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ZAP-70 and SLP-76 Regulate Protein Kinase C- and NF-B Activation in Response to Engagement of CD3 and CD28

Thomas M. Herndon^*,,, Xiaochuan C. Shan^1,*, George C. Tsokos^, and Ronald L. Wange^2,*

^* Laboratory of Biological Chemistry, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224; Department of Cellular Injury, Walter Reed Army Institute of Research, Washington, DC 20307; and Department of Medicine, Uniform Services University of the Health Sciences, Bethesda, MD 20814

	Abstract

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The transcription factor NF-B is a critical regulator of T cell function that becomes strongly activated in response to coengagement of TCR and CD28. Although events immediately proximal to NF-B activation are well understood, uncertainty remains over which upstream signaling pathways engaged by TCR and CD28 lead to NF-B activation. By using Jurkat T cell lines that are deficient or replete for either the protein tyrosine kinase ZAP-70 or the cytosolic adapter molecule SLP-76, the role of these proteins in modulating NF-B activation was examined. NF-B was not activated in response to coengagement of TCR and CD28 in either the ZAP-70- or SLP-76-negative cells, whereas stimuli that bypass these receptors (PMA plus A23187, or TNF-) activated NF-B normally. Protein kinase C (PKC) activation, which is required for NF-B activation, also was defective in these cells. Reexpression of ZAP-70 restored PKC and NF-B activation in response to TCR and CD28 coengagement. p95^vav (Vav)-1 tyrosine phosphorylation was largely unperturbed in the ZAP-70-negative cells; however, receptor-stimulated SLP-76/Vav-1 coassociation was greatly reduced. Wild-type SLP-76 fully restored PKC and NF-B activation in the SLP-76-negative cells, whereas 3YF-SLP-76, which lacks the sites of tyrosine phosphorylation required for Vav-1 binding, only partially rescued signaling. These data illustrate the importance of the ZAP-70/SLP-76 signaling pathway in CD3/CD28-stimulated activation of PKC and NF-B, and suggest that Vav-1 association with SLP-76 may be important in this pathway.

	Introduction

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The "two-signal" hypothesis of T cell activation contends that for a naive T cell to proliferate, engagement of the TCR by Ag-MHC (signal one) must be accompanied by a stimulus through a coreceptor (signal two) (1). Over the past several years, substantial progress has been made in understanding the nature of signal one, and this area has been extensively reviewed (2, 3, 4, 5). One of the key signaling proteins involved in mediating signal one is ZAP-70,³ a 70-kDa Syk family protein tyrosine kinase (PTK) expressed in T cells and NK cells (6). The importance of ZAP-70 in normal T cell development and TCR signaling was illustrated by the discovery that some cases of human severe combined immunodeficiency are caused by an inability to express functional ZAP-70 protein (6). In addition, Jurkat T cell mutants that fail to express ZAP-70 show multiple signaling deficiencies at the level of protein tyrosine phosphorylation, Ca²⁺ mobilization and mitogen-activated protein kinase (MAPK) activation, and are unable to activate the transcription factors NF-AT and AP-1 (7, 8, 9, 10).

ZAP-70 couples to downstream signaling events by phosphorylating the linker/adapter proteins Lat and SLP-76 (11, 12, 13, 14, 15). Lat is a 36-kDa transmembrane protein that becomes rapidly tyrosine phosphorylated on TCR engagement and plays an important role in organizing multimolecular signaling complexes in the lipid raft subdomains of the plasma membrane. These clusters contain key signaling proteins, including phospholipase C (PLC)1, growth factor receptor binding protein 2, Gads, SLP-76, phosphatidylinositol 3-kinase, Vav-1 and IL-2-inducible T cell kinase (Itk; Refs. 11, 12, 16, 17, 18). SLP-76 is a cytosolic adapter protein that mediates the formation of additional multimolecular signaling complexes thought to be involved in TCR signaling (3, 4, 19). Jurkat T cells lacking SLP-76 are defective in activation of both the calcium mobilization and p21^ras oncoprotein (Ras) pathways in response to TCR engagement and are unable to activate AP-1 or NF-AT reporter constructs (20).

