T cell antagonism is functionally uncoupled from the 21- and 23-kDa tyrosine-phosphorylated TCR zeta subunits

The functional effects of altered peptide ligands on T cells is proposed to involve differential intracellular signaling mediated by the 21- and 23-kDa tyrosine-phosphorylated derivatives of the TCR zeta subunit (p21 and p23). To understand the functional contribution of p21 and p23 to T cell development and T cell antagonism, we generated selected TCR zeta transgenic mice maintained on the P14 alphabeta TCR transgenic line such that p23 or both p21 and p23 were selectively eliminated. Importantly, one line (YF1,2) retains the constitutively tyrosine-phosphorylated p21 in the complete absence of inducible p23. We determined that T cell development was uncoupled from p21 and/or p23. Using a series of agonist, weak agonist, and antagonist peptides, we analyzed the role of each of the phosphorylated forms of TCR zeta on T cell activation and antagonism. In this study, we report that the proliferative responses of alphabeta P14 T cells to agonist peptides and the inhibition of proliferation resulting from antagonist peptide treatments was functionally uncoupled from p21 and/or p23. These results suggest that the mechanism of T cell antagonism is independent of the two phosphorylated TCR zeta derivatives.     

A tyrosine-phosphorylated 70-kDa protein binds a photoaffinity analogue of ATP and associates with both the zeta chain and CD3 components of the activated T cell antigen receptor.

An early event in T cell antigen receptor (TCR)-mediated signal transduction is the activation of a protein tyrosine kinase (PTK) pathway. An unidentified PTK activity and a kinase substrate termed ZAP-70 have previously been shown to associate with TCR zeta upon cross-linking of TCR beta. Here we report that TCR activation, by antibody cross-linking of either TCR beta or CD3 epsilon, results in the association of a PTK activity with both CD3 and TCR zeta. A number of in vitro PTK substrates are also associated with CD3 and TCR zeta, including CD3 epsilon, TCR zeta, p60fyn, p62yes, and a predominant 70-kDa protein (ZAP-70). The shared PTK activity and PTK substrates suggest that both CD3 and TCR zeta are involved in signal transduction via a shared pathway. We used [alpha-32P]gamma-azidoanilido ATP, a photoreactive analogue of ATP, to detect CD3-associated proteins that bound ATP upon TCR activation, reasoning that such proteins could represent PTKs. A 70-kDa protein bound [alpha-32P]gamma-azidoanilido ATP only upon TCR activation, and we propose that this protein and the 70-kDa PTK substrate are the same protein. Furthermore, we propose that this protein is responsible for the PTK activity observed to be associated with TCR zeta and CD3 upon TCR activation.     

The constitutive tyrosine phosphorylation of CD3zeta results from TCR-MHC interactions that are independent of thymic selection

The TCR complex, when isolated from thymocytes and peripheral T cells, contains a constitutively tyrosine-phosphorylated CD3zeta molecule termed p21. Previous investigations have shown that the constitutive phosphorylation of CD3zeta results from TCR interactions with MHC molecules occurring in both the thymus and the periphery. To determine what contribution the selection environment had on this constitutive phosphorylation, we analyzed CD3zeta from several distinct class I- and II-restricted TCR-transgenic mice where thymocyte development occurred in either a selecting or a nonselecting MHC environment. Herein, we report that constitutively phosphorylated CD3zeta (p21) was present in thymocytes that developed under nonselecting peptide-MHC conditions. These findings strongly support the model that the TCR has an inherent avidity for MHC molecules before repertoire selection. Biochemical analyses of the TCR complex before and after TCR stimulation suggested that the constitutively phosphorylated CD3zeta subunit did not contribute to de novo TCR signals. These findings may have important implications for T cell functions during self-MHC recognition under normal and autoimmune circumstances.     

Restoration of T cell receptor-mediated signal transduction by transfection of CD45 cDNA into a CD45-deficient variant of the Jurkat T cell line.

