©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
F(Pmp)-TAM, a Novel Competitive Inhibitor of the Binding of ZAP-70 to the T Cell Antigen Receptor, Blocks Early T Cell Signaling (*)

(Received for publication, June 24, 1994; and in revised form, September 9, 1994)

Ronald L. Wange (1) Noah Isakov (1) Terrence R. Burke Jr. (2) Akira Otaka (2)(§) Peter P. Roller (2) Julian D. Watts (3)(¶) Ruedi Aebersold (3)(¶)(**) Lawrence E. Samelson (1)(§§)

From the  (1)Cell Biology and Metabolism Branch, NICHD, and the (2)Laboratory of Medicinal Chemistry, DTP, DCT, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the (3)Biomedical Research Centre, University of British Columbia, Vancouver, BC, V6T 1Z3 Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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 (F(2)Pmp) 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.


INTRODUCTION

The T cell-mediated immune response depends upon the ability of the T cell antigen receptor (TCR) (^1)to recognize specific antigen and respond by initiating an intracellular signaling cascade. The alpha and beta chains of the multisubunit TCR determine the antigen specificity of the TCR, while the , , and CD3 chains and the TCR 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 chain and as a single copy in each of the CD3 chains, for initiating T cell signaling(1, 2, 3) . This sequence has many descriptive names including the tyrosine-based activation motif (TAM) and has the general structure YXX(L/I)XYXX(L/I) (4) . In chimeric receptor cross-linking studies, TAMs have been shown to be necessary and sufficient for initiation of T cell activation (1, 2, 3) .

The earliest biochemical event detected upon TCR stimulation is increased tyrosine phosphorylation of a number of cellular substrates (5) , including the TCR chain TAMs(6, 7, 8) , ZAP-70(9) , Vav(10, 11) , Shc(12) , phospholipase C1(13, 14) , and VCP (15) . 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 (16, 17, 18) .

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 chimeric protein (19) . In this system, Lck is required to tyrosine phosphorylate the TAMs of CD8 which permits ZAP-70 binding. Upon association with CD8, 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(20, 21) . 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 and CD3 chains via its SH2 domains; both SH2 domains are required for high affinity binding(22) . That the TCR TAM motifs mediate this interaction was demonstrated in studies showing binding of ZAP-70 to transmembrane chimeric receptors with a single chain TAM as the cytoplasmic tail(3) . In this study, we report that a novel phosphatase-resistant synthetic peptide based on the C-terminal (pTyr)(2)-TAM of the TCR 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 PROCEDURES

Cells, Antibodies, and Miscellaneous Reagents

Jurkat T cells were cultured as described previously(23) . The C305 anti-Jurkat beta chain mAb, the OKT3 anti-CD3 mAb, and the 4G10 anti-pTyr mAb have been described previously(22) . 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. (^2)Tetanolysin was a gift of E. Bonvini (FDA).

Fusion Proteins and Synthetic Peptides

Preparation of glutathione S-transferase-ZAP-70(SH2)(2) has been described(22) . The cold (pTyr)(2)-TAM(3) peptide has the sequence LpYQGLSTATKDTpYDALH and was purchased from M. Berne (Tufts University, Boston, MA). F(2)Pmp was synthesized as described previously(24, 25) . The synthesis of the F(2)Pmp-containing peptides was accomplished essentially according to methodologies previously described(26) . The TAM peptide, HDGLYQGLSTATKDTYDALHM, was purchased from Peptide Technologies Corp. (Gaithersburg, MD). This peptide was tyrosine-phosphorylated with [-P]ATP in vitro using purified recombinant Lck, as described(27) . 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 Immunoblotting

TCR stimulation was for 2 min at 37 °C with either the anti-beta 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(22) . In some experiments, Na(3)VO(4) 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 Na(3)VO(4), 5 mM EDTA, 10 µg/ml aprotinin and leupeptin, and 25 µMp-nitrophenyl p`-quanidinobenzoate). Brij 96 maintains association of CD3 and TCR chains(28) . Methods for immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and immunoblotting have been described(22) . Immunoblots were developed by enhanced chemiluminescence (Amersham).

Tetanolysin Permeabilization and Peptide Incubation

The method for tetanolysin permeabilization of T lymphocytes has been published previously(29, 30) . 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 Assay

Prior 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 MnCl(2), 1 µM ATP, 0.5 µg of cfb3, 10 µCi of [-P]ATP. The reaction was terminated by addition of one-third volume of 95 °C 4times-reducing Laemmli sample buffer and incubation for 5 min at 95 °C.


