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F2 (Pmp)2 -TAM ζ3 , a Novel Competitive Inhibitor of the Binding of ZAP-70 to the T Cell Antigen Receptor, Blocks Early T Cell Signaling

1995; Elsevier BV; Volume: 270; Issue: 2 Linguagem: Inglês

10.1074/jbc.270.2.944

ISSN

1083-351X

Autores

Ronald L. Wange, Noah Isakov, Terrence R. Burke, Akira Otaka, Peter P. Roller, Julian D. Watts, Ruedi Aebersold, Lawrence E. Samelson,

Tópico(s)

Protein Tyrosine Phosphatases

Resumo

Signaling by the T cell antigen receptor (TCR) is mediated by 17-residue tyrosine-based activation motifs (TAM) present in the cytoplasmic tails of the TCR ζ and CD3 chains. TAMs become tyrosine-phosphorylated upon TCR stimulation, creating a high affinity binding site for the tandem SH2 domains of ZAP-70. In permeabilized T cells, the association of TCR and ZAP-70 was inhibited by a protein tyrosine phosphatase (PTPase)-resistant TAM peptide analog, in which difluorophosphonomethyl phenylalanyl (F2Pmp) residues replaced phosphotyrosine. Inhibition of this association prevented TCR-stimulated tyrosine phosphorylation of ZAP-70 and reduced ZAP-70 kinase activity to basal levels. The reduction in ZAP-70 activity coincided with reduced tyrosine phosphorylation of a number of substrates. Such PTPase-resistant peptides, capable of disrupting SH2 domain-mediated protein-protein interactions, should prove useful in further dissection of multiple signaling pathways and may serve as models for rationally designed chemotherapeutic agents for the treatment of autoimmune and neoplastic disorders. Signaling by the T cell antigen receptor (TCR) is mediated by 17-residue tyrosine-based activation motifs (TAM) present in the cytoplasmic tails of the TCR ζ and CD3 chains. TAMs become tyrosine-phosphorylated upon TCR stimulation, creating a high affinity binding site for the tandem SH2 domains of ZAP-70. In permeabilized T cells, the association of TCR and ZAP-70 was inhibited by a protein tyrosine phosphatase (PTPase)-resistant TAM peptide analog, in which difluorophosphonomethyl phenylalanyl (F2Pmp) residues replaced phosphotyrosine. Inhibition of this association prevented TCR-stimulated tyrosine phosphorylation of ZAP-70 and reduced ZAP-70 kinase activity to basal levels. The reduction in ZAP-70 activity coincided with reduced tyrosine phosphorylation of a number of substrates. Such PTPase-resistant peptides, capable of disrupting SH2 domain-mediated protein-protein interactions, should prove useful in further dissection of multiple signaling pathways and may serve as models for rationally designed chemotherapeutic agents for the treatment of autoimmune and neoplastic disorders. INTRODUCTIONThe T cell-mediated immune response depends upon the ability of the T cell antigen receptor (TCR) 1The abbreviations used are: TCRT cell antigen receptorTAMtyrosine-based activation motifPTPaseprotein tyrosine phosphataseF2Pmpdifluorophosphonomethyl phenylalanineSH2Src-homology 2pTyrphosphotyrosinemAbmonoclonal antibodycfb3cytosolic fragment of erythrocyte band 3. to recognize specific antigen and respond by initiating an intracellular signaling cascade. The α and β chains of the multisubunit TCR determine the antigen specificity of the TCR, while the γ , δ , and ε CD3 chains and the TCR ζ chain are coupled to intracellular signaling molecules. A number of studies have demonstrated the importance of a short amino acid sequence, present as three copies in the cytoplasmic domain of the TCR ζ chain and as a single copy in each of the CD3 chains, for initiating T cell signaling(1Letourneur F. Klausner R.D. Science. 1992; 255: 79-82Crossref PubMed Scopus (342) Google Scholar, 2Romeo C. Amiot M. Seed B. Cell. 1992; 68: 889-897Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 3Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (251) Google Scholar). This sequence has many descriptive names including the tyrosine-based activation motif (TAM) and has the general structure YXX(L/I)X (6-8) YXX(L/I) (4Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1154) Google Scholar). In chimeric receptor cross-linking studies, TAMs have been shown to be necessary and sufficient for initiation of T cell activation (1Letourneur F. Klausner R.D. Science. 1992; 255: 79-82Crossref PubMed Scopus (342) Google Scholar, 2Romeo C. Amiot M. Seed B. Cell. 1992; 68: 889-897Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 3Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (251) Google Scholar).The earliest biochemical event detected upon TCR stimulation is increased tyrosine phosphorylation of a number of cellular substrates (5June C.H. Fletcher M.C. Ledbetter J.A. Samelson L.E. J. Immunol. 