The nature of signal two has been more difficult to ascertain, despite recognition for many years that the major initiator of this signal is the 44-kDa transmembrane protein CD28. CD28 is engaged by its ligands B7-1 (CD80) and B7-2 (CD86), which show restricted expression on activated APCs. CD28 engagement has been reported to increase the enzymatic activities of Lck, Fyn, Itk, phosphatidylinositol 3-kinase, sphingomyelinase, Vav-1, and mitogen-activated protein/extracellular signal-related kinase kinase-1 and results in tyrosine phosphorylation of several proteins, including the cytoplasmic domain of CD28 itself (21, 22, 23). However, the role of any of these events in providing signal two remains controversial. There are some recent suggestions that CD28 may also play a role in augmenting signals delivered through the TCR rather than delivering a unique second signal (24, 25).

Most of the signaling pathways that are required to initiate T cell proliferation can be activated in in vitro model systems by TCR engagement alone; however, certain key signaling events require concurrent engagement of both the TCR and CD28. These events are presumed to either represent integration points for signals one and two or to lie downstream of a signal integrator. At the transcription factor level, this is seen with AP-1 and NF-B, which are required for efficient transcription of IL-2 message in T cells. The dependence of AP-1 activation on both signals one and two has been traced largely to the requirement for activation of c-Jun amino-terminal kinase, which requires signals from the TCR and CD28 for full activation (26). The precise signals leading to NF-B activation in response to coengagement of the TCR and CD28 are less well defined, although compelling data recently has been presented to show that the -isoform of protein kinase C (PKC) is required for NF-B activation in response to these stimuli (27, 28, 29, 30). Like NF-B, PKC requires signaling through both the TCR and CD28 for full activation.

NF-B is a member of the Rel family of proteins and is regulated by numerous stimuli in different cell types (31). Regulatory proteins called IBs, of which IB is the best characterized, bind NF-B in an inactive form in the cytosol. After phosphorylation of serines 32 and 36 on IB by the IB kinase (IKK), IB is proteolyzed, allowing NF-B to migrate into the nucleus, where it binds to and modulates the transcription efficiency of its specific target genes. There is as yet little consensus on how IKK activity is regulated, and many candidate regulators have been proposed (32, 33).

Another area in which consensus has yet to emerge is in the mechanism of NF-B activation in T cells responding to coengagement of TCR and CD28. As in other systems, IKK activation and degradation of IB is the principle inducer of NF-B activation (34, 35), but knowledge about the intervening steps between receptor engagement and IKK activation remains limited. There is a required role for Ca²⁺ influx or activated calcineurin in NF-B activation in T cells stimulated through the TCR, but not for stimulation through the TNF- receptor (36, 37, 38). More recently, Vav-1 and PKC have been found to play a role in NF-B activation in TCR-stimulated T cells. T cells isolated from mice genetically manipulated to be defective for expression of either of these two proteins fail to proteolyze IB and fail to activate NF-B in response to stimulation through the TCR and CD28 (29, 39). These proteins appear to act together in supporting NF-B activation, with Vav-1 acting upstream of PKC (30, 40). Additional evidence for a key role in mediating TCR-stimulated NF-B activation comes from the observation that overexpression of either Vav-1 or PKC (but not other PKC isoforms) augments TCR/CD28-stimulated NF-B activation (27, 28, 30). In addition, dominant negative PKC or addition of PKC antisense-RNA inhibits NF-B activation in response to these stimuli (28, 30).