The TCR is a multimeric structure comprised of distinct Ag recognition and signal transduction components. Although none of the molecules that make up the TCR possess intrinsic protein tyrosine kinase (PTK) activity, stimulation of T cells via the TCR results in the rapid appearance of newly tyrosine phosphorylated proteins in cell lysates. Evidence suggests ligation of the TCR induces activation of a PTK that may be a member of the src family. One early consequence of this TCR-mediated PTK activation is the phosphorylation of the gamma 1 isoform of phospholipase C. This phosphorylation event is associated with increased enzymatic activity resulting in the hydrolysis of phosphatidylinositol 4,5 bisphosphate into two second messengers, inositol 1,4,5 trisphosphate and diacylglycerol. Recently, our laboratory and others have isolated mutant T cells that lack surface expression of CD45, the major surface tyrosine phosphatase expressed on lymphoid cells. Stimulation of the TCR on these cells fails to result in the expected activation events. We demonstrate that reconstitution of surface expression of the 180-kDa isoform of CD45 by gene transfer into a CD45-deficient mutant of the Jurkat T cell leukemic line restores the ability of the TCR to couple fully to its signal transduction machinery. These results support the role of CD45 tyrosine phosphatase activity in regulating the TCR-activated PTK.     

Phosphorylation of multiple CD3 zeta tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation.

T lymphocyte activation resulting from antigen recognition involves a protein tyrosine kinase pathway which triggers phosphorylation of several cellular substrates including the CD3 zeta subunit of the T cell receptor (TCR) to form pp21. The homologous TCR-associated protein, CD3 eta, is an alternatively spliced product of the same gene locus as CD3 zeta. CD3 eta lacks one of six cytoplasmic tyrosine residues (Tyr-132) found in CD3 zeta and is itself not phosphorylated. Site-directed mutagenesis in conjunction with in vitro and in vivo phosphorylation studies herein demonstrates that Tyr-132 is required for the formation of pp21. Moreover, the differential phosphorylation of CD3 zeta versus CD3 eta is not due to a selective association of the known TCR-associated protein tyrosine kinase, p59fyn; p59fyn but not p56lck or p62yes is associated with each of the three TCR isoforms containing CD3 zeta 2, or CD3 eta 2, or CD3 zeta-eta. This association occurs through components of the TCR complex distinct from CD3 zeta or CD3 eta. In addition, we show that pp21 formation is not only dependent on Tyr-132 but results from concomitant phosphorylation of other CD3 zeta residues including Tyr-121. Mutation of Tyr-90, -121, or -132 does not alter primary signal transduction as shown by the ability of individual CD3 zeta Tyr----Phe mutants to produce interleukin-2 upon TCR stimulation. Thus, the substantial structural changes in CD3 zeta upon TCR stimulation as reflected by alteration in its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis may affect subsequent events such as receptor desensitization, receptor movement, and/or protein associations.     

PTPH1 is a predominant protein-tyrosine phosphatase capable of interacting with and dephosphorylating the T cell receptor zeta subunit

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor zeta subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor zeta subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR zeta substrate (specific activity ranging from 0.23 to 40 pmol/min/microg). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-zeta. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR zeta ITAMs, with PTPH1 directly dephosphorylating zeta. This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR zeta.     

ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain.

Protein-tyrosine kinases (PTKs) play an integral role in T cell activation. Stimulation of the T cell antigen receptor (TCR) results in tyrosine phosphorylation of a number of cellular substrates. One of these is the TCR zeta chain, which can mediate the transduction of extracellular stimuli into cellular effector functions. We have recently identified a 70 kd tyrosine phosphoprotein (ZAP-70) that associates with zeta and undergoes tyrosine phosphorylation following TCR stimulation. Here we report the isolation of a cDNA clone encoding ZAP-70. ZAP-70 represents a novel PTK and is expressed in T and natural killer cells. Moreover, tyrosine phosphorylation and association of ZAP-70 with zeta require the presence of src family PTKs and provide a potential mechanism by which the src family PTKs and ZAP-70 may interact to mediate TCR signal transduction.     