1990; 144: 1591-1599PubMed Google Scholar), including the TCR chain TAMs(6Baniyash M. Garcia-Morales P. Luong E. Samelson L.E. Klausner R.D. J. Biol. Chem. 1988; 263: 18225-18230Abstract Full Text PDF PubMed Google Scholar, 7Qian D. Griswold-Prenner I. Rosner M.R. Fitch F.W. J. Biol. Chem. 1993; 268: 4488-4493Abstract Full Text PDF PubMed Google Scholar, 8Sancho J. Franco R. Chatila T. Hall C. Terhorst C. Eur. J. Immunol. 1993; 23: 1636-1642Crossref PubMed Scopus (31) Google Scholar), ZAP-70(9Chan A.C. Irving B.A. Fraser J.D. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9166-9170Crossref PubMed Scopus (334) Google Scholar), Vav(10Bustelo X.R. Ledbetter J.A. Barbacid M. Nature. 1992; 356: 68-71Crossref PubMed Scopus (240) Google Scholar, 11Margolis B. Hu P. Katzav S. Li W. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (302) Google Scholar), Shc(12Ravichandran K.S. Lee K.K. Songyang Z. Cantley L.C. Burn P. Burakoff S.J. Science. 1993; 262: 902-905Crossref PubMed Scopus (293) Google Scholar), phospholipase C γ 1(13Park D.J. Rho H.W. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5453-5466Crossref PubMed Scopus (207) Google Scholar, 14Weiss A. Koretzky G. Schatzman R.C. Kadlecek T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5484-5488Crossref PubMed Scopus (275) Google Scholar), and VCP (15Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (121) Google Scholar). Many other substrates have yet to be identified. Three protein tyrosine kinases that have been implicated as being involved in this early phase of TCR-mediated signaling are the Src-family members Fyn and Lck and the Syk-family member ZAP-70. The evidence for the involvement of these protein tyrosine kinases in TCR signaling has been extensively reviewed (16Samelson L.E. Klausner R.D. J. Biol. Chem. 1992; 267: 24913-24916Abstract Full Text PDF PubMed Google Scholar, 17Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1943) Google Scholar, 18Isakov N. Wange R.L. Samelson L.E. J. Leukocyte Biol. 1994; 55: 265-271Crossref PubMed Scopus (49) Google Scholar).Recently it was shown that Lck and ZAP-70 can act sequentially to tyrosine phosphorylate various substrates in COS cells transfected with Lck, ZAP-70, and a CD8 ζ chimeric protein (19Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). In this system, Lck is required to tyrosine phosphorylate the TAMs of CD8 ζ which permits ZAP-70 binding. Upon association with CD8 ζ , ZAP-70 is tyrosine-phosphorylated, becomes activated, and induces tyrosine phosphorylation of substrates. One would predict from such a model that agents that block the association of ZAP-70 with the TCR could prove useful in identifying the downstream effectors of ZAP-70 and in blocking T cell activation. Recent insights into the mechanism of the association of ZAP-70 with the TCR provide the basis for the design of such inhibitory agents.Src-homology 2 (SH2) domains mediate protein-protein interactions by binding to pTyr-containing sequences(20Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Rotnosfsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar, 21Pawson T. Schlessinger J. Curr. Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (574) Google Scholar). The presence of two SH2 domains in ZAP-70 and 2 pTyr residues in stimulated TCR TAMs suggested a possible mechanism of interaction. Experiments using glutathione S-transferase fusion proteins containing the SH2 domains of ZAP-70 show that ZAP-70 binds to the tyrosine-phosphorylated TCR ζ and CD3 ε chains via its SH2 domains; both SH2 domains are required for high affinity binding(22Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar). That the TCR TAM motifs mediate this interaction was demonstrated in studies showing binding of ZAP-70 to transmembrane chimeric receptors with a single ζ chain TAM as the cytoplasmic tail(3Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (251) Google Scholar). In this study, we report that a novel phosphatase-resistant synthetic peptide based on the C-terminal (pTyr)2-TAM of the TCR ζ chain competitively blocks ZAP-70 binding to activated TCR. As a consequence, TCR stimulation fails to induce tyrosine phosphorylation of ZAP-70 or activation of its kinase activity. Coincident with this loss of ZAP-70 kinase activity is reduced tyrosine phosphorylation of a number of substrates. This result suggests that the peptide blocks early TCR-mediated signaling events through a ZAP-70 dependent mechanism, as would be predicted by the model of early activation events mentioned above.EXPERIMENTAL PROCEDURESCells, Antibodies, and