©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Zap-70 by Src Family Tyrosine Protein Kinases in an Antigen-specific T-cell Line (*)

(Received for publication, October 24, 1994)

Robert Weil (1)(§) Jean-François Cloutier (1) (2)(¶) Marielle Fournel (1) André Veillette (1) (2) (3) (4)(**)

From the  (1)McGill Cancer Centre and Departments of (2)Medicine and (3)Biochemistry and Oncology, McGill University, Montréal H3G 1Y6, Canada, and the (4)Departments of Medicine and Oncology, Montreal General Hospital, Montréal H3G 1A4, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To further understand the interactions between Zap-70, Src family kinases, and other T-cell proteins, we have examined the regulation of Zap-70 in the antigen-specific T-cell line BI-141. By analyzing derivatives containing an activated version of either p56 or p59, it was observed that the two Src-related enzymes augmented T-cell receptor (TCR)-mediated tyrosine phosphorylation of Zap-70, as well as its association with components of the antigen receptor complex. Importantly, the accumulation of TCRbulletZap-70 complexes quantitatively and temporally correlated with the induction of tyrosine phosphorylation of the CD3 and chains of TCR. Using a CD4-positive variant of BI-141, we also found that the ability of Zap-70 to undergo tyrosine phosphorylation and associate with TCR was enhanced by aggregation of TCR with the CD4 co-receptor. Further studies allowed the identification of two distinct pools of tyrosine-phosphorylated Zap-70 in activated T-cells. While one population was associated with TCR, the other was co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated protein of unknown identity. In addition to supporting the notion that Src-related enzymes regulate the recruitment of Zap-70 in TCR signaling, these data added further complexity to previous models of regulation of Zap-70. Furthermore, they suggested that p120 may be an effector and/or a regulator of Zap-70 in activated T-lymphocytes.


INTRODUCTION

Stimulation of T-lymphocytes by antigen or anti-T-cell receptor (TCR) (^1)antibodies results in a rapid rise in intracellular tyrosine protein phosphorylation (reviewed in Samelson and Klausner, 1992; Veillette and Davidson, 1992; Perlmutter et al., 1993; Weiss and Littman, 1994). This early biochemical signal leads to a series of changes that includes elevation of intracellular calcium, activation of the serine/threonine-specific protein kinase C, and production of lymphokines such as interleukin (IL)-2 and IL-4. Ultimately, these modifications allow T-cell mitogenesis and clonal expansion. The antigen receptor, as well as the associated CD3 and chains do not possess intrinsic catalytic properties capable of inducing tyrosine protein phosphorylation. However, the CD3 and subunits have the ability to promote interactions between cytoplasmic tyrosine protein kinases and tyrosine phosphorylation substrates, thereby inducing the TCR signaling cascade.

Accumulating data indicate that antigen receptor-triggered tyrosine protein phosphorylation is initiated by p56 and p59, two Src-related tyrosine protein kinases abundantly expressed in T-cells (reviewed in Samelson and Klausner, 1992; Veillette and Davidson, 1992; Perlmutter et al., 1993; Weiss and Littman, 1994). Upon TCR stimulation, Lck and FynT are postulated to cause phosphorylation of critical tyrosine residues located within the tyrosine-based activation motifs of CD3 and . This phosphorylation presumably creates docking sites for the recruitment of molecules involved in signal amplification.

Recently, significant attention has been directed toward a 70-kDa tyrosine-phosphorylated protein undergoing association with TCR in activated T-cells (Chan et al., 1991; Wange et al., 1992). Through protein purification and cDNA cloning, this polypeptide (termed Zap-70) was shown to be a novel member of the Syk family of tyrosine protein kinases, exclusively expressed in T-cells and natural killer cells (Chan et al., 1992). Like Syk, Zap-70 possesses two amino-terminal Src homology 2 (SH2) domains, as well as a carboxyl-terminal catalytic region. Reconstitution experiments in Cos-7 cells have indicated that the association of Zap-70 with TCR requires signals provided by Lck or FynT (Chan et al., 1992). This idea is also supported by the finding that TCR stimulation on Jurkat T-cells failed to provoke association of Zap-70 with TCR in the absence of p56 expression (Iwashima et al., 1994). Seemingly, the Src-related enzymes have the ability to trigger binding of the SH2 domains of Zap-70 to TCR by phosphorylating CD3 and (Wange et al., 1993; Iwashima et al., 1994; Timson-Gauen et al., 1994). Finally, as kinase-defective variants of Zap-70 are still efficiently tyrosine phosphorylated in the Cos-7 system, the tyrosine phosphorylation of Zap-70 is thought not to be the result of autophosphorylation. Instead, it is presumed to be mediated by Src family kinases (Iwashima et al., 1994).

The importance of Zap-70 in normal T-cell physiology has been demonstrated by two lines of experimental evidence. First, Kolanus et al.(1993) reported that antibody-mediated aggregation of chimeric molecules bearing the sequence of Zap-70 linked to the transmembrane and extracellular domains of unrelated molecules is sufficient to cause an elevation of cytoplasmic calcium in T-cells. Hence, Zap-70-mediated signals are likely involved in the regulation of cytosolic calcium during T-cell activation. Second, several groups have demonstrated that the zap-70 gene is mutated in humans with selective T-cell immunodeficiency (Arpaia et al., 1994; Elder et al., 1994; Chan et al., 1994). As Zap-70-deficient individuals exhibit marked alterations in both T-cell differentiation and mature T-cell functions (Arpaia et al., 1994), this observation provides the clearest indication of the central role played by Zap-70 in TCR signaling.

