(Received for publication, May 5, 1995; and in revised form, June 2, 1995)
From the
ZAP-70 is a 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that it is positively regulated by tyrosine phosphorylation. Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement. To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for the tyrosine phosphorylation-induced activation of ZAP-70. The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation. These results reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.
ZAP-70 is a protein tyrosine kinase (PTK) ()that
plays an essential role in T lymphocyte development and activation. Its
role in these processes has been demonstrated in recent studies showing
a loss of T cell signaling upon disruption of ZAP-70 recruitment to the
activated T cell antigen receptor (TCR) and by the identification of
patients who lack ZAP-70 and exhibit a severe combined
immunodeficiency(1, 2, 3, 4) .
ZAP-70 is 620 amino acids in length and is composed of 3 identifiable
domains, a tyrosine kinase domain and two SH2 domains that mediate
association of ZAP-70 with the activated
TCR(5, 6, 7, 8) .
Because of the important role played by ZAP-70 in regulating T lymphocyte activation and development, there is much interest in the investigation of the mechanism of regulation of ZAP-70 kinase activity. It has been shown that recruitment to the activated TCR is necessary (4) , but insufficient for ZAP-70 activation(9, 10) . There is a strong correlation between tyrosine phosphorylation of ZAP-70 and activation of its kinase activity(4, 10, 11) , and it is likely that after recruitment to the TCR one or more tyrosine residues of ZAP-70 must become phosphorylated in order for ZAP-70 to become activated. We have recently identified the tyrosine residues in ZAP-70 that become phosphorylated in vivo in Jurkat T cells upon TCR stimulation. These are tyrosine 292, which lies in the region between the C-terminal SH2 domain and the kinase domain, and tyrosines 492 and 493, within the kinase domain(12) .
Activation of PTKs by phosphorylation of critical tyrosine residues within the kinase domain is a well recognized mechanism of positive regulation in virtually all PTKs (for review, see (13) and (14) ). A common approach for studying the effects of phosphorylation of a particular tyrosine upon the activity of a kinase is to mutate the tyrosine residue to phenylalanine. For example, using this approach, tyrosine 416 of Src, tyrosine 394 of Lck, and tyrosines 1162 and 1163 of the insulin receptor kinase (IRK) were all identified as being critical sites of autophosphorylation and positive regulation within these kinases(15, 16, 17, 18, 19, 20, 21) .
To study the role of phosphorylation of tyrosines within the kinase domain of ZAP-70 upon its activity, tyrosines 492 and 493 in ZAP-70 were individually mutated to phenylalanine. The mutated enzymes were expressed in COS cells either alone or with constitutively activated Y505F-Lck, and the ZAP-70-associated activity was accessed via in vitro kinase assays. We found that tyrosine 493 is necessary for phosphorylation-induced activation of ZAP-70 kinase activity and appears to be requisite for additional tyrosine phosphorylation events. Interestingly, mutation of tyrosine 492 to phenylalanine activates ZAP-70.
To examine the role played by tyrosines 492 and 493 in the regulation of ZAP-70 kinase activity, these residues were individually mutated to phenylalanine by site-directed mutagenesis. As COS cell transfections have previously been shown to be useful in the study of ZAP-70 activity(5, 7) , wild type (WT) and mutated ZAP-70 were expressed in COS cells and precipitated from cell lysates via a C-terminal epitopic tag containing 9 amino acids from c-Myc, recognized by the mAb 9E10. An immune-complex kinase assay was performed on these immunoprecipitates. Anti-ZAP-70 immunoblotting was used to show that comparable amounts of ZAP-70 were recovered in the 9E10 immunoprecipitates from the different transfections (Fig.1, upper panel). ZAP-70 expressed in COS cells had basal kinase activity, as evidenced by the appearance of autophosphorylated ZAP-70 and increased phosphorylation of the exogenous substrate, cfb3, in the immune-complex kinase assay (Fig.1, lower panel). Surprisingly, the Y492F-mutated ZAP-70 showed markedly increased specific activity compared to WT. Densitometric analysis of the data in Fig.1(normalized for the amount of ZAP-70 in each reaction), as well as data from three similar experiments (not shown), indicates that mutation of Tyr-492 to phenylalanine resulted in an approximately 4-fold increase in basal kinase activity. The Y493F-mutated ZAP-70, on the other hand, had the same specific activity as WT ZAP-70.
Figure 1:
Basal kinase activity of mutated
ZAP-70. COS cells were transiently transfected with 3 µg of
pSXSR, pSXSR
-ZAP-Myc (wild type), pSXSR
-ZAP-Myc (Y492F), or pSXSR
-ZAP-Myc (Y493F). Anti-Myc
immunoprecipitates from cell lysates were subjected to an in vitro kinase assay. The products were then separated by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose. ZAP-70 was detected by anti-ZAP-70 immunoblotting (top panel). In vitro phosphorylated proteins were
detected by autoradiography (lower panel). Results are
representative of more than 3 similar
experiments.