Miscellaneous ReagentsJurkat T cells were cultured as described previously(23Wange R.L. Kong A.N. Samelson L.E. J. Biol. Chem. 1992; 267: 11685-11688Abstract Full Text PDF PubMed Google Scholar). The C305 anti-Jurkat β chain mAb, the OKT3 anti-CD3 ε mAb, and the 4G10 anti-pTyr mAb have been described previously(22Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar). The polyclonal antisera to ZAP-70 was produced in rabbits against a glutathione S-transferase fusion protein containing amino acids 255-345 of ZAP-70 and was a gift from J. Bolen (Bristol-Myers Squibb). This antiserum is specific for ZAP-70 and does not recognize Syk. 2R. L. Wange, unpublished observation. Tetanolysin was a gift of E. Bonvini (FDA).Fusion Proteins and Synthetic PeptidesPreparation of glutathione S-transferase-ZAP-70(SH2)2 has been described(22Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar). The cold (pTyr)2-TAM ζ3 peptide has the sequence LpYQGLSTATKDTpYDALH and was purchased from M. Berne (Tufts University, Boston, MA). F2Pmp was synthesized as described previously(24Burke Jr., T.R Smyth M.S. Otaka A. Roller P.P. Tetrahedron Lett. 1993; 34: 4125-4128Crossref Scopus (86) Google Scholar, 25Smyth M.S. Burke Jr., T.R. Tetrahedron Lett. 1994; 35: 551-553Crossref Scopus (93) Google Scholar). The synthesis of the F2Pmp-containing peptides was accomplished essentially according to methodologies previously described(26Otaka A. Burke Jr., T.R. Smyth M.S. Nomizu M. Roller P.P. Tetrahedron Lett. 1993; 34: 7039-7042Crossref Scopus (33) Google Scholar). The TAM ζ peptide, HDGLYQGLSTATKDTYDALHM, was purchased from Peptide Technologies Corp. (Gaithersburg, MD). This peptide was tyrosine-phosphorylated with [ γ - 32 P]ATP in vitro using purified recombinant Lck, as described(27Watts J.D. Wilson G.M. Ettenhadieh E. Clark-Lewis I. Kubanek C.A. Astell C.R. Marth J.D. Aebersold R. J. Biol. Chem. 1992; 267: 901-907Abstract Full Text PDF PubMed Google Scholar). Peptides in which both tyrosine residues were phosphorylated were purified by reverse phase high performance liquid chromatography, and their purity was confirmed by mass spectroscopy.TCR Stimulation, Immunoprecipitation, and ImmunoblottingTCR stimulation was for 2 min at 37°C with either the anti-β chain mAb C305 (1:50 dilution of culture supernatant) or the anti-CD3 mAb OKT3 (1:100 dilution of purified mAb). Both mAbs give equivalent stimulation(22Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar). In some experiments, Na3VO4 was added to the permeabilized cells to a final concentration of 1 mM and followed 10 s later by addition of stimulatory C305 mAb. Stimulation was terminated in a Brij 96 lysis buffer (1% Brij 96, 25 mM Tris (pH 7.6), 150 mM NaCl, 1 mM Na3VO4, 5 mM EDTA, 10 μg/ml aprotinin and leupeptin, and 25 μMp-nitrophenyl p′-quanidinobenzoate). Brij 96 maintains association of CD3 and TCR ζ chains(28Beyers A.D. Spruyt L.L. Williams A.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2945-2949Crossref PubMed Scopus (205) Google Scholar). Methods for immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and immunoblotting have been described(22Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar). Immunoblots were developed by enhanced chemiluminescence (Amersham).Tetanolysin Permeabilization and Peptide IncubationThe method for tetanolysin permeabilization of T lymphocytes has been published previously(29Conti A. Brando C. DeBell K.E. Alava M.A. Hoffman T. Bonvini E. J. Biol. Chem. 1993; 268: 783-791Abstract Full Text PDF PubMed Google Scholar, 30Sarosi G.A. Thomas P.M. Egerton M. Phillips A.F. Kim K.W. Bonvini E. Samelson L.E. Int. Immunol. 1992; 4: 1211-1217Crossref PubMed Scopus (40) Google Scholar). 95% of cells were permeable by trypan blue uptake. Peptide (4 μM) was added to the cells on ice, just prior to transfer to 37°C (initiation of permeabilization). Permeabilization/peptide incubation was for 10 min at 37°C and was followed by TCR stimulation and/or lysis.Immune Complex Kinase AssayPrior to the kinase reaction, immunoprecipitates were washed twice with lysis buffer. The kinase reaction was carried out at 25°C for 5 min on the beads in 20 mM Tris (pH 7.6), 10 mM MnCl2, 1 μM ATP, 0.5 μg of cfb3, 10 μCi of [ γ - 32 P]ATP. The reaction was terminated by addition of one-third volume of 95°C 4×-reducing Laemmli sample buffer and incubation for 5 min at 95°C.RESULTS AND DISCUSSIONBecause of the presence of active protein tyrosine phosphatases (PTPases) in T cells(31Garcia-Morales P. Minami Y. Luong E. Klausner R.D. Samelson L.