In this report, we have examined the regulation of Zap-70 in an antigen-specific T-cell line. By studying various BI-141 derivatives, we found that expression of activated forms of p56 or p59 or co-aggregation of TCR with CD4 caused an increase in the rapidity and extent of Zap-70 tyrosine phosphorylation during T-cell activation. The activated Src-related enzymes also facilitated the association of Zap-70 with the TCR complex, a likely consequence of their ability to promote tyrosine phosphorylation of CD3 and . Evidence was also obtained that two distinct pools of tyrosine-phosphorylated Zap-70 molecules existed in activated T-cells. While one pool was stably associated with TCR, the other was complexed with a 120-kDa tyrosine-phosphorylated protein (p120) of yet unknown identity. In addition to supporting models of Zap-70 regulation based on studies in heterologous systems, these data indicated that the regulation of Zap-70 is more complex than initially suspected. Furthermore, they raised the possibility that p120 may be an effector and/or a regulator of Zap-70 during T-lymphocyte activation.


MATERIALS AND METHODS

Cells

BI-141 is a CD4-negative, CD8-negative mouse T-cell hybridoma specific for the antigen beef insulin (Reske-Kunz and Rüde, 1985). BI-141 derivatives expressing the neomycin phosphotransferase alone or in combination with tyrosine 505 to phenylalanine 505 (F505) Lck, tyrosine 528 to phenylalanine 528 (F528) FynT, or mouse CD4 were previously reported (Abraham et al., 1991; Caron et al., 1992; Davidson et al., 1992).

Antibody-mediated T-cell Activation

T-cells were activated by stimulation with anti-TCR Vbeta8 mouse monoclonal antibody (MAb) F23.1 (Staerz et al., 1985) followed by sheep anti-mouse (SAM) IgG, as outlined elsewhere (Abraham et al., 1991; Davidson et al., 1992). To ligate CD4, cells were incubated with anti-CD4 rat MAb GK1.5 (Wilde et al., 1983) and rabbit anti-rat IgG. Finally, for co-aggregation of TCR and CD4, cells were treated with MAb F23.1-MAb GK1.5 heteroconjugates (kindly given by Dr. Trevor Owens, Montreal Neurological Institute, Montréal, Québec, Canada), followed by SAM IgG. Following activation, cells were lysed by adding an equal volume of 2 times TNE buffer (100 mM Tris, pH 8.0, 2% Nonidet P-40, 40 mM EDTA), supplemented with 20 µg/ml each of the protease inhibitors leupeptin, aprotinin, N-tosyl-L-phenylalanine chloromethyl ketone, N-p-tosyl-L-lysine chloromethyl ketone, and phenylmethylsulfonyl fluoride, as well as the phosphatase inhibitors sodium fluoride (100 mM) and sodium orthovanadate (2 mM).

Immunoprecipitations

After lysis, specific polypeptides were recovered by immunoprecipitation, using lysates corresponding to equivalent cell numbers. The following antibodies were utilized in our studies: rabbit anti-Zap-70 sera directed either against amino acids 253-329 (``linker'' region) of Zap-70 (918 and 956) (^2)or against the tandem SH2 domains of Zap-70 (provided by Dr. A. S. Shaw, Washington University School of Medicine, St. Louis, MO); rabbit anti-Lck serum 7229 (Abraham et al., 1991); rabbit anti-Fyn serum 428 (Davidson et al., 1992); rabbit anti- serum 387 (provided by Dr. R. D. Klausner, National Institutes of Health, Bethesda, MD); hamster anti- MAb H-146 (provided by Dr. R. T. Kubo, Cytel Corporation, San Diego, CA); hamster anti-CD3 MAb 145-2C11 (Leo et al., 1987); rabbit anti-FAK serum BC3, mouse anti-Fak MAb 2A7, and mouse anti-p110 MAb F1 (provided by Dr. J. T. Parsons, University of Virginia Cancer Center, Charlottesville, VA); rabbit anti-GTPase activating protein serum and rabbit anti-p85 serum (provided by Dr. T. Pawson, Mount Sinai Hospital Research Institute, Toronto, Ontario, Canada); and rabbit anti-Cbl serum R2 (provided by Dr. L. E. Samelson, National Institutes of Health, Bethesda, MD).

Immune complexes were collected with Staphylococcus aureus protein A (Pansorbin, Calbiochem) coupled, if indicated, to the appropriate second step antibody (either rabbit anti-mouse (RAM) IgG or rabbit anti-hamster IgG). After being recovered by centrifugation, immunoprecipitates were washed several times in lysis buffer, eluted in sample buffer, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE).

To immunoprecipitate the TCR complex, cells were stimulated with MAb F23.1 and RAM IgG. After lysis, immune complexes were collected by the addition of S. aureus protein A (Pansorbin, Calbiochem). Control cells were treated in a similar manner, except that lysis was performed prior to the addition of RAM IgG. For these samples, TCR immunoprecipitation was achieved after supplementing the cell lysates with RAM IgG. In some cases, the TCR-depleted lysates were also subjected to a second round of immunoprecipitation, using standard protocols.

When specified, cells were lysed in boiling 2 times TNE buffer supplemented with 1% SDS to disrupt non-covalent complexes. After shearing the DNA and removing particulate matters by centrifugation, lysates were adjusted to a final concentration of 0.1% SDS, using 1 times TNE buffer supplemented with the protease and phosphatase inhibitors outlined above. Subsequent immunoprecipitations were conducted as usual.

Immunoblots

Immunoblots were performed according to a previously described protocol (Veillette et al., 1988a). Anti-phosphotyrosine immunoblots were done using affinity-purified rabbit anti-phosphotyrosine antibodies.^2 The antiserum directed against the linker region of Zap-70 was used for immunoblotting of Zap-70. After incubation of the Immobilon membranes (Millipore) with I-protein A (Amersham Corp.), immunoreactive products were detected by autoradiography and quantitated with a phosphorImager (BAS 2000, Fuji).


