- cascade culminating in the induction of a variety of T cell tained within the cytoplasmic domains of the invariant functions (1). One of the earliest detectable signaling events TCR subunits. This phosphorylation leads to the recruit- after TCR stimulation is the tyrosine phosphorylation of a ment of the Syk family PTK, ZAP-70, to the TCR (4-6). number of protein substrates, including the invariant sub- The association of ZAP-70 with the TCR is mediated by a units of the TCR complex, phospholipase C (PLC)I~/1, high affinity interaction between the two tandemly ar- p95 ray, zeta-associated protein (ZAP)-70, Shc, and valosin- ranged SH2 domains of the ZAP-70 molecule and the two containing protein (VCP) (1). However, unlike many phosphorylated tyrosines in the ITAM (5, 7-9). Both SH2 growth factor receptors, the TCR lacks intrinsic protein domains of ZAP-70 are required for this association. Ty- tyrosine kinase (PTK) activity. Numerous biochemical and rosine phosphorylation of ZAP-70 itself also takes place af- ter its binding to the TCR, a process dependent on the Src family PTKs (5, 8, 10). The functional significance of 1Abbreviations used in this paper: ERK2, extraceUularsignal-regulated kinase ZAP-70 tyrosine phosphorylation is still not clear, but may 2; GRB2, growth factorreceptor-binding protein 2; HM1, human mus- relate to its catalytic activation and interaction with other carinic subtype 1 receptor; ITAM, immunoreceptortyrosine-based acti- vation motifi KI, inactive kinase; NFAT, nuclear factor of activated T proteins (11, 12). The combined activation of both Src and cells; PLC, phospholipase C; PTK, protein tyrosinekinase; TAg, large T Syk family PTKs results in tyrosine phosphorylation of cel- antigen; ZAP-70, zeta-associatedprotein 70. lular substrates, which in turn contributes to the activation 611 J. Exp. Med. 9The Rockefeller University Press 90022-1007/96/02/611/10 $2.00 Volume 183 February 1996 611--620 of several signaling pathways, including phospholipid me- subcloned into pEF-BOS, was kindly provided by D. Motto and tabolism, elevation of cytoplasmic free calcium, P, as-GTP Dr. G. Koretzky (University of Iowa, Iowa City, IA). All expres- accumulation, and an extracellular signal-regulated kinase sion plasmids contain an SV40 origin of replication to allow high cascade involving several cytoplasmic serine/threonine ki- level gene expression in TAgJurkat cells (25). nases (1-3). These signaling events ultimately lead to T cell Antibodies. The following mAbs (and their specificity) were used: C305 (mouse anti-Jurkat Ti [3 chain) (32), 9E10 (mouse differentiation, proliferation, and induction of effector func- anti-Myc epitope) (kindly provided by J. M. Bishop, University tions. The requisite role of ZAP-70 in TCR signaling has of California, San Francisco) (33), 4G10 (mouse antiphosphoty- been established by recent studies (13-16) on human pa- rosine) (purchased from Upstate Biotechnology, Inc., Lake tients with an immunodeficiency disease caused by loss-of- Placid, NY), OKT8 (mouse anti-human CD8) (obtained from function mutations of ZAP-70. CD4 + T cells from these American Type Culture Collection, R.ockville, MD), 2F3.2 patients are defective in both early and late signaling through (mouse anti-human ZAP-70) (8), 6Bl0.2 (mouse anti-TCR~ the TCR. Moreover, these patients do not have CD8 + T chain) (34), and M2 (mouse anti-FLAG epitope) (obtained from cells because of a developmental arrest. These findings pro- Eastman Kodak Co., New Haven, CT). The rabbit polyclonal vide strong genetic evidence that ZAP-70 is a critical mol- anti-GRB2 serum was purchased from Santa Cruz Biotechnol- ecule involved in T cell signahng and development. ogy, Inc. (Santa Cruz, CA). Transfections, Stimulations, Lucferase Assay, and Solubilization.fi~r Although null mutations in patients or animals can pro- Biochemical Analysis. Cells (107) were transiently transfected by vide insights into the importance of molecules involved in electroporation using the Gene Pulser (Bio-Rad Laboratories, signal transduction, the resultant phenotype is a conse- Hercules, CA) as previously described (25). 40 h later, transfected quence of a complex developmental program and adaptive cells were aliquoted into a U-bottom 96-well plate (10S/well) mechanisms so that the precise function of the affected and cultured with various stinmli (1:500 dilution ofC305 ascites, molecules is often obscured. The use of dominant-negative 500 IxM carbachol, or 50 ng/ml PMA plus 1.0 txM ionomycin) mutations in signaling molecules is an alternatitive ap- in P, PMI 1640 medium supplemented with 10% FCS in a final proach to investigate signal transduction pathways. In fact, volume of 90 Ixl. After a 6-h stinmlation period at 37~ cells dominant-negative mutants of Src family PTKs (Lck and were lysed in a buffer containing 1% Triton X-100, 100 mM Fyn), Ser/Thr kinases (MAP kinase or ERK kinase [MEK] KPO4, pH 7.8, and 1.0 mM dithiothreitol. Lysates (0.1 ml) were mixed with 0.1 ml assay buffer (200 mM KPO 4, pH 7.8, 10 mM and Rat'), and the small GTP-binding protein t<as, have all ATP, 20 mM MgC12) and 0.1 ml 1.0 mM luciferin; luciferase ac- been successfully used to dissect T cell signaling pathways tivity was measured in the MONOLIGHT 2001 Luminometer in T cell lines or in transgenic mice (17-24). In this report, (Analytical Luminescence Laboratory, Inc., San Diego, CA). For we show that two ZAP-70 mutants can specifically sup- biochemical analysis, transfected cells were resuspended in PBS, press TCR-mediated early and late sigalal transduction equilibrated at 37~ for 15 min, stinmlated at 37~ with a 1:50(I leading to nuclear factor of activated T cells (NFAT) acti- dilution ofC305 ascites, 50 ng/ml PMA, or a 1:1,000 dilution of vation inJurkat T cells. OKT8 ascites for 2 min, and lysed immediately in a buffer con- taining 1% NP-40, 10 mM Tris, pH 7.6, 15(1 mM NaCI, 2 mM EDTA, protease, and phosphatase inhibitors, as previously de- Materials and Methods scribed (35). Cells. Large T antigen (TAg) Jurkat cells, described previ- Immunoprecipitation and Immunoblot Analysis. Inmmnoprecipi- ously (25), are human leukemia Jurkat T cells stably transfected tation was carried out as described earlier (36). Immunoblot anal- with SV40 TAg (kindly provided by Dr. G. Crabtree, Stanford ysis using the commercial enhanced chelmluminescence (ECI.) University, Palo Alto, CA). J.HM1.2.2 cells have been described (Amersham Corp., Arlington Heights, I1) detection kit was per- previously (26). Cells were maintained as described (25, 26). formed as previously described (37). Stripping and reprobing Plasmids. The NFAT-luciferase reporter construct, in which blots was carried out according to the ECL kit instruction pro- the expression of luciferase is driven by multiple copies of NFAT vided by Amersham Corp. P, eprobed blots were developed using DNA binding element, was a gift from Dr. G. Crabtree. The 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium col- gys 369 to Ala point mutant (inactive kinase [K1]) of ZAP-70, orimetric development reagents as described (36). described previously (8), was subcloned into pBJl, a mamma- lian expression vector containing the SRo~ promoter (27). The SH2(N+C) mutant was generated by digesting the wild-type ZAP-70 cDNA with HindlI at nucleotide position 1039 and Results then adding a stop codon next to this HindlI site.
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