The increased basal specific activity of the Y492F mutation was unanticipated, and we were concerned about the possibility that the mutation of Tyr-492 to phenylalanine had facilitated an association of ZAP-70 with a COS cell protein that either allosterically activated the kinase activity of ZAP-70 or that itself possessed kinase activity. To test this possibility, we repeated the experiment in Fig.1, comparing either mild or stringent wash conditions. The mild wash condition entails 3 washes with Brij 96 lysis buffer, while the stringent wash condition employs 2 washes with Brij 96 lysis buffer and additional washes with LiCl and water (see ``Materials and Methods''). Comparable amounts of ZAP-70 were recovered for each of the samples (not shown). Surprisingly, immune-complex kinase assays on anti-Myc immunoprecipitates prepared in this manner showed that the stringent wash condition lead to higher, not lower, kinase activity (Fig.2). Possible mechanisms of this stringent wash activation are discussed below. This result suggests that a co-precipitating COS cell protein was not responsible for the increased kinase activity of the Y492F-mutated ZAP-70.
Figure 2:
Effect of wash stringency or pervanadate
treatment on ZAP-70-associated kinase activity. COS cells were
transiently transfected with 3 µg of pSXSR, pSXSR
-ZAP-Myc (wild type), pSXSR
-ZAP-Myc (Y492F), or
pSXSR
-ZAP-Myc (Y493F). One-half of the COS cells were
treated with pervanadate for 20 min at 37 °C, while the other half
were incubated without pervanadate. The anti-Myc immunoprecipitates
were split and subjected to either mild or stringent wash conditions,
followed by an in vitro kinase assay and autoradiography or
anti-phosphotyrosine immunoblotting. Results are representative of 3
similar experiments.
Previous studies have demonstrated that tyrosine phosphorylation of ZAP-70 is correlated with increased ZAP-70 kinase activity(4, 10, 11) . To examine whether or not the Y492F and Y493F mutants of ZAP-70 could be activated by tyrosine phosphorylation in vivo, transfected COS cells were incubated with pervanadate, a potent membrane-permeant protein tyrosine phosphatase inhibitor(30, 31, 32) . Pervanadate incubation caused an increase in the accumulation of phosphotyrosine in WT and in Y492F-mutated ZAP-70, while relatively little tyrosine phosphorylation was induced in Y493F-mutated ZAP-70. This finding is consistent either with phosphorylation of Tyr-493 being requisite for phosphorylation of other tyrosine residues in ZAP-70 or with Tyr-493 being the major site of tyrosine phosphorylation in ZAP-70. This latter possibility is not supported by the mapping studies of the sites of tyrosine phosphorylation in ZAP-70(12) . The increased tyrosine phosphorylation of WT and Y492F-mutated ZAP-70 induced by pervanadate treatment was correlated with increased kinase activity, while the Y493F-mutated ZAP-70 was poorly tyrosine-phosphorylated, and its activity was indistinguishable from basal levels.
As Lck has been shown to tyrosine-phosphorylate ZAP-70 in vitro and to be required for optimal ZAP-70 activity in COS cell transfections(5, 7, 12) , we examined the effect of co-transfection of WT ZAP-70 and constitutively activated Y505F-Lck on the phosphotyrosine content and kinase activity of ZAP-70. Co-transfection of ZAP-70 and Y505F-Lck resulted in increased tyrosine phosphorylation of ZAP-70 and increased ZAP-70 kinase activity compared to ZAP-70 expressed alone (Fig.3), consistent with previous findings(5, 7) . Different amounts of ZAP-70 DNA were compared in the transfections, so as to achieve expression levels of ZAP-70 that would be comparable between the single and co-transfections. The amount of ZAP-70 recovered from the transfected cell lysates was determined by immunoblotting for ZAP-70 (top panel). Tyrosine phosphorylation of ZAP-70, as detected by anti-phosphotyrosine blotting, was observed only in ZAP-70 precipitated from COS cells co-transfected with Lck (second panel). In this experiment, little or no ZAP-70-associated kinase activity (as measured by cfb3 phosphorylation) was measured above background (vector alone) in the absence of Lck co-expression (third panel), even when ZAP-70 was expressed alone at relatively high levels (lane 3). Anti-phosphotyrosine blotting of anti-Lck immunoprecipitates demonstrated the presence of Y505F-Lck expression in the appropriate samples (lanes 4 and 5, bottom panel). Under the conditions employed in this experiment, Y505F-Lck does not co-precipitate with ZAP-70 in the 9E10 immunoprecipitations and does not contribute toward the accumulation of phosphate on cfb3 (not shown).