RESULTS

Regulation of Zap-70 by Src-related Enzymes in BI-141 T-cells

In the past, the regulation of Zap-70 was primarily examined in non-lymphoid cell systems such as Cos-7 cells and HeLa cells (Chan et al., 1992; Straus and Weiss, 1993, Wange et al., 1993; Iwashima et al., 1994; Timson-Gauen et al., 1994). Thus, to further our comprehension of the interactions between Zap-70 and Src family kinases and to identify potential interactions with other T-cell proteins, we have evaluated the regulation of Zap-70 in the antigen-specific mouse T-cell line BI-141. Previously described derivatives expressing the neomycin phosphotransferase alone or in combination with an activated version of either p56 (tyrosine 505 to phenylalanine 505 (F505) Lck mutant) or p59 (tyrosine 528 to phenylalanine 528 (F528) FynT mutant) were utilized in these studies. Consistent with the proposed roles of Lck and FynT in TCR signaling, BI-141 cells containing activated forms of these Src-like enzymes have been shown to exhibit enhanced TCR-induced tyrosine protein phosphorylation (Abraham et al., 1991; Davidson et al., 1992) (Fig. 1B) and antigen-triggered lymphokine secretion (Abraham et al., 1991; Davidson et al., 1992).


Figure 1: Effects of activated p56 and p59 on Zap-70 tyrosine phosphorylation. Cells were stimulated for the indicated periods of time with anti-TCR MAb F23.1 and SAM IgG and were then lysed in Nonidet P-40-containing buffer. A, anti-Zap-70 immunoprecipitations. Toppanel, anti-phosphotyrosine immunoblot; bottompanel, anti-Zap-70 immunoblot. Exposures: toppanel, 16 h; bottompanel, 6 h. B, total cell lysates. Anti-phosphotyrosine immunoblot is shown. Exposure, 20 h. The positions of prestained molecular mass markers are shown on the right, while those of p120, Zap-70, and heavy chain of IgG (Ig) are indicated on the left. Neo, neomycin-resistant cells.



We first tested the impact of F505 Lck and F528 FynT on the state of tyrosine phosphorylation of Zap-70. After incubation with anti-TCR MAb F23.1, BI-141 cells were activated for variable lengths of time by the addition of SAM IgG. After lysis in non-ionic detergent-containing buffer, Zap-70 was immunoprecipitated using a rabbit serum against its linker region. Zap-70 tyrosine phosphorylation was then ascertained by anti-phosphotyrosine immunoblotting (Fig. 1A, toppanel). This experiment showed that cells bearing activated p56 (lanes7-12) exhibited a marked increase in TCR-induced tyrosine phosphorylation of Zap-70 when compared with cells expressing the neomycin phosphotransferase alone (Neo, lanes1-6). Cells containing activated p59 (lanes 13-18) also demonstrated an enhanced Zap-70 tyrosine phosphorylation response, albeit to a lesser extent than cells expressing F505 Lck. In all cell lines tested, no significant amount of phosphotyrosine was noted in Zap-70 in the absence of TCR stimulation (lanes1, 7, and 13). Furthermore, the induction of Zap-70 tyrosine phosphorylation was proportional to the increase in overall tyrosine protein phosphorylation observed in the various cell types (Fig. 1B). Importantly, an anti-Zap-70 immunoblot of Zap-70 immunoprecipitates verified that the augmented tyrosine phosphorylation of Zap-70 was not due to a change in its cellular abundance (Fig. 1A, bottompanel).

It should be pointed out that Zap-70 immunoprecipitates from TCR-stimulated cells also contained a tyrosine-phosphorylated polypeptide of approximately 120 kDa (p120) (Fig. 1A). The accumulation of p120 was clearly greater in cells containing F505 Lck (lanes 8-12) when compared with neomycin-resistant cells (lanes 1-6). Moreover, it tended to be more rapid and more sustained in F528 FynT-expressing cells (lanes 14-18). The nature of p120 will be further discussed below. In addition, immunoprecipitates of Zap-70 possessed tyrosine-phosphorylated products of 85, 78, and 60 kDa. These polypeptides represent post-translationally modified versions of Zap-70.^2

The impact of the activated Src-like enzymes on the ability of Zap-70 to associate with TCR was also determined. Cells were stimulated with anti-TCR MAb F23.1 and RAM IgG, as described under ``Materials and Methods.'' Following cell lysis, TCR complexes were collected by the addition of S. aureus protein A. After several washes, associated Zap-70 molecules were detected by immunoblotting with anti-Zap-70 antibodies. Untreated control cells were lysed immediately after incubation with MAb F23.1 prior to the addition of the second step antibody. In this case, immunoprecipitation of TCR was achieved by adding RAM IgG and S. aureus protein A to the cell lysate. In the absence of TCR stimulation, no or little Zap-70 was associated with TCR (Fig. 2A, lanes1, 3, 5, 7, 9, and 11). However, upon engagement of TCR, there was easily appreciable binding of Zap-70 to TCR (lanes2, 4, 6, 8, 10, and 12). In comparison with expression of the neomycin phosphotransferase alone (lanes 1-4), introduction of activated p56 (lanes 5-8) or activated p59 (lanes 9-12) caused a 10-fold enhancement of the extent of Zap-70 association with TCR.