RESULTS AND DISCUSSION

Because of the presence of active protein tyrosine phosphatases (PTPases) in T cells(31, 32, 33) , the peptides were synthesized with a phosphotyrosyl analog that is resistant to hydrolysis. In this analog, difluorophosphonomethyl phenylalanine (F(2)Pmp), a difluoromethylene group replaces the phenolic oxygen of phosphotyrosine (24, 25) . In previous studies, F(2)Pmp- and pTyr-containing hexapeptides have been shown to bind their complementary SH2 domains with similar affinities(34) . Four different F(2)Pmp-containing peptides were synthesized based on the sequence LYQGLSTATKDTYDALH, which is the C-terminal (third) TAM of the human TCR chain. The peptide in which both Tyr residues are replaced with F(2)Pmp groups is referred to as (F(2)Pmp)(2)-TAM(3), while peptides in which either the N- or C-terminal Tyr is replaced with F(2)Pmp are (F(2)Pmp)(N)-TAM or (F(2)Pmp)(C)-TAM(3), respectively. The fourth peptide is a control in which the phosphonate oxygens of (F(2)Pmp)(2)-TAM(3) 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 P-labeled (pTyr)(2)-TAM(3) 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 F(2)Pmp 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). (F(2)Pmp)(2)-TAM(3) bound the tandem SH2 domains of ZAP-70 with an affinity similar to the (pTyr)(2)-TAM(3) itself. A higher concentration of cold (pTyr)(2)-TAM(3) (1 µM) was required to give 50% competition of 4 nMP-labeled (pTyr)(2)-TAM(3). 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 IC values, but these values remain useful in deriving relative affinities(35) . In additional binding studies, (F(2)Pmp)(2)-TAM(3) cross-competed with (pTyr)(2)-TAM(1), (pTyr)(2)-TAM(2), and (pTyr)(2)-TAM indicating that ZAP-70 binding to all TCR and CD3 TAMs can be inhibited by (F(2)Pmp)(2)-TAM(3) .^2


Figure 1: Competitive binding of F(2)Pmp-containing peptides to glutathione S-transferase-ZAP-70(SH2)(2). Three µg of glutathione S-transferase-ZAP-70-(SH2)(2) immobilized on glutathione agarose were incubated with -P-labeled (pTyr)(2)-TAM(3) (4 nM) together with increasing concentrations of the indicated peptides. After 1 h of incubation at 4 °C, the beads were washed extensively, and bound radioactivity was measured by scintillation counting. circle, (F(2)Pmp)(2)-TAM(3); box, (F(2)Pmp)(N)-TAM(3); bullet, (F(2)Pmp)(C)-TAM(3); Delta, control peptide; , (pTyr)(2)-TAM(3). Data are from one experiment and are representative of three experiments.



Having found that (F(2)Pmp)(2)-TAM(3) could bind ZAP-70 in vitro, the ability of (F(2)Pmp)(2)-TAM(3) 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(9, 23) . The association of ZAP-70 with TCR was markedly inhibited by (F(2)Pmp)(2)-TAM(3), but not by the ether-blocked control peptide or the singly F(2)Pmp-substituted peptides. The ZAP-70 co-precipitated with TCR was tyrosine-phosphorylated (Fig. 2A). In addition, co-recovery of phospho- or phospho- with ZAP-70 immunoprecipitates was also inhibited by (F(2)Pmp)(2)-TAM(3) (not shown). (F(2)Pmp)(2)-TAM(3) 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 (F(2)Pmp)(2)-TAM(3) is to block initial assembly of ZAP-70 with the TCR (not shown).


Figure 2: (F(2)Pmp)(2)-TAM(3) 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).



Recent studies would predict that inhibition of ZAP-70 association with the TCR would reduce TCR-stimulated tyrosine phosphorylation of ZAP-70 (19) . To test this prediction, anti-ZAP-70 immunoprecipitates from permeabilized Jurkat T cells, incubated with (F(2)Pmp)(2)-TAM(3), were blotted for pTyr (Fig. 2B). (F(2)Pmp)(2)-TAM(3) blocked the TCR-stimulated tyrosine phosphorylation of total ZAP-70, while the control peptide and the singly F(2)Pmp-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 (F(2)Pmp)(2)-TAM(3) 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 (F(2)Pmp)(2)-TAM(3) 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: (F(2)Pmp)(2)-TAM(3) 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 (F(2)Pmp)(2)-TAM(3) (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 (F(2)Pmp)(2)-TAM(3) 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.