Figure 3:
Effect of Y505F-Lck co-expression on wild
type ZAP-70 kinase activity. COS cells were transiently transfected
with 3 µg of pSXSR (lane 1), 3 µg of
pSXSR
-ZAP-Myc (lane 2), 10 µg of pSXSR
-ZAP-Myc (lane 3), 1 µg of pSXSR
-ZAP-Myc and 1 µg of
pSXSR
-Lck (Y505F) (lane 4), or 3 µg of
pSXSR
-ZAP-Myc and 3 µg of pSXSR
-Lck (Y505F) (lane
5). Anti-Myc or anti-Lck immunoprecipitates were prepared from the
cell lysates. The anti-Myc immunoprecipitates were subjected to ZAP-70
immunoblotting (top panel), phosphotyrosine immunoblotting (second panel), or an in vitro kinase assay (third panel). Anti-Lck immunoprecipitates were immunoblotted
for phosphotyrosine (bottom panel). Results are representative
of 3 similar experiments.
The mutant ZAP-70 molecules were similarly analyzed with and without Y505F-Lck co-transfection (Fig.4). The Y492F mutant of ZAP-70 exhibited increased tyrosine phosphorylation and increased specific activity toward the exogenous substrate cfb3, in response to co-transfection with Y505F-Lck. The specific activity of WT ZAP-70 also increased in response to Y505F-Lck co-transfection; however, in this experiment, this increase was masked by a 2.5-fold lower level of expression of WT ZAP-70 in the co-transfected COS cells (bottom panel). While the Y493F mutant of ZAP-70 showed increased incorporation of phosphate on tyrosine, its kinase activity did not increase in response to Y505F-Lck co-transfection. Since these results are from overexpressed proteins in COS cells, it is possible that some of the tyrosine phosphorylation in Y493F-ZAP-70 is due to phosphorylation of tyrosine residues irrelevant to ZAP-70 activation, as Lck has been shown to be able to phosphorylate numerous residues in vitro which are not phosphorylated in vivo(12) .
Figure 4:
Effect of Y505F-Lck co-expression on
mutant ZAP-70 kinase activity. COS cells were transiently transfected
with 3 µg of pSXSR, pSXSR
-ZAP-Myc (wild type),
pSXSR
-ZAP-Myc (Y492F), or pSXSR
-ZAP-Myc (Y493F) ± 3 µg of pSXSR
-Lck (Y505F).
Anti-Myc immunoprecipitates from cell lysates were subjected to an in vitro kinase assay (top panel),
anti-phosphotyrosine immunoblotting (middle panel), or
anti-ZAP-70 immunoblotting (bottom panel). Results are
representative of more than 3 similar
experiments.
Virtually all PTKs are themselves substrates for tyrosine phosphorylation. In some PTKs, such as the Src family, there are tyrosine residues, generally outside the kinase domain, which inhibit kinase activity when phosphorylated, and are largely the substrate of other regulatory PTKs. Other sites include critical tyrosine residues present in the kinase domain of all PTKs. Phosphorylation of these sites is usually by autophosphorylation (either trans or cis) and is required for full activation of the kinase(13, 14) .
These latter sites of
phosphorylation have been studied extensively in the Src family of PTKs
and in the insulin receptor kinase (IRK). In Src, the principle site of
autophosphorylation is Tyr-416, which is found in the sequence context
of EYTAR(33) . The insulin receptor undergoes
autophosphorylation on three residues within its kinase domain:
Tyr-1158, Tyr-1162, and Tyr-1163, which are present within the sequence
context:
Y
ETDY
Y
RK(34) . The
second of these, Tyr-1162, appears to be phosphorylated first and is
thought to be equivalent to Src-Tyr-416(21, 35) . In
general, phosphorylation of these residues is associated with greatly
increased kinase activity, and mutation of these tyrosines to
phenylalanine produces a kinase that cannot be
activated(13, 14) .
The recent solution of the x-ray crystal structure of the apo form of the IRK shows that Tyr-1158, Tyr-1162, and Tyr-1163 are all on the activation loop of the kinase domain. Hubbard et al.(36) postulate that this activation loop blocks access of substrate to the binding site and that it must be shifted, as a consequence of phosphorylation of the activation loop tyrosines, into a different position in order for the kinase to become activated. The structure also shows that Tyr-1162 (and therefore presumably Src-Tyr-416) actually sits in the active site and is hydrogen-bonded to the catalytic base, aspartate 1132, further blocking substrate access to the active site.