Figure 2: Effects of activated p56 and p59 on Zap-70 association with TCR. A, effects of activated Lck and FynT on Zap-70 association with TCR. Cells (5 times 10^6) were activated for 2 min with anti-TCR MAb F23.1 and RAM IgG. After lysis, immune complexes were collected with S. aureus protein A and were probed by anti-Zap-70 immunoblotting. Untreated controls were processed as described under ``Materials and Methods.'' Two different clones of each type were analyzed in these assays. Lanes1-12, anti-TCR immunoprecipitations; lanes13 and 14, anti-Zap-70 immunoprecipitations (obtained from 2 times 10^6 cells stimulated with MAb F23.1 and SAM IgG). Exposure, 18 h. B, titration. Various numbers of F505 Lck-expressing cells were activated for 2 min with MAb F23.1 and SAM IgG (lanes 2-6) or MAb F23.1 and RAM IgG (lanes 8-12). Untreated controls are in lanes1 and 7. Lysates were subjected to either anti-Zap-70 (lanes1-6) or anti-TCR (lanes 7-12) immunoprecipitations and probed by anti-Zap-70 immunoblotting. Exposure, 20 h. The migrations of prestained molecular size markers are shown on the right, and that of Zap-70 is indicated on the left. Neo, neomycin-resistant cells.



To determine the stoichiometry of these interactions, Zap-70 (Fig. 2B, lanes1-6) and TCR (lanes 7-12) were individually immunoprecipitated from increasing numbers of MAb F23.1-stimulated F505 p56-expressing cells. The abundance of Zap-70 in each of these immunoprecipitates was measured by anti-Zap-70 immunoblotting and was quantitated using a PhosphorImager. These data demonstrated that approximately 10% of Zap-70 was complexed to TCR in activated F505 p56-bearing cells. This quantitation is clearly exemplified by comparing the intensity of the signal in lane12 (which corresponds to 2 times 10^6 cells) with those in lanes3 and 4 (which correspond to 0.25 times 10^6 and 0.5 times 10^6 cells, respectively). A similar proportion of Zap-70 bound the TCR in F528 FynT-bearing cells (Fig. 2A, lanes10 and 12). In contrast, however, only 1% of Zap-70 molecules became associated with TCR in neomycin-resistant cells (Neo, lanes2 and 4).

Augmented Association of Zap-70 with TCR Correlates with Enhanced Tyrosine Phosphorylation of CD3 and

The association of Zap-70 with TCR is based on interactions between its two SH2 domains and the tyrosine-phosphorylated CD3 and/or subunits of TCR (Wange et al., 1993; Iwashima et al., 1994; Timson-Gauen et al., 1994). In light of this notion, we wanted to confirm that Lck and FynT increased the formation of TCR-Zap-70 complexes by promoting the tyrosine phosphorylation of CD3/. Cells were stimulated with MAb F23.1 and RAM IgG and lysed in non-ionic detergent-containing buffer; TCR complexes were recovered by adsorption onto S. aureus protein A. After resolving immune complexes in 12% SDS-PAGE gels, tyrosine-phosphorylated polypeptides were detected by anti-phosphotyrosine immunoblotting (Fig. 3A). This assay revealed that the activated versions of Lck (lane4) and FynT (lane6) enhanced the TCR-induced tyrosine phosphorylation of products of 16-20 kDa, consistent with the various phosphorylated forms of (Baniyash et al., 1988; Koyasu et al., 1992). TCR immunoprecipitates from activated cells also contained tyrosine-phosphorylated polypeptides of 23-28 kDa, which likely corresponded to components of the CD3 complex (Wange et al., 1992; Qian et al., 1993).


Figure 3: Impact of activated p56 and p59 on tyrosine phosphorylation of CD3 and . A, anti-phosphotyrosine immunoblot. Cells were stimulated for 2 min with anti-TCR MAb F23.1 and RAM IgG, as described in the legend of Fig. 2A. After immunoprecipitation, TCR complexes were separated in 12% SDS-PAGE gels and probed by anti-phosphotyrosine immunoblotting. Lanes1, 3, and 5, untreated controls; lanes2, 4, and 6, anti-TCR-stimulated cells. Exposure, 6 days. B, time-course experiment. Cells were activated for the indicated periods of time with anti-TCR MAb F23.1 and RAM IgG. After lysis, TCR immunoprecipitates were resolved by 12% SDS-PAGE and probed by immunoblotting with anti-Zap-70 (toppanel) or anti-phosphotyrosine (bottompanel) antibodies. These two immunoblots were derived from the same gel. Exposure: toppanel, 22 h; bottompanel, 5 days. The positions of prestained molecular mass markers are shown on the right, while those of Zap-70, heavy chain of IgG (Ig), CD3, and are indicated on the left. Neo, neomycin-resistant cells.



These findings suggested that the ability of activated Src-related enzymes to facilitate the association of Zap-70 with TCR was indeed related to their efficiency at phosphorylating CD3/. To obtain further support for this conclusion, the kinetics and extent of CD3/ tyrosine phosphorylation were compared with those of Zap-70 binding to TCR. Thus, TCR immunoprecipitates from resting or activated cells were immunoblotted with either anti-Zap-70 (Fig. 3B, toppanel) or anti-phosphotyrosine (bottompanel) antibodies. These assays showed that, both in neomycin-resistant cells (lanes1-6) and in cells containing F505 p56 (lanes7-12), the onset and extent of Zap-70 association with TCR closely followed those of tyrosine phosphorylation of CD3/. Moreover, the disappearance of TCR-bound Zap-70 (toppanel) clearly coincided with the loss of tyrosine-phosphorylated CD3/ (bottompanel).