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9255-9259Crossref PubMed Scopus (233) Google Scholar, 32O'Shea J.J. McVicar D.W. Bailey T.L. Burns C. Smyth M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10306-10310Crossref PubMed Scopus (141) Google Scholar, 33Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar), the peptides were synthesized with a phosphotyrosyl analog that is resistant to hydrolysis. In this analog, difluorophosphonomethyl phenylalanine (F2Pmp), a difluoromethylene group replaces the phenolic oxygen of phosphotyrosine (24Burke Jr., T.R Smyth M.S. Otaka A. Roller P.P. Tetrahedron Lett. 1993; 34: 4125-4128Crossref Scopus (86) Google Scholar, 25Smyth M.S. Burke Jr., T.R. Tetrahedron Lett. 1994; 35: 551-553Crossref Scopus (93) Google Scholar). In previous studies, F2Pmp- and pTyr-containing hexapeptides have been shown to bind their complementary SH2 domains with similar affinities(34Burke Jr., T.R. Smyth M.S. Otaka A. Nomizu M. Roller P.P. Wolf G. Case R. Shoelson S.E. Biochemistry. 1994; 33: 6490-6494Crossref PubMed Scopus (197) Google Scholar). Four different F2Pmp-containing peptides were synthesized based on the sequence LYQGLSTATKDTYDALH, which is the C-terminal (third) TAM of the human TCR ζ chain. The peptide in which both Tyr residues are replaced with F2Pmp groups is referred to as (F2Pmp)2-TAM ζ3, while peptides in which either the N- or C-terminal Tyr is replaced with F2Pmp are (F2Pmp)N-TAM ζ or (F2Pmp)C-TAM ζ3, respectively. The fourth peptide is a control in which the phosphonate oxygens of (F2Pmp)2-TAM ζ3 remain blocked with ethoxy groups from the synthesis.The ability of these synthetic peptides to bind to ZAP-70 was assessed in competition binding studies with a synthetic 32 P-labeled (pTyr)2-TAM ζ3 peptide that binds a glutathione S-transferase fusion protein containing both SH2 domains of ZAP-70 (Fig. 1). Only those peptides containing two pTyr or F2Pmp groups competed for binding. The ether-blocked control peptide did not compete. No peptide binding was observed to glutathione S-transferase alone or fusion proteins containing a single C- or N-terminal ZAP-70 SH2 domain (not shown). (F2Pmp)2-TAM ζ3 bound the tandem SH2 domains of ZAP-70 with an affinity similar to the (pTyr)2-TAM ζ3 itself. A higher concentration of cold (pTyr)2-TAM ζ3 (1 μM) was required to give 50% competition of 4 nM 32 P-labeled (pTyr)2-TAM ζ3. This disparity reflects the fact that the effective concentration of the binding site, glutathione/agarose-adsorbed glutathione S-transferase-ZAP-70-(SH2)2, is much higher than that of the labeled peptide. The excess of binding sites over ligand in this assay precludes the determination of absolute affinities from the IC50 values, but these values remain useful in deriving relative affinities(35Isakov N. Wange R.L. Burgess W.H. Watts J.D. Aebersold R. Samelson L.E. J. Exp. Med. 1995; (in press)PubMed Google Scholar). In additional binding studies, (F2Pmp)2-TAM ζ3 cross-competed with (pTyr)2-TAM ζ1, (pTyr)2-TAM ζ2, and (pTyr)2-TAM ε indicating that ZAP-70 binding to all TCR ζ and CD3 ε TAMs can be inhibited by (F2Pmp)2-TAM ζ3.2Having found that (F2Pmp)2-TAM ζ3 could bind ZAP-70 in vitro, the ability of (F2Pmp)2-TAM ζ3 to bind ZAP-70 and block its association with TCR was tested in Jurkat T cells permeabilized with the bacterial toxin tetanolysin. The TCR from peptide-treated permeabilized Jurkat T cells was immunoprecipitated and examined for associated ZAP-70, as detected by anti-ZAP-70 or anti-pTyr blotting (Fig. 2A). As demonstrated previously, ZAP-70 bound the TCR only upon TCR stimulation(9Chan A.C. Irving B.A. Fraser J.D. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9166-9170Crossref PubMed Scopus (334) Google Scholar, 23Wange R.L. Kong A.N. Samelson L.E. J. Biol. Chem. 1992; 267: 11685-11688Abstract Full Text PDF PubMed Google Scholar). The association of ZAP-70 with TCR was markedly inhibited by (F2Pmp)2-TAM ζ3, but not by the ether-blocked control peptide or the singly F2Pmp-substituted peptides. The ZAP-70 co-precipitated with TCR was tyrosine-phosphorylated (Fig. 2A). In addition, co-recovery of phospho- ζ or phospho- ε with ZAP-70 immunoprecipitates was also inhibited by (F2Pmp)2-TAM ζ3 (not shown). (F2Pmp)2-TAM ζ3 could be acting to block co-recovery of ZAP-70 with TCR either by blocking initial TCR-stimulated binding or by competing after cell lysis. To test the latter possibility, the peptide was added after termination of TCR stimulation, such that the peptide could not block initial assembly, but could compete with the TCR for ZAP-70 during immunoprecipitation. Only a minimal reduction in ZAP-70 co-recovery with the TCR was observed, suggesting that the major