The effect of (F(2)Pmp)(2)-TAM(3) 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(36) . cfb3 is known to be a substrate for the related protein tyrosine kinase, Syk(37) . ZAP-70 was immunoprecipitated from lysates of intact or permeabilized cells incubated with and without (F(2)Pmp)(2)-TAM(3). 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- 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(38, 39, 40) ; however, neither Lck activity nor Syk protein is detected in the ZAP-70 immunoprecipitates^2(41) , suggesting that the kinase activity observed is predominantly due to ZAP-70. Incubation of permeabilized Jurkat with (F(2)Pmp)(2)-TAM(3) 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: (F(2)Pmp)(2)-TAM(3) 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 (F(2)Pmp)(2)-TAM(3) (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).



The results show a direct correlation between (F(2)Pmp)(2)-TAM(3)-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 (F(2)Pmp)(2)-TAM 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 (F(2)Pmp)(2)-TAM(3) 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 (F(2)Pmp)(2)-TAM(3)(19) . Also, elimination of the binding capacity of the SH2 domain of Lck by mutation does not affect its ability to tyrosine-phosphorylate or ZAP-70(19) . In addition, the F(2)Pmp-containing peptides have no effect on Lck kinase activity toward a synthetic peptide, containing residues 52-164 of the cytoplasmic tail of in an in vitro kinase assay^2(27) .

To determine whether (F(2)Pmp)(2)-TAM(3)-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. 4and 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 (F(2)Pmp)(2)-TAM(3) decreased tyrosine phosphorylation of a number of proteins, while control peptide had no effect. As was seen in Fig. 2B, the (F(2)Pmp)(2)-TAM(3) 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 (F(2)Pmp)(2)-TAM(3). The identification of these phosphoproteins, which might be ZAP-70 substrates, and the effect of (F(2)Pmp)(2)-TAM(3) 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(40, 41) . In these cells, Syk is recruited to phospho- concurrently with ZAP-70(41) . Thus, it remains formally possible that part of the effect of (F(2)Pmp)(2)-TAM(3) on substrate phosphorylation may be due to the blockade of Syk association with the TCR.

Tyrosine phosphorylation of and CD3 receptor chains was also decreased in (F(2)Pmp)(2)-TAM(3)-treated Jurkat T cells (Fig. 4A). If our model of (F(2)Pmp)(2)-TAM(3) action is correct, then reduced tyrosine phosphorylation of and CD3 is not likely to be due to reduced kinase activity, as ZAP-70 exhibits no activity for TCR subunits, and (F(2)Pmp)(2)-TAM(3) fails to block Lck activity^2(19) . One possibility to explain the decreased TCR tyrosine phosphorylation is that (F(2)Pmp)(2)-TAM 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(19) . Such a mechanism has been demonstrated for phospholipase C1 binding to the epidermal growth factor receptor(42) . 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 (F(2)Pmp)(2)-TAM(3) peptide to reduce tyrosine phosphorylation of and CD3. 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 (F(2)Pmp)(2)-TAM(3)-mediated reduction in tyrosine phosphorylation of other substrates (not shown). Therefore, we favor the interpretation that the principle action of (F(2)Pmp)(2)-TAM(3) 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(43) . 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.

Funded by grants from the Medical Research Council of Canada.

**
Current address: Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195.

§§
To whom correspondence and reprint requests should be addressed. Tel.: 301-402-1400; Fax: 301-402-0078.

(^1)
The abbreviations used are: TCR, T cell antigen receptor; TAM, tyrosine-based activation motif; PTPase, protein tyrosine phosphatase; F(2)Pmp, difluorophosphonomethyl phenylalanine; SH2, Src-homology 2; pTyr, phosphotyrosine; mAb, monoclonal antibody; cfb3, cytosolic fragment of erythrocyte band 3.

(^2)
R. L. Wange, unpublished observation.


ACKNOWLEDGEMENTS

We thank J. Bolen, E. Bonvini, I. Clark-Lewis, R. Knowles, P. Low, and A. Weiss for providing reagents. We also thank J. Donovan and R. Klausner for critical review of this manuscript. R. A. and J. D. W. also thank D. Krebs for technical assistance.

Note Added in Proof-An agreement was reached recently by a group of researchers in the field concerning the nomenclature of the motif referred to in this manuscript as a TAM and which has been alternatively named an ARH1, ARAM, YXXL, or Reth motif. The newly accepted term for this motif is ITAM for Immunoreceptor Tyrosine-based Activation Motif (submitted for publication).


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