Regulation of Zap-70 by Engagement of the CD4 Co-receptor

Previous studies have shown that expression of CD4 greatly enhanced the antigen responsiveness of BI-141 cells (Ballhausen et al., 1988), a likely consequence of the ability of this cell surface molecule to bind and activate p56 (Veillette et al., 1988b; Rudd et al., 1988; Veillette et al., 1989; Luo and Sefton, 1990). Therefore, to study the impact of p56 activation in a more physiological context, we tested the effects of antibody-mediated ligation of CD4 on the properties of Zap-70. CD4-positive BI-141 derivatives were incubated with anti-TCR MAb F23.1, anti-CD4 MAb GK1.5, or MAb F23.1-MAb GK1.5 heteroconjugates and subsequently activated by addition of the appropriate second step antibody. Under these conditions, little or no tyrosine phosphorylation of cellular proteins was induced by ligation of TCR or CD4 alone (Fig. 4A, lanes12 and 13, respectively). In contrast, however, marked accumulation of phosphotyrosine-containing proteins was provoked by co-aggregating TCR and CD4 (lane14).


Figure 4: Regulation of Zap-70 by engagement of the CD4 co-receptor. A, Zap-70 tyrosine phosphorylation. Cells were stimulated for 2 min by incubation with the indicated antibodies, followed by SAM IgG (except for lanes5 and 13, where rabbit anti-rat IgG was used). After lysis, Zap-70 was immunoprecipitated and probed by immunoblotting with either anti-phosphotyrosine (toppanel) or anti-Zap-70 (bottompanel) antibodies. Lanes9-16 represent total cell lysates. Exposure: toppanel, 20 h; bottompanel, 15 h. B, Zap-70 association with TCR. Cells (5 times 10^6) were stimulated as described for A, except that RAM IgG was used as a second-step antibody. TCR complexes were collected with S. aureus protein A and were probed by anti-Zap-70 immunoblotting. Lanes9 and 10 were obtained from 2 times 10^6 cells immunoprecipitated with anti-Zap-70 antibodies. Exposure, 14 h. The migrations of prestained molecular size markers are shown on the right, and those of p120, Zap-70, and heavy chain of IgG (Ig) are indicated on the left. Neo, neomycin-resistant cells.



Changes in Zap-70 tyrosine phosphorylation were monitored by anti-phosphotyrosine immunoblotting of Zap-70 immunoprecipitates (Fig. 4A, toppanel). Whereas ligation with anti-TCR MAb F23.1 alone (lane4) or anti-CD4 MAb GK1.5 alone (lane5) did not cause tyrosine phosphorylation of Zap-70, co-aggregation of TCR and CD4 (lane6) promptly increased the phosphotyrosine content of Zap-70 in a manner analogous to expression of F505 p56 (lane8). Quantitative analyses of these data indicated that Zap-70 was seven times more tyrosine phosphorylated after co-aggregation of TCR and CD4 (lane6), when compared with stimulation of TCR alone (lane4). Co-ligation of TCR with CD4 also increased the recovery of tyrosine-phosphorylated p120 by approximately 5-fold (lane6).

The extent of association of Zap-70 with TCR was also determined (Fig. 4B). Antibody-mediated co-aggregation of TCR and CD4 significantly enhanced the binding of Zap-70 to TCR (lane6). Indeed, while essentially no detectable Zap-70 was bound to TCR in MAb F23.1-stimulated cells (lane4), approximately 5% of these molecules became associated with TCR in cells treated with MAb F23.1-MAb GK1.5 heteroconjugates (lane6).

Two populations of Tyrosine-phosphorylated Zap-70 Molecules Are Detected in Activated BI-141 T-cells

Next, we surveyed the relative distribution of tyrosine-phosphorylated Zap-70 molecules in activated BI-141 cells. TCR- and non-TCR-associated Zap-70 were fractionated by sequential immunoprecipitation with anti-TCR and anti-Zap-70 antibodies. The phosphotyrosine content of these two populations was then determined by anti-phosphotyrosine immunoblotting (Fig. 5A, toppanel). The abundance of Zap-70 in these two fractions was also measured by anti-Zap-70 immunoblotting (bottompanel). In the various BI-141 derivatives tested, the majority of tyrosine-phosphorylated Zap-70 molecules was not stably associated with TCR (compare lane2 with lane8; lane4 with lane10; and lane6 with lane12). A time course of activation of F505 Lck-containing cells revealed that the association of tyrosine-phosphorylated Zap-70 with TCR was transient, peaking at 1 min after TCR stimulation (Fig. 5B, toppanel, lanes1-6). In contrast, the accumulation of tyrosine-phosphorylated Zap-70 was more sustained in TCR-depleted lysates (lanes 7-12, toppanel). Indeed, it persisted up to 10 min after the onset of TCR stimulation (lane12). These studies also showed that tyrosine-phosphorylated p120 was present in Zap-70 immunoprecipitates obtained from TCR-depleted lysates (lanes7-12). However, it was not detected in anti-TCR immunoprecipitates (lanes 1-6), even upon longer autoradiographic exposure (data not shown).


Figure 5: Identification of two populations of tyrosine-phosphorylated Zap-70 in activated BI-141 T-cells. Cells were stimulated with anti-TCR MAb F23.1 and RAM IgG and lysed, and TCR complexes were immunoprecipitated by the addition of S. aureus protein A. TCR-depleted lysates were subjected to re-immunoprecipitation with anti-Zap-70 antibodies. Immunoprecipitates were blotted with either anti-phosphotyrosine (toppanels) or anti-Zap-70 (bottompanels) antibodies. A, characterization of cells expressing F505 Lck and F528 FynT. All cells were stimulated for 2 min with anti-TCR MAb F23.1 and RAM IgG. Exposures: toppanel, 16 h; bottompanel, 16 h. B, time course of TCR stimulation on F505 Lck-expressing cells. Exposures: toppanel, 16 h; bottompanel, 14 h. The positions of prestained molecular mass markers are shown on the right, and those of p120, Zap-70, and heavy chain of IgG (Ig) are indicated on the left. Neo, neomycin-resistant cells.