Vav-1 is a rho-family guanine nucleotide exchange factor that becomes rapidly tyrosine phosphorylated in T cells on stimulation of the TCR. TCR-stimulated Vav-1 tyrosine phosphorylation has been reported to be a ZAP-70-dependent process, although it is unlikely that Vav-1 is a direct ZAP-70 substrate (10, 41, 42, 43). Vav-1 also can be tyrosine phosphorylated in response to CD28 engagement (44, 45), and it has been suggested that tyrosine phosphorylation of Vav-1 may represent a point of integration for TCR and CD28-mediated signaling events (10, 46, 47). In general, increased tyrosine phosphorylation of Vav-1 has been considered to positively affect its activity (48, 49), but recently it has been shown that some sites of phosphorylation negatively regulate its activity (50). Vav-1 has been shown to form a stable complex, both in vivo and in vitro with tyrosine-phosphorylated SLP-76 (15, 51, 52, 53). The formation of this complex is mediated by the Src homology domain 2 (SH2) domain of Vav-1 binding the sequences surrounding Y113 and Y128 in the N-terminal region of SLP-76 when either of these residues becomes phosphorylated by ZAP-70 (14, 15, 54). SLP-76 has been shown to synergize with Vav-1 in activating effectors downstream of TCR engagement, such as NF-AT, Rac, and Cdc42 (51, 53), indicating that these two proteins cooperate in regulating certain signaling pathways. It remains unclear to what extent the signaling cooperativity of these proteins is dependent on their ability to physically associate (53, 54).

PKC is a member of the "novel" class of protein kinase C enzymes, being sensitive to activation by diacylglycerol, but not Ca²⁺. PKC has a restricted expression pattern, with high expression levels in T cells (55, 56, 57). Interest in the role of PKC in TCR signaling piqued when it was noted that of the many different PKC isoforms expressed in T cells, only PKC translocates to the site of contact between APCs and T cells undergoing stimulation (58).

In this study, mutants of the Jurkat T cell line that are either deficient or replete for expression of ZAP-70 or SLP-76 were used to examine the question of whether or not these two proteins are required for activation of NF-B in T cells undergoing stimulation through CD3 and CD28. Jurkat cells lacking either one of these proteins failed to activate NF-B or the upstream kinase PKC in response to stimulation through CD3 and CD28 while remaining competent for NF-B activation by other stimuli. Vav-1 tyrosine phosphorylation was generally unperturbed in the ZAP-70-negative cells, but the ability of Vav-1 and SLP-76 to coassociate was greatly reduced. This coupled with the observation that SLP-76 mutated at the sites of Vav-1 interaction was defective in restoring NFB activation suggests a model in which ZAP-70-mediated SLP-76 phosphorylation and consequent Vav-1 association may be required for NF-B activation in response to coengagement of CD3 and CD28.

	Materials and Methods

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Cells and reagents

The Jurkat, P116, P116.c39, J14-76-11, and J14-v-29 T cell lines have been described (7, 20) and were the kind gifts of R. Abraham (Duke University, Durham, NC) (Jurkat, P116, and P116.c39) and A. Weiss (University of California, San Francisco, CA) (J14-76-11 and J14-v-29). The cells were maintained in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 10% FBS (Biofluids, Rockville, MD), 2 mM L-glutamine, and 10 µg/ml ciprofloxacin. Where indicated, cells were subjected to serum starvation for 3 to 5 h before harvesting for experiments. The OKT3, anti-CD3 mAb, and polyclonal antisera to ZAP-70 have been described (59). The anti-CD28 Ab 9.3 was a gift from Carl June (University of Pennsylvania, Philadelphia, PA). Rabbit anti-mouse IgG was obtained from Southern Biotechnology Associates (Birmingham, AL). PMA and A23187 were purchased from Sigma (St. Louis, MO). TNF- was obtained from Endogen (Woburn, MA). Abs to IB and to the NF-B subunits (cRel, p50, p52, and p65) and PKC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phosphotyrosine mAb, 4G10, and Abs to Vav-1 were obtained from Upstate Biotechnology (Lake Placid, NY). The NF-B and AP2 consensus oligonucleotides were purchased from Promega (Madison, WI). The SLP-76 mammalian expression plasmids pEF-SLP-76-flag, and pEF-SLP-76(3YF)-flag (tyrosines 113, 128, and 145 mutated to phenylalanine) and the bacterial expression plasmid encoding the GST-Vav-1(SH2) fusion protein were gifts from Gary Koretzky (University of Pennsylvania). The anti-SLP-76 mAb was a gift from Paul Findell (Roche Biosciences, Palo Alto, CA).