The T cell antigen receptor zeta chain is tyrosine phosphorylated upon activation

The T cell antigen receptor is composed of at least seven chains derived from six different gene products. Upon stimulation, several chains can be phosphorylated. Two of these, CD3-gamma and CD3-epsilon are phosphorylated on serine residues. In addition, a 21-kDa nonglycosylated receptor component is phosphorylated, upon activation, on tyrosine residues. We have referred to this phosphoprotein as p21 because we have previously not been able to assign the tyrosine phosphorylation to any of the described receptor subunits (Samelson, L. E., Patel, M. D., Weissman, A. M., Harford, J. B., and Klausner, R. D. (1986) Cell 46, 1083-1090). In this paper, we demonstrate that it is the 16-kDa zeta chain which is the tyrosine phosphorylated subunit, and thus the p21 nomenclature can be replaced. This phosphorylation results in a shift of the apparent Mr of zeta to 21 kDa. Proof that p21 is tyrosine phosphorylated zeta was afforded by a number of approaches. Specific anti-zeta antibodies directly precipitated phospho-p21. Metabolically labeled protein corresponding to p21 could only be observed after activation. When this 21-kDa band was isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment with alkaline phosphatase, its migration was identical with that of zeta. Furthermore, peptide mapping of metabolically labeled p21 (after gel isolation and dephosphorylation) showed it to be indistinguishable from p21. Thus, one of the early events of T cell activation is the tyrosine phosphorylation of the zeta chain of the T cell antigen receptor.     

A cyclic adenosine 3',5'-monophosphate-induced tyrosine phosphorylation of Syk protein tyrosine kinase in the flagella of boar spermatozoa

A cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein tyrosine phosphorylation is involved in the expression of fertilizing ability in mammalian spermatozoa. However, there are only limited data concerning the identification of protein tyrosine kinase (PTK) that is activated by the cAMP signaling. In this study, we have shown data supporting that boar sperm flagellum possesses a unique cAMP-protein kinase A (PKA) signaling cascade leading to phosphorylation of Syk PTK at the tyrosine residues of the activation loop. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium (mKRH) containing polyvinyl alcohol (PVA) plus 0.1 mM cBiMPS (a cell-permeable cAMP analog), 0.25 mM sodium orthovanadate (Na3VO4) (a protein tyrosine phosphatase (PTP) inhibitor) or both at 38.5 degrees C for 180 min. Aliquots of the sperm suspensions were recovered before and after incubation and then used to detect sperm tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence. In the Western blotting, the anti-phosphotyrosine monoclonal antibody (4G10) recognized several bands including 72-kDa protein in the protein extracts from spermatozoa that were incubated solely with cBiMPS. The tyrosine phosphorylation in these sperm proteins was dependent on cBiMPS and enhanced by the addition of Na3VO4. The 72-kDa tyrosine-phosphorylated protein was apparently reacted with the anti-phospho-Syk antibody (Tyr525/526). Indirect immunofluorescence revealed that the connecting and principal pieces of spermatozoa incubated with cBiMPS and Na3VO4 were stained with the anti-phospho-Syk antibody. However, the reactivity of the 72-kDa protein with the anti-phospho-Syk antibody was reduced by the addition of H-89 (a PKA inhibitor, 0.01-0.1 mM) to the sperm suspensions but not affected by the pretreatment of spermatozoa with BAPTA-AM (an intracellular Ca2+ chelator, 0.1 mM). Fractionation of phosphorylated proteins from the spermatozoa with a detergent Nonidet P-40 suggested that the 72-kDa tyrosine-phosphorylated protein might be a cytoskeletal component. Based on these findings, we have concluded that the cAMP-PKA signaling is linked to the Ca2+-independent tyrosine phosphorylation of Syk in the connecting and principal pieces of boar spermatozoa.     

Binding of ZAP-70 to phosphorylated T-cell receptor zeta and eta enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins.

ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.     

TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex

We show in this study that human T cells purified from peripheral blood, T cell clones, and Jurkat T cells release microvesicles in the culture medium. These microvesicles have a diameter of 50-100 nm, are delimited by a lipidic bilayer membrane, and bear TCR beta, CD3epsilon, and zeta. This microvesicle production is regulated because it is highly increased upon TCR activation, whereas another mitogenic signal, such as PMA and ionomycin, does not induce any release. T cell-derived microvesicles also contain the tetraspan protein CD63, suggesting that they originate from endocytic compartments. They contain adhesion molecules such as CD2 and LFA-1, MHC class I and class II, and the chemokine receptor CXCR4. These transmembrane proteins are selectively sorted in microvesicles because CD28 and CD45, which are highly expressed at the plasma membrane, are not found. The presence of phosphorylated zeta in these microvesicles suggests that the CD3/TCR found in the microvesicles come from the pool of complexes that have been activated. Proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the T cell-derived microvesicles. Our data demonstrate that T lymphocytes produce, upon TCR triggering, vesicles whose morphology and phenotype are reminiscent of vesicles of endocytic origin produced by many cell types and called exosomes. Although the exact content of T cell-derived exosomes remains to be determined, we suggest that the presence of TCR/CD3 at their surface makes them powerful vehicles to specifically deliver signals to cells bearing the right combination of peptide/MHC complexes.     

Interleukin-3 stimulates the tyrosine phosphorylation of the 140-kilodalton interleukin-3 receptor

Murine interleukin-3 (mIL-3) stimulates the rapid and transient tyrosine phosphorylation of a number of proteins in mIL-3-dependent B6SUtA1 cells. Two of these proteins, p68 and p140, are maximally phosphorylated at tyrosine residues within 2 min of addition of mIL-3. Because 125I-mIL-3 can be cross-linked to both 70- and 140-kDa proteins on intact B6SUtA1 cells, we investigated whether the tyrosine phosphorylated p68 and p140 were these two mIL-3 receptor proteins. Addition of antiphosphotyrosine antibodies (alpha PTyr Abs) to cell lysates from B6SUtA1 cells, to which 125I-mIL-3 had been disuccinimidyl suberate-cross-linked, resulted in the immunoprecipitation of 125I-mIL-3 complexed to both 70- and 140-kDa proteins. To determine if the observed immunoprecipitation pattern was due to the direct interaction of alpha-PTyr Abs with these two mIL-3 receptor proteins or with tyrosine-phosphorylated proteins that were associated with the receptor proteins, cell lysates were treated with 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, and boiled for 1 min. After removal of sodium dodecyl sulfate and 2-mercaptoethanol, alpha PTyr Abs immunoprecipitated 125I-mIL-3 cross-linked to only the 140-kDa protein. To confirm this finding, 32P-labeled B6SUtA1 cells were treated with biotinylated or fluoresceinated mIL-3. Addition of immobilized streptavidin or antifluorescein antibodies, respectively, to cell lysates from these cells resulted in the enrichment of only a 140-kDa tyrosine phosphorylated protein. Taken together, these results strongly suggest that only the 140-kDa receptor protein is tyrosine phosphorylated upon mIL-3 binding.     

Differential Src family kinase activity requirements for CD3 zeta phosphorylation/ZAP70 recruitment and CD3 epsilon phosphorylation