Because the association of CD3/ with TCR can be dissociated by non-ionic detergents such as Nonidet P-40, we wished to verify that the detection of a non-TCR-bound pool of tyrosine-phosphorylated Zap-70 was not consequent to artificial dissociation of CD3/ from TCR. To this end, TCR-depleted lysates were re-immunoprecipitated with antibodies directed against Zap-70 (Fig. 6, lane3), (lanes4 and 5), or CD3- (lane6), and recovered polypeptides were probed by anti-phosphotyrosine immunoblotting. While abundant quantities of phosphotyrosine-containing Zap-70 were present in TCR-depleted lysates (lane3, toppanel), these lysates contained little or no tyrosine-phosphorylated CD3 or (lanes4, 5, and 6, bottompanel). Since the association of Zap-70 with CD3/ is known to be resistant to the presence of Nonidet P-40 (Chan et al., 1991), this observation implied that the accumulation of tyrosine-phosphorylated Zap-70 in TCR-depleted lysates was not due to post-lysis dissociation of CD3/ from TCR.


Figure 6: Characterization of TCR-depleted lysates. TCR-depleted lysates from F505 p56-containing cells were re-immunoprecipitated with the indicated antibodies. After separation in 12% SDS-PAGE gels, polypeptides were detected by immunoblotting with anti-phosphotyrosine antibodies. The top and bottompanels correspond to different exposures of the same immunoblot. The absence of a detectable heavy chain of IgG in lane5 is due to the fact that MAb H146 does not require a second-step antibody for binding S. aureus protein A. The 28-kDa immunoreactive product present in anti-Zap-70 immunoprecipitates (lane3) is seemingly a degradation product of Zap-70.^2 Exposures: toppanel, 16 h; bottompanel, 6 days. The positions of prestained molecular mass markers are shown on the right, and those of p120, Zap-70, heavy chain of IgG (Ig), CD3, and are indicated on the left.



Characterization of the 120-kDa Tyrosine-phosphorylated Protein Observed in Anti-Zap-70 Immunoprecipitates

The experiments depicted above indicated that a 120-kDa tyrosine-phosphorylated polypeptide (p120) was present in anti-Zap-70, but not anti-TCR, immunoprecipitates from activated BI-141 cells (Fig. 1A, Fig. 4A, and Fig. 5B). To characterize this product, we first wanted to ensure that it was specifically co-immunoprecipitated with Zap-70. In the study shown in Fig. 7A, no appreciable amount of p120 was present in either anti-Lck (lane2) or anti-Fyn (lane3) immunoprecipitates or in immune complexes obtained using pre-immune serum (lane1). In keeping with former reports, however (da Silva et al., 1993; Tsygankov et al., 1994; Reedquist et al., 1994), small quantities of a tyrosine-phosphorylated p120 were observed in anti-Fyn immunoprecipitates upon longer autoradiographic exposures of this immunoblot (data not shown). It is not known whether this polypeptide was identical to the p120 found in anti-Zap-70 immunoprecipitates. In a previous publication (Weil and Veillette, 1994), we also demonstrated that p120 was not present in immunoprecipitates of phospholipase C-1, Vav, ezrin, Hcp, and Shc.


Figure 7: Characterization of p120. F505 p56-bearing cells were stimulated for 2 min with anti-TCR MAb F23.1 and SAM IgG. Immunoprecipitates were then analyzed by anti-phosphotyrosine immunoblotting. A, specificity of co-immunoprecipitation of Zap-70 and p120. Exposure, 16 h. B, p120 is co-immunoprecipitated by multiple anti-Zap-70 sera. Exposure, 4 days. C, effects of denaturation on the Zap-70-p120 interaction. Cells were stimulated for the indicated periods of time with anti-TCR MAb F23.1 and SAM IgG. They were then lysed in either standard Nonidet P-40-containing lysis buffer (lanes1-6) or in boiling SDS-containing buffer (lanes 7-12). Exposure, 2 days. D, effects of phenylphosphate on the Zap-70-p120 interaction. After lysis in Nonidet P-40-containing buffer, Zap-70 immunoprecipitations were conducted in the presence of progressively higher concentrations of phenylphosphate (lanes1-6) or phosphoserine (lanes7-12). Exposure, 2 days. The positions of prestained molecular mass markers are shown on the right, while those of p120, Zap-70, and heavy chain of IgG (Ig) are indicated on the left.



Two independent rabbit antisera generated against the linker region of Zap-70, as well as a polyclonal rabbit serum recognizing the SH2 domains of Zap-70, were evaluated in our assays (Fig. 7B). The two anti-linker sera were equally efficient at recovering the tyrosine-phosphorylated p120 (lanes2 and 4). The antiserum reacting with the Zap-70 SH2 domains also precipitated p120 (lane6), albeit at a lower efficiency. This serum was also less adequate at immunoprecipitating Zap-70 (lane6; data not shown). Similar data were obtained using an antiserum against the carboxyl terminus of Zap-70 (data not shown). Perhaps the antibodies directed against the linker region (which lies between the end of the second SH2 domain and the beginning of the catalytic region) stabilized the interaction of Zap-70 with p120, thus facilitating its recovery. Alternatively, the anti-SH2 domains and anti-carboxyl terminus sera may have lowered the immunoprecipitation of p120 by causing its displacement from Zap-70.