effect of (F2Pmp)2-TAM ζ3 is to block initial assembly of ZAP-70 with the TCR (not shown).Figure 2:(F2Pmp)2-TAM ζ3 inhibition of TCR-stimulated ZAP-70/TCR association and ZAP-70 tyrosine phosphorylation. A, TCR was immunoprecipitated from unstimulated(-) or TCR-stimulated (+) tetanolysin-permeabilized Jurkat T cells incubated with 4 μM concentrations of the indicated peptides. The immunoprecipitates were analyzed for co-precipitating ZAP-70 with a mAb to pTyr (4G10) (upper panel) or a polyclonal antiserum to ZAP-70 (lower panel). B, ZAP-70 was immunoprecipitated from intact Jurkat T cells and Jurkat T cells treated as in A. The immunoprecipitates were analyzed for ZAP-70 with 4G10 (upper panel) or a polyclonal antiserum to ZAP-70 (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Recent studies would predict that inhibition of ZAP-70 association with the TCR would reduce TCR-stimulated tyrosine phosphorylation of ZAP-70 (19Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). To test this prediction, anti-ZAP-70 immunoprecipitates from permeabilized Jurkat T cells, incubated with (F2Pmp)2-TAM ζ3, were blotted for pTyr (Fig. 2B). (F2Pmp)2-TAM ζ3 blocked the TCR-stimulated tyrosine phosphorylation of total ZAP-70, while the control peptide and the singly F2Pmp-substituted peptides did not. The amount of ZAP-70 immunoprecipitated was the same in each lane as shown in the anti-ZAP-70 blot of a replicate gel (Fig. 2B). The same effect of (F2Pmp)2-TAM ζ3 on ZAP-70 tyrosine phosphorylation was also seen in anti-pTyr blots of whole cell lysates from peptide-treated permeable Jurkat cells (see Fig. 4A). The inhibitory effect of (F2Pmp)2-TAM ζ3 on ZAP-70 tyrosine phosphorylation is not mediated by activation of a PTPase activity, since this effect is unaffected by orthovanadate (not shown).Figure 4:(F2Pmp)2-TAM ζ3 inhibition of tyrosine phosphorylation of Jurkat T cell substrates. A, whole cell lysates were collected from unstimulated(-) or TCR-stimulated (+) tetanolysin-permeabilized Jurkat T cells incubated without added peptide(-), with control peptide (C), or with (F2Pmp)2-TAM ζ3 (T). Lysates were also collected from unstimulated(-) or TCR-stimulated (+) intact Jurkat T cells. Lysates were analyzed for pTyr-containing proteins by immunoblotting with 4G10. The arrowhead indicates the position of ZAP-70. The arrow shows the position of TCR chains. The dots indicate the position of other proteins with reduced tyrosine phosphorylation upon (F2Pmp)2-TAM ζ3 incubation. B, TCR was immunoprecipitated as in Fig. 2, except that cells were also incubated with (+) or without(-) 1 mM orthovanadate. TCR subunits were analyzed for pTyr by 4G10 immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The effect of (F2Pmp)2-TAM ζ3 incubation on ZAP-70 kinase activity was determined in an immune complex kinase assay. Since no substrates of the ZAP-70 kinase have yet been identified, we used a cytosolic fragment of the erythrocyte band 3 protein (cfb3) as a substrate in the kinase assay(36Wang C.C. Badylak J.A. Lux S.E. Moriyama R. Dixon J.E. Low P.S. Protein Sci. 1992; 1: 1206-1214Crossref PubMed Scopus (42) Google Scholar). cfb3 is known to be a substrate for the related protein tyrosine kinase, Syk(37Harrison M.L. Isaacson C.C. Burg D.L. Geahlen R.L. Low P.S. J. Biol. Chem. 1994; 269: 955-959Abstract Full Text PDF PubMed Google Scholar). ZAP-70 was immunoprecipitated from lysates of intact or permeabilized cells incubated with and without (F2Pmp)2-TAM ζ3. The immune complex kinase assay was carried out on a portion of the immunoprecipitated ZAP-70, while the remainder was analyzed for the pTyr content of ZAP-70 by anti-pTyr blotting (Fig. 3, upper and middle panels, respectively). Stimulation of the TCR by mAb ligation increased the kinase activity associated with ZAP-70, as reflected in increased phosphorylation of cfb3 and ZAP-70 itself. The predominant kinase activity associated with the ZAP-70 immunoprecipitate is ZAP-70 itself, although other kinases that associate with either ZAP-70 or phospho- ζ could also contribute activity in this assay. Two kinases that would be most likely to co-precipitate with ZAP-70 or associated molecules are Lck and Syk(38Duplay P. Thome M. Herve F. Acuto O. J. Exp. Med. 1994; 179: 1163-1172Crossref PubMed Scopus (153) Google Scholar, 39Couture C. Baier G. Altman A. Mustelin T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5301-5305Crossref PubMed Scopus (113) Google Scholar, 40Chan A.C. van Oers N.S.C. Tran A. Turka L. Law C.-L. Ryan J.C. Clark E.A. Weiss A. J. Immunol. 