The current model of T cell activation is that TCR engagement stimulates Src family tyrosine kinases (SFK) to phosphorylate CD3zeta. CD3zeta phosphorylation allows for the recruitment of the tyrosine kinase ZAP70, which is phosphorylated and activated by SFK, leading to the phosphorylation of downstream targets. We stimulated mouse CTLs with plate-bound anti-CD3 and, after cell lysis, recovered proteins that associated with the CD3 complex. The protein complexes were not preformed, and a number of tyrosine-phosphorylated proteins were inducibly and specifically associated with the TCR/CD3 complex. These results suggest that complex formation only occurs at the site of TCR engagement. The recruitment and tyrosine phosphorylation of most proteins were abolished when T cells were stimulated in the presence of the SFK inhibitor PP2. Surprisingly, CD3zeta, but not CD3epsilon, was inducibly tyrosine phosphorylated in the presence of PP2. Furthermore, ZAP70 was recruited, but not phosphorylated, after TCR stimulation in the presence of PP2, thus confirming the phosphorylation status of CD3zeta. These data suggest that there is a differential requirement for SFK activity in phosphorylation of CD3zeta vs CD3epsilon. Consistent with this possibility, ZAP70 recruitment was also detected with anti-CD3-stimulated, Lck-deficient human Jurkat T cells. We conclude that TCR/CD3-induced CD3zeta phosphorylation and ZAP70 recruitment do not absolutely require Lck or other PP2-inhibitable SFK activity, but that SFK activity is absolutely required for CD3epsilon and ZAP70 phosphorylation. These data reveal the potential for regulation of signaling through the TCR complex by the differential recruitment or activation of SFK.     

Architectural changes in the TCR:CD3 complex induced by MHC:peptide ligation

A hallmark of T cell activation is the ligation-induced down-modulation of the TCR:CD3 complex. However, little is known about the molecular events that drive this process. The CD3 zeta-chain has been shown to play a unique role in regulating the assembly, transport, and cell surface expression of the TCR:CD3 complex. In this study we have investigated the relationship between CD3zeta and the TCRalphabetaCD3epsilondeltagamma complex after ligation by MHC:peptide complexes. Our results show that there is a significant increase in free surface CD3zeta, which is not associated with the TCR:CD3 complex, after T cell stimulation. This may reflect dissociation of CD3zeta from the TCRalphabetaCD3epsilondeltagamma complex or transport of intracellular CD3zeta directly to the cell surface. We also show that MHC:peptide ligation also results in exposure of the TCR-associated CD3zeta NH2 terminus, which is ordinarily buried in the complex. These observations appears to be dependent on Src family protein tyrosine kinases, which are known to be critical for efficient T cell activation. These data suggest a mechanism by which ligated TCR may be differentiated from unligated TCR and selectively down-modulated.     

Tyrosine phosphorylation of a novel 100-kDa protein coupled to CD28 in resting human T cells is enhanced by a signal through TCR/CD3 complex

For T cell activation, two signals are required, i.e., a T cell receptor (TCR)/CD3-mediated main signal and a CD28-mediated costimulatory signal. CD28 binds to its ligand (CD80 or CD86) and transduces the most important costimulatory signal. The cytoplasmic domain of the CD28 molecule, composed of 41 amino acids, does not contain any intrinsic enzyme activity. The cytoplasmic domain of CD28 is remarkably conserved among species and is associated with a number of signaling molecules that affect the main signal. We report here that a tyrosine phosphorylated 100-kDa protein (ppl00) was coupled to the CD28 cytoplasmic domain in Jurkat and human peripheral T cells. The pp100 was distinguished from other CD28 associated molecules such as Vav, STAT5, PI 3-kinase, Valosin-containing protein (VCP), Nucleolin, Gab2 (Grb2-associated binding protein 2), and STAT6. The tyrosine phosphorylation of pp100 coprecipitated with CD28 was enhanced by CD3 stimulation by the specific antibody, tyrosine phosphatase inhibitor and PKC activator. Tyrosine phosphorylation of pp100 was attenuated by the prior addition of PKC inhibitor. These findings indicate that pp100 is a novel tyrosine phosphorylated protein coupled to CD28 under continuous control of tyrosine phosphatases and might play a role in T cell activation augmented by a TCR/CD3-mediated main signal.     