These results implied that p120 specifically interacted with Zap-70 in activated BI-141 cells. To better understand the basis for this interaction, the effects of denaturation were examined (Fig. 7C). Cells were lysed either in standard Nonidet P-40-containing buffer (lanes1-6) or in boiling SDS-containing buffer (lanes7-12) prior to immunoprecipitation with anti-Zap-70 antibodies. An anti-phosphotyrosine immunoblot of these immunoprecipitates revealed that the presence of SDS completely abolished the association of p120 with Zap-70 (lanes7-12). Although the ability to immunoprecipitate Zap-70 was also partially reduced in the SDS-containing buffer, this diminution was probably consequent to the added ionic strength provided by the detergent.

The potential involvement of tyrosine-phosphorylated residues in the formation of this complex was also evaluated (Fig. 7D). Zap-70 was immunoprecipitated in the absence or presence of progressively higher concentrations of either phenylphosphate, an analog of phosphotyrosine (lanes2-6), or phosphoserine (lanes 8-12). A subsequent anti-phosphotyrosine immunoblot showed that phenylphosphate caused a dose-dependent dissociation of p120 from Zap-70 (lanes 2-6), which was nearly complete at a concentration of 50 mM (lane5). A similar effect was obtained with phosphotyrosine (data not shown). In contrast, however, equivalent concentrations of phosphoserine failed to interfere with the binding of p120 to Zap-70 (lanes 8-12).

In an attempt to identify p120, lysates were immunoprecipitated with antibodies against known tyrosine phosphorylation substrates of approximately 120 kDa. The phosphotyrosine content of these products in activated BI-141 cells was then measured by anti-phosphotyrosine immunoblotting (Fig. 8). No significant tyrosine phosphorylation was detected in immunoprecipitates of the 110-kDa phosphatidylinositol 3` kinase (precipitated with anti-p85 antibodies) (lane3), the 110-kDa actin-binding protein p110 (lane4), the 120-kDa GTPase-activating protein of p21 (lane5), and the integrin-regulated tyrosine protein kinase p125 (lanes6 and 7). Recent data also demonstrated that p120, the cellular homolog of the Cbl oncoprotein, undergoes prominent tyrosine phosphorylation in activated Jurkat T-cells (Donovan et al., 1994). While anti-Cbl antibodies also identified a prominent 120-kDa tyrosine-phosphorylated protein in activated BI-141 cells (Fig. 8B, lane3), this polypeptide was not detectably associated with Zap-70. Based on this result, it appeared unlikely that the 120-kDa Zap-associated protein was Cbl.


Figure 8: The Zap-70-associated p120 is distinct from known tyrosine phosphorylation substrates. F505 p56-bearing cells were stimulated for 2 min with anti-TCR MAb F23.1 and SAM IgG. Various immunoprecipitates were then analyzed by anti-phosphotyrosine immunoblotting. Exposures, 17 h. NRS, normal rabbit serum.




DISCUSSION

In this paper, we have studied the regulation of Zap-70 in the antigen-specific T-cell line BI-141. We found that expression of activated versions of p56 or p59 caused an appreciable increase in the rapidity, intensity, and duration of Zap-70 tyrosine phosphorylation during T-cell activation (Fig. 1A). Furthermore, using CD4-positive BI-141 derivatives, it was observed that antibody-mediated aggregation of TCR with the CD4 co-receptor allowed a greater accumulation of tyrosine-phosphorylated Zap-70 (Fig. 4A). As ligation of CD4 is known to activate p56 (Veillette et al., 1989; Luo and Sefton, 1990), this observation further supported the notion that the recruitment of Zap-70 in TCR signaling is regulated by Src-like enzymes.

We also demonstrated that activated Lck and FynT, as well as co-ligation of TCR with CD4, increased the extent of Zap-70 association with TCR by 5-10-fold ( Fig. 2and Fig. 4B). Importantly, this enhancement paralleled the ability of Lck and FynT to promote CD3 and tyrosine phosphorylation (Fig. 3). Indeed, in careful time-course analyses, it was shown that the accumulation of TCRbulletZap-70 complexes in activated T-cells coincided with the induction of CD3/ tyrosine phosphorylation.

Given the sensitivity of the anti-Zap-70 antibodies used in our studies, we were able to obtain an assessment of the stoichiometry of association of Zap-70 with TCR. Under linear assay conditions, it was estimated that roughly 1% of Zap-70 molecules became bound to TCR in activated neomycin-resistant BI-141 T-cells. This proportion rose to 10% in cells bearing activated versions of p56 or p59. Furthermore, in CD4-positive BI-141 derivatives, approximately 5% of Zap-70 polypeptides became associated with TCR upon co-aggregation of TCR with CD4.

Together, these observations supported the model proposed by others (Chan et al., 1992; Iwashima et al., 1994; Timson-Gauen et al., 1994), which was primarily based on reconstitution experiments in non-lymphoid cells. Seemingly, engagement of TCR triggers the T-cell activation cascade by allowing Lck and/or FynT to phosphorylate CD3/. This phosphorylation allows the recruitment of Zap-70 at the membrane, where it is susceptible to phosphorylation by Src-related enzymes. Membrane-associated and tyrosine-phosphorylated Zap-70 is presumably able to phosphorylate its putative targets and/or to behave as a docking molecule, capable of recruiting additional signal transducers at the plasma membrane.

These concepts are somewhat complicated by the finding that two distinct pools of tyrosine-phosphorylated Zap-70 molecules were present in activated BI-141 cells (Fig. 5). Whereas one pool was stably associated with TCR, thus fulfilling the scheme described above, a second more prominent population was not detectably associated with TCR. In light of this observation, special efforts were made to rule out the possibility that the non-TCR-bound Zap-70 was produced as a result of artificial dissociation of CD3/ from TCR (Fig. 6). However, we felt confident that our TCR immunoprecipitates contained essentially all the tyrosine-phosphorylated CD3/ molecules from activated BI-141 cells. Coupled with the fact that the interaction between CD3/ and Zap-70 is not affected by lysis conditions similar to those used in our studies (Chan et al., 1991), it thus appeared unlikely that the existence of non-TCR-associated tyrosine-phosphorylated Zap-70 was consequent to in vitro disruption of the antigen receptor complex.