1994; 152: 4758-4766PubMed Google Scholar); however, neither Lck activity nor Syk protein is detected in the ZAP-70 immunoprecipitates2(41Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Pendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar), suggesting that the kinase activity observed is predominantly due to ZAP-70. Incubation of permeabilized Jurkat with (F2Pmp)2-TAM ζ3 reduced the kinase activity of ZAP-70 to the level observed in unstimulated intact cells, while control peptide had no effect. The level of tyrosine phosphorylation of the ZAP-70 used in the kinase assay shows that the basal kinase activity is associated with nonphosphorylated ZAP-70, while increased kinase activity is associated with tyrosine-phosphorylated ZAP-70 (Fig. 3, middle panel). The amount of ZAP-70 in each lane was the same, as is shown by anti-ZAP-70 blotting (lower panel). The actual mechanism of ZAP-70 tyrosine phosphorylation remains to be determined, and, while ZAP-70 activation coincides with both TCR binding and tyrosine phosphorylation of ZAP-70, it is still unclear which, if either, of these events activates the kinase.Figure 3:(F2Pmp)2-TAM ζ3 inhibition of TCR-stimulated ZAP-70 kinase activity. ZAP-70 was immunoprecipitated from TCR-stimulated (+), tetanolysin-permeabilized Jurkat T cells incubated without peptide, or with either the (F2Pmp)2-TAM ζ3 (T) or the ether-blocked control peptide (C). For comparison, ZAP-70 was also immunoprecipitated from unstimulated(-) or TCR-stimulated (+) intact Jurkat T cells. The immunoprecipitates were analyzed for in vitro kinase activity (upper panel). A portion of the immunoprecipitates were analyzed for tyrosine-phosphorylated ZAP-70 by 4G10 immunoblotting (middle panel). The relative amount of ZAP-70 in each immunoprecipitate was analyzed by immunoblotting the nitrocellulose membrane from the kinase assay (upper panel) with a polyclonal antiserum to ZAP-70 (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The results show a direct correlation between (F2Pmp)2-TAM ζ3-induced inhibition of ZAP-70 association with activated TCR, loss of tyrosine phosphorylation of ZAP-70, and loss of TCR-stimulated ZAP-70 kinase activity. These findings are consistent with a model whereby (F2Pmp)2-TAM ζ binds to ZAP-70, occupies the tandem SH2 domains of ZAP-70, and thereby prevents ZAP-70 association with tyrosine-phosphorylated TCR. Inhibition of ZAP-70 binding to the TCR then prevents tyrosine phosphorylation and activation of ZAP-70. An alternate model in which (F2Pmp)2-TAM ζ3 binds to Lck, prevents Lck from tyrosine-phosphorylating the TCR, and thus prevents association of ZAP-70 with the TCR, is unlikely, since Lck does not bind a synthetic peptide with a similar structure to (F2Pmp)2-TAM ζ3(19Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). Also, elimination of the binding capacity of the SH2 domain of Lck by mutation does not affect its ability to tyrosine-phosphorylate ζ or ZAP-70(19Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). In addition, the F2Pmp-containing peptides have no effect on Lck kinase activity toward a synthetic peptide, containing residues 52-164 of the cytoplasmic tail of ζ in an in vitro kinase assay2(27Watts J.D. Wilson G.M. Ettenhadieh E. Clark-Lewis I. Kubanek C.A. Astell C.R. Marth J.D. Aebersold R. J. Biol. Chem. 1992; 267: 901-907Abstract Full Text PDF PubMed Google Scholar).To determine whether (F2Pmp)2-TAM ζ3-mediated inhibition of ZAP-70 association with the TCR had an effect on TCR-stimulated tyrosine phosphorylation of Jurkat T cell substrates, lysates from Jurkat T cells were treated as indicated in Fig. 4 and blotted for pTyr. In intact cells there was a marked increase in tyrosine phosphorylation of many proteins in response to mAb stimulation of the TCR. Although tetanolysin permeabilization, itself, caused increased tyrosine phosphorylation of some proteins, stimulation of the TCR in the permeabilized cells caused a further increase in tyrosine phosphorylation. Addition of (F2Pmp)2-TAM ζ3 decreased tyrosine phosphorylation of a number of proteins, while control peptide had no effect. As was seen in Fig. 2B, the (F2Pmp)2-TAM ζ3 peptide almost completely blocked tyrosine phosphorylation of ZAP-70. Several other unidentified proteins with molecular masses of 120, 80, and 35-40 kDa showed reduced tyrosine phosphorylation after incubation with (F2Pmp)2-TAM ζ3. The identification of these phosphoproteins, which might be ZAP-70 substrates, and the effect of (F2Pmp)2-TAM ζ3 on distal signaling molecules in viable T cells are the subjects of continuing investigation. Jurkat T cells have recently been shown to contain the protein tyrosine kinase Syk(40Chan A.C. van Oers N.S.C. Tran A. Turka L. Law C.