ZAP-70 contains the
sequence SYY
TAR within the region predicted
to contain the activation loop. Because ZAP-70 has 2 adjoining
tyrosines, it has been difficult to say unambiguously which tyrosine is
actually the Src416/IRK1162 equivalent. Our results suggest that ZAP-70
Tyr-493 is analogous to Tyr-416 and Tyr-1162 of Src and IRK, in that
phosphorylation of this residue seems to be required in order to fully
activate the kinase activity of ZAP-70, and mutation of Tyr-493 to
phenylalanine blocks activation in response to tyrosine
phosphorylation.
The finding that phosphorylation of Tyr-493 is
required for full activation of ZAP-70 is particularly interesting in
light of our recent phosphopeptide mapping data which show that ZAP-70
cannot phosphorylate itself on Tyr-493, whereas addition of Lck results
in phosphorylation of this site. There is, in fact, considerable
evidence in support of a role for a PTK, perhaps Lck, for activating
ZAP-70 after recruitment of ZAP-70 to the TCR. For instance, while
recruitment of ZAP-70 to the activated TCR is required for ZAP-70
activation(4) , mere recruitment is insufficient to induce
activation(9, 10) . In addition, studies by Kolanus et al.(37) and Chan et al.(5) have
shown that a Src family kinase member (either Lck or Fyn) must be
co-expressed for observation of ZAP-70 activity in COS cells. Also, Lck
phosphorylates in vivo the sequence EYTAR within
its own kinase domain(38) ; this sequence is similar to that
surrounding Tyr-493 of ZAP-70 (SY
Y
TAR).
Our finding that mutation of Tyr-492 to phenylalanine causes basal activation of ZAP-70 was unexpected, although not without precedent. Activating point mutations in the kinase domains of many different PTKs have been reported, including E378G, I441F, and Y416Q in c-Src, V157I in erb-B, Y1162F/Y1163F (double mutant) in IRK, and D814V in c-kit(20, 39, 40, 41, 42) . These mutations do not seem to cluster in any particular region of the kinase domain and may simply indicate that the kinase domain is susceptible to changes in activity in response to small structural changes that increase the access of substrates to the active site. This phenomenon may also explain the activation of ZAP-70 that we observed in Fig.2when the immunoprecipitates were stringently washed. This activating effect of the stringent wash may be due to subtle structural changes in the kinase domain induced by these mildly denaturing wash conditions that provide greater accessibility of substrate for the active site. It is unlikely that stringent washing removes an inhibitory protein from the immune complex, as additional washes of these immunoprecipitates with 150 mM NaCl in 25 mM Tris-HCl, pH 7.6, reverses the activating effect of stringent washing.
As mentioned above, the mechanism by which replacement of tyrosine with phenylalanine at position 492 results in activation of ZAP-70 kinase activity is unclear. The net effect of this mutation is, of course, the loss of a phenolic hydroxyl group, with the consequent loss of activity as a phosphate acceptor and the loss of hydrogen bonding capability. The activating effect of the Y492F mutation is probably not due to the loss of a negatively regulating phosphorylation event at Tyr-492, as we observed no significant basal tyrosine phosphorylation of ZAP-70, and Tyr-492 is phosphorylated under conditions in which ZAP-70 is fully activated(12) . This suggests that the gain of activity may be due to lost capacity to hydrogen bond with some other residue in the kinase domain, which could make the active site more accessible to substrate. In support of this, the basal activation of the IRK in response to mutating tyrosines 1162 and 1163 to phenylalanine has been postulated to occur through a similar mechanism(20) . Also, it is interesting that the analogous double mutation in ZAP-70, Y492F/Y493F, also gives increased basal kinase activity, but cannot be stimulated by tyrosine phosphorylation (not shown). This is precisely the behavior exhibited by the Y1162F/Y1163F-mutated IRK(20) . It remains to be established whether phosphorylation of Tyr-492 also causes activation, and, if so, whether it does so by the same mechanism as the Y492F mutation.
In summary, our results are consistent with Tyr-493 of ZAP-70 being the Src-Tyr-416/IRK-Tyr-1162 equivalent and is probably the initial site of phosphorylation in the kinase domain. Mutation of Tyr-492 to phenylalanine results in constitutive activation of ZAP-70. Both the mechanism of this activation and the normal contribution of Tyr-492 phosphorylation to the kinase activity of ZAP-70 remains to be determined. It is tempting, however, to speculate that ZAP-70 may first be phosphorylated at Tyr-493 via the action of another kinase, presumably Lck. Phosphorylation at Tyr-493 permits phosphorylation at 492, either by ZAP-70 itself or by another kinase, which results in full activation of ZAP-70. Evaluation of this model is the focus of our current investigation.