The mechanisms of production, as well as the functions of these two groups of tyrosine-phosphorylated Zap-70 remain to be clarified. Our kinetics analyses indicated that the presence of TCR-associated tyrosine-phosphorylated Zap-70 was transient in activated T-cells (Fig. 5B). In contrast, the accumulation of non-TCR-associated polypeptides tended to be more sustained. Because tyrosine phosphorylation of both pools of Zap-70 was increased by activated Src-related enzymes (Fig. 5A), it is conceivable that they had a common origin. Possibly, the non-TCR-associated population was generated following in vivo dissociation of the TCR-bound pool, perhaps as a consequence of CD3/ dephosphorylation. Although it seems somewhat less likely, it remains also possible that the two populations of Zap-70 were produced through distinct processes. Future studies will be necessary to test these possibilities.

Interestingly, we noted that non-TCR-associated Zap-70 molecules from activated BI-141 cells specifically co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated polypeptide. In addition to lending credence to the notion that the two pools of Zap-70 may serve distinct functions during TCR signaling, this finding intimated that p120 may be either a substrate or a regulator of Zap-70. Unfortunately, however, we have not yet been able to define the identity of p120. Our results indicated that the Zap-70-associated p120 was not the catalytic subunit of phosphatidylinositol 3` kinase, GTPase-activating protein, p125, or the 110-kDa actin-binding protein (p110) (Fig. 8A). Furthermore, it did not appear to be Cbl, a 120-kDa polypeptide recently shown to undergo prominent tyrosine phosphorylation in activated T-lymphocytes (Fig. 8B) (Donovan et al., 1994). While not formally excluded, it is also improbable that p120 was a protein kinase, as it failed to undergo phosphorylation during immune complex kinase reactions.^2

Additional biochemical characterizations showed that the Zap-70bulletp120 complex was dissociated by boiling in the presence of SDS, implying that it was non-covalent in nature (Fig. 7C). Moreover, it could be disrupted by either phenylphosphate (Fig. 7D) or phosphotyrosine (data not shown), suggesting that it was dependent on the presence of phosphorylated tyrosine residues. However, the complex did not apparently involve the SH2 domains of Zap-70, as recombinant fusion proteins bearing this portion of Zap-70 could not recover tyrosine-phosphorylated p120 from activated T-cell lysates.^2 Therefore, the putative critical sites of tyrosine phosphorylation may be situated on Zap-70 itself. Obviously, further characterization of p120 is likely to provide useful insights into the regulation and function(s) of Zap-70 in T-lymphocytes.

A recent report suggested that Syk, a Zap-70-related tyrosine protein kinase expressed in at least some T-cells, also plays a prominent role in T-cell receptor signaling (Couture et al., 1994). It was observed that, unlike Zap-70, Syk is constitutively associated with TCR. Furthermore, the authors reported that TCR stimulation caused an activation of the catalytic function of Syk. They proposed that Syk-mediated signals during T-cell activation may be more proximal than those provided by Src-related enzymes or Zap-70. While it is possible that this scheme applies to a subset of T-cells, it should be pointed out that we have been unable to detect expression of Syk in BI-141 T-cells, using at least two different anti-Syk sera.^2 Hence, based on our data, Syk is clearly not an essential component of the TCR signaling pathway.

In summary, we have shown that the ability of Zap-70 to undergo tyrosine phosphorylation and associate with TCR is markedly enhanced by expression of activated forms of p56 and p59 or by ligation of TCR with the CD4 co-receptor. The extent and duration of Zap-70 association with TCR correlated with those of tyrosine phosphorylation of CD3 and , thus supporting the notion that Src-related enzymes initiate TCR-mediated signals by catalyzing the tyrosine phosphorylation of these TCR components. Our studies also allowed the identification of two distinct pools of tyrosine-phosphorylated Zap-70 in activated T-cells. Whereas one pool was associated with TCR, the other was devoid of demonstrable interactions with this structure. Even though the nature and function of these two populations were not determined, it was observed that the non-TCR-associated fraction specifically co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated polypeptide of yet undefined identity. These data definitely add further complexity to previous models of regulation of Zap-70. Furthermore, they suggest that p120 may be a target and/or a regulator of Zap-70 in activated T-lymphocytes.


FOOTNOTES

*
This work was supported by grants from the Cancer Research Society and the Medical Research Council of Canada. 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.

§
The recipient of a Fellowship from the Cancer Research Society. Present address: Département de Biologie Moléculaire, Institut Pasteur, 75724 Paris, Cédex 15 France.

Holds a Studentship from the Fonds Formation de Chercheurs et L'aide À La Recherche.

**
A Scientist of the Medical Research Council of Canada. To whom correspondence should be addressed: Rm. 715, McIntyre Medical Sciences Bldg., McGill University, 3655 Drummond St., Montréal H3G 1Y6, Canada. Tel.: 514-398-8936; Fax: 514-398-6769; veillette{at}medcor.mcgill.ca.

(^1)
The abbreviations used are: TCR, T-cell receptor; SAM, sheep anti-mouse; RAM, rabbit anti-mouse; IL, interleukin; SH2, Src homology 2; MAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.

(^2)
R. Weil, J.-F. Cloutier, M. Fournel, and A. Veillette, unpublished data.


ACKNOWLEDGEMENTS

We acknowledge Drs. Tom Parsons, Tony Pawson, Trevor Owens, Rick Klausner, Ralph Kubo, Andrey Shaw, and Larry Samelson for gifts of antibodies.


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