-L. Ryan J.C. Clark E.A. Weiss A. J. Immunol. 1994; 152: 4758-4766PubMed Google Scholar, 41Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Pendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar). In these cells, Syk is recruited to phospho- ζ concurrently with ZAP-70(41Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Pendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar). Thus, it remains formally possible that part of the effect of (F2Pmp)2-TAM ζ3 on substrate phosphorylation may be due to the blockade of Syk association with the TCR.Tyrosine phosphorylation of ζ and CD3 ε receptor chains was also decreased in (F2Pmp)2-TAM ζ3-treated Jurkat T cells (Fig. 4A). If our model of (F2Pmp)2-TAM ζ3 action is correct, then reduced tyrosine phosphorylation of ζ and CD3 ε is not likely to be due to reduced kinase activity, as ZAP-70 exhibits no activity for TCR subunits, and (F2Pmp)2-TAM ζ3 fails to block Lck activity2(19Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). One possibility to explain the decreased TCR tyrosine phosphorylation is that (F2Pmp)2-TAM ζ could increase the effective PTPase activity toward the TCR by blocking the ability of ZAP-70 to bind to and protect the TAM pTyr residues from dephosphorylation(19Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). Such a mechanism has been demonstrated for phospholipase C γ 1 binding to the epidermal growth factor receptor(42Rotin D. Margolis B. Mohammadi M. Daly R.J. Daum G. Li N. Fischer E.H. Burgess W.H. Ullrich A. Schlessinger J. EMBO J. 1992; 11: 559-567Crossref PubMed Scopus (250) Google Scholar). To test this possibility, the TCR was immunoprecipitated from peptide-treated Jurkat T cells in the presence or absence of orthovanadate during TCR stimulation and analyzed by anti-pTyr blotting (Fig. 4B). Orthovanadate increased the overall pTyr signal, but also inhibited the ability of the (F2Pmp)2-TAM ζ3 peptide to reduce tyrosine phosphorylation of ζ and CD3 ε . This suggests that, in the absence of orthovanadate, the TAMs are tyrosine-phosphorylated normally, but are dephosphorylated rapidly in the absence of ZAP-70 binding. Orthovanadate did not block the (F2Pmp)2-TAM ζ3-mediated reduction in tyrosine phosphorylation of other substrates (not shown). Therefore, we favor the interpretation that the principle action of (F2Pmp)2-TAM ζ3 is to bind to ZAP-70 and block its association with the TCR. Failure of ZAP-70 binding to the TCR blocks the tyrosine phosphorylation and activation of ZAP-70 and results in reduced tyrosine phosphorylation of cellular substrates.The ability to target and disrupt specific phosphoprotein-SH2 domain interactions holds tremendous potential in the development of reagents to aid in further analysis of signaling pathways and, perhaps more importantly, in the development of rationally designed chemotherapeutic agents, which could see application as novel immunosuppressive or antineoplastic drugs(43Brugge J.S. Science. 1993; 260: 918-919Crossref PubMed Scopus (94) Google Scholar). These studies represent a significant step toward this goal and demonstrate that synthetic PTPase-resistant phosphotyrosyl analog-containing peptides, based on SH2 domain binding sequences, can be successfully introduced into cellular systems to disrupt signaling pathways. INTRODUCTIONThe T cell-mediated immune response depends upon the ability of the T cell antigen receptor (TCR) 1The abbreviations used are: TCRT cell antigen receptorTAMtyrosine-based activation motifPTPaseprotein tyrosine phosphataseF2Pmpdifluorophosphonomethyl phenylalanineSH2Src-homology 2pTyrphosphotyrosinemAbmonoclonal antibodycfb3cytosolic fragment of erythrocyte band 3. to recognize specific antigen and respond by initiating an intracellular signaling cascade. The α and β chains of the multisubunit TCR determine the antigen specificity of the TCR, while the γ , δ , and ε CD3 chains and the TCR ζ chain are coupled to intracellular signaling molecules. A number of studies have demonstrated the importance of a short amino acid sequence, present as three copies in the cytoplasmic domain of the TCR ζ chain and as a single copy in each of the CD3 chains, for initiating T cell signaling(1Letourneur F. Klausner R.D. Science. 1992; 255: 79-82Crossref PubMed Scopus (342) Google Scholar, 2Romeo C. Amiot M. Seed B. Cell. 1992; 68: 889-897Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 3Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (251) Google Scholar). This sequence has many descriptive names including the tyrosine-based activation motif (TAM) and has the general structure YXX(L/I)X (6-8) YXX(L/I) (4Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1154) Google Scholar). In chimeric receptor cross-linking studies, TAMs have been shown to be necessary and sufficient for initiation of T cell activation (1Letourneur F. Klausner R.D. Science. 