Flow cytometric analysis

For analysis of CD3 and CD28 surface expression on Jurkat, P116, P116.c39, J14-76-11, and J14-v-29 cell lines, 1 x 10⁶ washed cells were suspended in RPMI 1640 plus 2% FBS with a 1:100 dilution of the appropriate Ab and incubated on ice for 45 min. The cells were washed three times then resuspended in RPMI 1640 with 2% FBS and a 1:100 dilution of FITC-conjugated sheep-anti mouse Ab (Amersham, Arlington Heights, IL) and incubated on ice in the dark for 30 min. The samples were washed three times, suspended in PBS and analyzed by using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).

Cell stimulations and sample preparation

The cells were harvested and washed in 4°C RPMI 1640, resuspended in 4°C RPMI 1640 with 2 mM glutamine, and maintained on ice until stimulated. After equilibration to 37°C for 5–10 min, the cells were incubated with the indicated stimulants. It should be noted that when CD28 was stimulated in the absence of CD3 stimulation, a secondary rabbit anti-mouse IgG antisera was used to cross-link the anti-CD28 mAb to generate a maximal CD28 signal. Similar results were obtained with and without secondary Ab cross-linking. At each time point, 2 x 10⁶ cells were washed with ice-cold PBS, resuspended in ice-cold lysis buffer (20 mM HEPES, pH 7.4, 50 mM -glycerophosphate, 2 mM EGTA, 10 mM NaF, 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and 1 mM sodium orthovanadate) and then processed as postnuclear lysates, as previously described (8). The remainder of the cells (1.0 x 10⁷ for each time point) were processed for extraction of nuclear proteins as previously described (60, 61).

Immunoprecipitations, affinity precipitations, and immunoblotting

The postnuclear lysates were normalized to equal amounts of total protein by using the Bradford assay (Bio-Rad, Hercules, CA) before being subjected to SDS-PAGE. Immunoprecipitations, affinity precipitations, electrophoresis, and immunoblotting were conducted as described previously (8).

EMSAs

Oligonucleotides containing the consensus sequence of the NF-B binding site and the complement strand were obtained from Promega. These were end-labeled with [-³²P]ATP (Amersham) with T4 polynucleotide kinase (Promega). Samples were normalized to equal amounts of protein as above. The samples were incubated for 15 min at room temperature in binding buffer containing poly[dIdC] (Promega) with or without excess unlabeled NF-B oligo, AP-2 oligo, Rel protein Abs, or normal rabbit serum before the addition of labeled NF-B oligo for an additional 20 min. The reaction mixture was loaded onto a 3% nondenaturing polyacrylamide gel in 0.5 x thiobarbituric acid buffer. The gel was dried and visualized by autoradiography.

In vitro PKC kinase assay

Anti-PKC immunoprecipitates from lysates containing 1.25 x 10⁶ cell equivalents were washed three times with PKC kinase assay buffer (20 mM HEPES, pH 7.2, 137 mM NaCl, 5.4 mM KCl, 0.3 mM NaH₂PO₄, 0.4 mM KH₂PO₄, 25 mM -glycerophosphate, 10 mM MgCl₂, 5 mM EGTA, and 2.5 mM CaCl₂; Ref. 58). Samples were divided into four aliquots, three for PKC kinase assay analysis in triplicate and one aliquot to determine relative PKC immunoprecipitation between groups. To each kinase assay replicate, 35 µl of PKC kinase assay buffer supplemented with 0.1 mM ATP, 5 µCi [-³²P]ATP, and 0.1 mM selectide PKC substrate (Calbiochem, La Jolla, CA) was added for 20 min at 30°C with frequent mixing. The reaction was terminated by the addition of 10 µl of 25% trichloroacetic acid. After brief centrifugation, the supernatants were spotted onto p81 phosphocellulose discs (Life Technologies). These were washed once in 10% acetic acid and three times in 75 mM phosphoric acid, and the ³²P incorporation was measured by liquid scintillation counting.