TCR/CD3 down-modulation and zeta degradation are regulated by ZAP-70

TCR down-modulation following binding to MHC/peptide complexes is considered to be instrumental for T cell activation because it allows serial triggering of receptors and the desensitization of stimulated cells. We studied CD3/TCR down-modulation and zeta degradation in T cells from two ZAP-70-immunodeficient patients. We show that, at high occupancy of the TCR, down-modulation of the CD3/TCR is comparable whether T cells express or do not express ZAP-70. However, if TCR occupancy was low, we found that CD3/TCR was down-regulated to a lesser extent in ZAP-70-negative than in ZAP-70-positive T cells. We studied CD3/TCR down-modulation in P116 (a ZAP-70-negative Jurkat cell-derived clone) and in P116 transfected with genes encoding the wild-type or a kinase-dead form of ZAP-70. Down-modulation of the TCR at high occupancy did not require ZAP-70, whereas at low TCR occupancy down-modulation was markedly reduced in the absence of ZAP-70 and in cells expressing a dead kinase mutant of ZAP-70. Thus, the presence of ZAP-70 alone is not sufficient for down-modulation; the kinase activity of this molecule is also required. The degradation of zeta induced by TCR triggering is also severely impaired in T cells from ZAP-70-deficient patients, P116 cells, and P116 cells expressing a kinase-dead form of ZAP-70. This defect in TCR-induced zeta degradation is observed at low and high levels of TCR occupancy. Our results identify ZAP-70, a tyrosine kinase known to be crucial for T cell activation, as a key player in TCR down-modulation and zeta degradation.     

TCR zeta mRNA with an alternatively spliced 3'-untranslated region detected in systemic lupus erythematosus patients leads to the down-regulation of TCR zeta and TCR/CD3 complex

The reduction or absence of TCR zeta-chain (zeta) expression in systemic lupus erythematosus (SLE) patients is thought to be related to the pathogenesis of SLE. Recently, we reported the predominant expression of zeta mRNA containing an alternatively spliced 3'-untranslated region (3'UTR; zetamRNA/as-3'UTR) and a reduction in the expression of zeta mRNA containing the wild-type 3'UTR (zetamRNA/w-3'UTR) in T cells from SLE patients. Here we show that AS3'UTR mutants (MA5.8 cells deficient in zeta protein that have been transfected with zetamRNA/as-3'UTR) exhibit a reduction in the expression of TCR/CD3 complex and zeta protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab compared with that in wild-type 3'UTR mutants (MA5.8 cells transfected with zetamRNA/w-3'UTR). Furthermore, the real-time PCR analyses demonstrated that the half-life of zetamRNA/as-3'UTR in AS3'UTR mutants (3 h) was much shorter than that of zetamRNA/w-3'UTR in wild-type 3'UTR mutants (15 h). Thus, the lower stability of zetamRNA/as-3'UTR, which is predominant in SLE T cells, may be responsible for the reduced expression of the TCR/CD3 complex, including zeta protein, in SLE T cells.     

Genetic Evidence of a Role for Lck in T-Cell Receptor Function Independent or Downstream of ZAP-70/Syk Protein Tyrosine Kinases

T-cell antigen receptor (TCR) engagement results in sequential activation of the Src protein tyrosine kinases (PTKs) Lck and Fyn and the Syk PTKs, ZAP-70 and Syk. While the Src PTKs mediate the phosphorylation of TCR-associated signaling subunits and the phosphorylation and activation of the Syk PTKs, the lack of a constitutively active Syk PTK has prohibited the analysis of Lck function downstream of these initiating signaling events. We describe here the generation of an activated Syk family PTK by substituting the kinase domain of Syk for the homologous region in ZAP-70 (designated as KS for kinase swap). Expression of the KS chimera resulted in its autophosphorylation, the phosphorylation of cellular proteins, the upregulation of T-cell activation markers, and the induction of interleukin-2 gene synthesis in a TCR-independent fashion. The KS chimera and downstream ZAP-70 or Syk substrates, such as SLP-76, were still phosphorylated when expressed in Lck-deficient JCaM1.6 T cells. However, expression of the KS chimera in JCaM1.6 cells failed to rescue downstream signaling events, demonstrating a functional role for Lck beyond the activation of the ZAP-70 and Syk PTKs. These results indicate that downstream TCR signaling pathways may be differentially regulated by ZAP-70 and Lck PTKs and provide a mechanism by which effector functions may be selectively activated in response to TCR stimulation.