1992; 255: 79-82Crossref PubMed Scopus (342) Google Scholar, 2Romeo C. Amiot M. Seed B. Cell. 1992; 68: 889-897Abstract Full Text PDF PubMed Scopus (260) Google Scholar, 3Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (251) Google Scholar).The earliest biochemical event detected upon TCR stimulation is increased tyrosine phosphorylation of a number of cellular substrates (5June C.H. Fletcher M.C. Ledbetter J.A. Samelson L.E. J. Immunol. 1990; 144: 1591-1599PubMed Google Scholar), including the TCR chain TAMs(6Baniyash M. Garcia-Morales P. Luong E. Samelson L.E. Klausner R.D. J. Biol. Chem. 1988; 263: 18225-18230Abstract Full Text PDF PubMed Google Scholar, 7Qian D. Griswold-Prenner I. Rosner M.R. Fitch F.W. J. Biol. Chem. 1993; 268: 4488-4493Abstract Full Text PDF PubMed Google Scholar, 8Sancho J. Franco R. Chatila T. Hall C. Terhorst C. Eur. J. Immunol. 1993; 23: 1636-1642Crossref PubMed Scopus (31) Google Scholar), ZAP-70(9Chan A.C. Irving B.A. Fraser J.D. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9166-9170Crossref PubMed Scopus (334) Google Scholar), Vav(10Bustelo X.R. Ledbetter J.A. Barbacid M. 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Three protein tyrosine kinases that have been implicated as being involved in this early phase of TCR-mediated signaling are the Src-family members Fyn and Lck and the Syk-family member ZAP-70. The evidence for the involvement of these protein tyrosine kinases in TCR signaling has been extensively reviewed (16Samelson L.E. Klausner R.D. J. Biol. Chem. 1992; 267: 24913-24916Abstract Full Text PDF PubMed Google Scholar, 17Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1943) Google Scholar, 18Isakov N. Wange R.L. Samelson L.E. J. Leukocyte Biol. 1994; 55: 265-271Crossref PubMed Scopus (49) Google Scholar).Recently it was shown that Lck and ZAP-70 can act sequentially to tyrosine phosphorylate various substrates in COS cells transfected with Lck, ZAP-70, and a CD8 ζ chimeric protein (19Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). In this system, Lck is required to tyrosine phosphorylate the TAMs of CD8 ζ which permits ZAP-70 binding. Upon association with CD8 ζ , ZAP-70 is tyrosine-phosphorylated, becomes activated, and induces tyrosine phosphorylation of substrates. One would predict from such a model that agents that block the association of ZAP-70 with the TCR could prove useful in identifying the downstream effectors of ZAP-70 and in blocking T cell activation. Recent insights into the mechanism of the association of ZAP-70 with the TCR provide the basis for the design of such inhibitory agents.Src-homology 2 (SH2) domains mediate protein-protein interactions by binding to pTyr-containing sequences(20Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Rotnosfsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar, 21Pawson T. Schlessinger J. Curr. Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (574) Google Scholar). The presence of two SH2 domains in ZAP-70 and 2 pTyr residues in stimulated TCR TAMs suggested a possible mechanism of interaction. Experiments using glutathione S-transferase fusion proteins containing the SH2 domains of ZAP-70 show that ZAP-70 binds to the tyrosine-phosphorylated TCR ζ and CD3 ε chains via its SH2 domains; both SH2 domains are required for high affinity binding(22Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Abstract Full Text PDF PubMed Google Scholar). That the TCR TAM motifs mediate this interaction was demonstrated in studies showing binding of ZAP-70 to transmembrane chimeric receptors with a single ζ chain TAM as the cytoplasmic tail(3Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (251) Google Scholar). In this study, we report that a novel phosphatase-resistant synthetic peptide based on the C-terminal (pTyr)2-TAM of the TCR ζ chain competitively blocks ZAP-70 binding to activated TCR. As a consequence, TCR stimulation fails to induce tyrosine phosphorylation of ZAP-70 or activation of its kinase activity. Coincident with this loss of ZAP-70 kinase activity is reduced tyrosine phosphorylation of a number of substrates. This result suggests that the peptide blocks early TCR-mediated signaling events through a ZAP-70 dependent mechanism, as would be predicted by the model of early activation events mentioned above.

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