Transient transfections

J14.v.29 cells in logarithmic-growth phase were transfected by electroporation as described previously (62). Briefly, cells were washed and resuspended in complete growth medium at a density of 4 x 10⁷ cells/ml, and 1.2 x 10⁷ cells were mixed with 35 µg of plasmid DNA in a 4-mm gap electroporation cuvette for 15 min before a 300 V pulse was applied for 10 ms with a BTX ECM 830 square wave electroporator (Genetronics, San Diego, CA). The cells were maintained overnight in complete media. Cell equivalents used in subsequent experiments were based on live cells as assessed by trypan blue staining.

Luciferase reporter assay

The NF-B luciferase reporter gene (Stratagene, La Jolla, CA) and -galactosidase gene were cotransfected with either pEF (empty vector), pEF-SLP-76-flag, or pEF-3YF-SLP-76-flag. After transfection, cells were cultured at 37°C for 15 h then washed and resuspended to 2 x 10⁶ cells/ml in complete medium. After stimulations, cells were washed two times in PBS, lysed, and analyzed by the luciferase assay system (Promega) and for -galactosidase activity (Tropix, Bedford, MA) with a model LB 953 Autolumat (Perkin-Elmer, Gaithersburg, MD).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
ZAP-70 is required for NFB activation in response to CD3 and CD28 coengagement

Although ZAP-70 has been shown to be a key mediator of TCR signaling, its role in regulating the activity of the transcription factor NF-B in response to concurrent signaling through the TCR and CD28 has not been assessed. The Jurkat T cell model system was used, in particular the ZAP-70-negative P116 subline, to test for such a role. The parental Jurkat T cells, the ZAP-70-negative P116 Jurkat T cells, and P116 T cells stably transfected with cDNA encoding ZAP-70 (P116.c39) all exhibit comparable levels of expression of CD3 and CD28 (Fig. 1). These three cell lines were stimulated with mAbs to CD3 and CD28. Samples were collected at 0, 40, and 120 min after addition of the mAbs (Fig. 2A, top). The first measure of NF-B activation assessed was the loss of IB protein from cellular lysates, as a function of its proteolytic degradation. In Jurkat cells, there was an almost complete loss of IB expression after 40 min of stimulation. By 120 min, IB expression had recovered and was consistently observed to be hyperexpressed compared with basal levels. This hyperexpression is consistent with NF-B activation because the promoter region controlling IB expression contains NF-B binding sites, and NF-B has been demonstrated to up-regulate IB expression (31). Notably, the P116 T cells show no loss of IB protein in response to coengagement of CD3 and CD28 at 40 min, nor hyperexpression at 120 min. In contrast, the P116.c39 T cell line in which ZAP-70 expression has been restored showed the same pattern of IB loss at 40 min and hyperexpression at 120 min exhibited by parental Jurkat. ZAP-70 expression levels were comparable in Jurkat and P116.c39 T cells and unmeasurable in P116 T cells (Fig. 2A, middle). As expected, IB degradation was negligible in both Jurkat and P116 T cells when stimulated through either CD3 or CD28 alone. (Fig. 2B). The above data are consistent with a requirement for ZAP-70 in the activation of NF-B in response to TCR and CD28 engagement.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. CD3 and CD28 expression levels are similar on Jurkat sublines. The levels of surface expression of CD3 and CD28 were measured on Jurkat, P116, P116.c39, J14.v.29, and J14.76.11 cells by flow cytometry as described in Materials and Methods. Control cells were stained only with the FITC-conjugated secondary Ab.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. ZAP-70 is required for IB- degradation and NF-B activation in response to CD3 and CD28 costimulation. Whole-cell lysates were prepared from serum-starved Jurkat, P116, and P116.c39 T cells that were incubated (A) in the presence of Abs to CD3 (1:50 OKT3 ascites) and CD28 (2 µg/ml 9.3), or (B) CD3 alone or CD28 cross-linked with rabbit-anti-mouse Ab (1 µg/ml) for 0, 40, or 120 min. Lysates were loaded at 10 µg/lane onto 4–12% NuPAGE gels in MOPS buffer, transferred to nitrocellulose, and immunoblotted with the indicated Ab. A, Membranes were blotted for IB, ZAP-70, and then stripped and blotted for SLP-76. B, Membranes were blotted for IB. C, Nuclear extracts were collected from serum-starved Jurkat, P116, and P116.c39 T cells that were incubated in the presence of Abs to CD3 (1:50 OKT3 ascites) and CD28 (2 µg/ml 9.3). Reactions were terminated at 0, 40, and 120 min. Equal amounts of protein were incubated in the presence of labeled NF-B consensus oligonucleotide followed by electrophoresis on a 3% polyacrylamide gel and autoradiography. D, Sample from the 40-min time point of Jurkat and P116.c39 T cells was incubated in the presence or absence of the indicated oligonucleotides or Abs before the addition of radiolabeled NF-B probe. NC, negative control; PC, positive control; SI, specific inhibitor, addition of greater than 100-fold excess NF-B consensus oligonucleotide; NI, nonspecific inhibitor, addition of greater than 100-fold excess AP-2 consensus oligonucleotide; cRel, addition of Ab to cRel; p50, addition of Ab to p50; p52, addition of Ab to p52; p65, addition of Ab to p65.

The Jurkat and P116.c39 nuclear DNA-binding activities were further analyzed with regard to specificity and composition (Fig. 2D). The extracts analyzed were from the 40-min time point after costimulation with anti-CD3 and anti-CD28 mAb. No band was detected in the negative control (NC, lane 1), which contains only the assay buffers and radiolabeled probe containing the NF-B binding site. Excess unlabeled oligonucleotide containing the NF-B binding site (specific inhibitor, SI), but not an excess of an unrelated oligo containing the AP-2 consensus-binding site (nonspecific inhibitor, NI) could prevent formation of the radiolabeled complex. Shown for comparison is the position of the complex formed in the absence of any competitors (positive control, PC). Abs to various Rel subunits were used in a supershift assay to identify the components of the DNA-binding activity present in the stimulated Jurkat and P116.c39 nuclear extracts. Antisera to p50 and p65 were found to shift the complex, whereas Abs to cRel and p52 did not produce a shift. The results for Jurkat and P116.c39 T cells were identical, with the principal oligonucleotide-binding complex formed in these cells in response to stimulation through CD3 and CD28 corresponding to the p50/p65 heterodimer, consistent with previous reports (63).

NF-B is activated normally in P116 T cells in response to distal or TCR-independent stimuli

To rule out the possibility that there could be a secondary deficit in P116 T cells that could lead to a defect in the NF-B pathway at a point distal to ZAP-70 action, we investigated the ability of the potent combined stimulus of PMA and the calcium ionophore A23187 to stimulate NF-B activation in P116 T cells (Fig. 3). Examining IB degradation (Fig. 3A, top), there was little difference between the pattern seen with Jurkat and that seen with P116 T cells. Both Jurkat and P116 T cells show a pronounced loss of IB protein expression 40 min after stimulation with PMA and calcium ionophore, and both show recovery of expression by 120 min. The ZAP-70-replete and -deficient status of Jurkat and P116 T cells, respectively, was confirmed by Western blotting for ZAP-70 (Fig. 3A, bottom).

View larger version (61K): [in this window] [in a new window]   FIGURE 3. NF-B is activated normally in response to distal or TCR-independent stimuli. A, Whole cell lysates were prepared from serum-starved Jurkat and P116 T cells that were incubated in the presence of PMA (50 ng/ml) and A23187 (500 ng/ml). Reactions were terminated at 0, 10, 40, and 120 min. Lysates were loaded at 10 µg protein/lane onto 4–12% NuPAGE gels in MOPS buffer, transferred to nitrocellulose, and immunoblotted with the indicated Ab. Membranes were blotted for IB and ZAP-70. B, Nuclear extracts were collected from serum-starved Jurkat and P116 T cells that were incubated in the presence of PMA and A23187 and were analyzed as in Fig. 2C. Sample from the EMSA 40-min time point of Jurkat and P116 T cells were analyzed as in Fig. 1. D, Nuclear extracts were collected from serum-starved Jurkat and P116 T cells stimulated with TNF- (10 ng/ml) and were analyzed as in Fig. 2C.

To further confirm that NF-B activation by TCR-independent pathways is normal in P116 T cells, NF-B activation was examined in Jurkat and P116 T cells in response to TNF- (Fig. 3D; Ref. 64). Nuclear extracts were prepared from Jurkat and P116 T cells that had been stimulated with TNF- for 0, 10, 40, and 120 min. Strong binding activity to the NF-B consensus binding site was observed after 40 min of stimulation in both P116 and Jurkat T cells. NF-B binding activity was reduced but still measurable after 120 min of stimulation in both cell lines. Taken together, the above data demonstrate that signaling events leading to NF-B activity that lie below the level of ZAP-70 or that lie in parallel pathways are intact in the P116 T cells. This further supports the idea that failure to activate NF-B in P116 T cells in response to concurrent stimulation of CD3 and CD28 results from the absence of ZAP-70 expression and not an undefined signaling defect.

Engagement of CD28 does not act by altering the kinetics of ZAP-70 activation

The above results identify ZAP-70 as a critical component of the TCR-initiated signaling pathway contributing to NF-B activation; however, the nature of the signal that must be supplied by CD28 to initiate full NF-B activation remains to be established. It has recently been proposed that the primary signal delivered by CD28 engagement may be an augmentation of signal one rather than a unique signal two. This was first suggested in response to the observation that CD28 engagement can increase the maximum amplitude and duration of tyrosine phosphorylation of proteins initiated by TCR engagement (24, 25). To test whether CD28 works by such a mechanism in our system, Jurkat T cells were stimulated for the times indicated with either anti-CD3 plus anti-CD28, or with anti-CD3 or anti-CD28 alone (Fig. 4, top). ZAP-70 immunoprecipitates were immunoblotted for phosphotyrosine, a marker for ZAP-70 activation in Jurkat T cells. The pattern of ZAP-70 tyrosine phosphorylation, both in terms of amplitude and kinetics is the same for both the CD3-stimulated and the CD3 plus CD28 costimulated samples. The 3-min pervanadate stimulation control demonstrates that the ZAP-70 tyrosine phosphorylation signal was not saturated at any of the CD3-stimulated or CD3 plus CD28-costimulated time-points, making it unlikely that a failure to detect a difference between the different stimulation conditions was the result of signal saturation. Comparable amounts of ZAP-70 were present in all of the samples (Fig. 4, bottom). ZAP-70 kinase activity also was measured under these conditions and similar results were obtained (data not shown).

View larger version (38K): [in this window] [in a new window]   FIGURE 4. CD28 does not alter ZAP-70 tyrosine phosphorylation kinetics. ZAP-70 immunoprecipitates were prepared from 2 x 106 Jurkat T cells after stimulation with both mAbs to CD3 (1:1000 of OKT3 ascites) and CD28 (2 µg/ml 9.3), CD3 alone, or CD28 cross-linked with rabbit-anti-mouse (1 µg/ml) Ab for 0, 3, 15, or 30 min or with pervanadate (PV) for 3 min at 37°C. Samples were loaded onto a 4–12% NuPAGE gel in MOPS buffer, transferred to nitrocellulose, and immunoblotted with the indicated Ab. The membrane was blotted for phosphotyrosine, stripped, and blotted for ZAP-70

ZAP-70 is required for PKC activation

PKC recently has been shown to be a key component of the signaling pathway coupling CD3/CD28 coengagement to NF-B activation (27, 28, 29, 30). Therefore, it would be predicted that PKC activation in response to CD3/CD28 stimulation also would be defective in the absence of ZAP-70. To test this, PKC was immunoprecipitated from the cellular lysates of P116 and P116.c39 Jurkat T cells. The intrinsic kinase activity of the immunoprecipitated enzyme was determined as described previously (58). There was little to no CD3/CD28-stimulated increase in kinase activity in PKC isolated from P116 cells, whereas the kinase was strongly stimulated in the CD3/CD28-activated P116.c39 cells (Fig. 5). This correlates well with what was observed for NF-B activation in these cells. Comparable amounts of PKC were used in the assays, as determined by immunoblotting for PKC (not shown).

View larger version (12K): [in this window] [in a new window]   FIGURE 5. CD3/CD28-stimulated activation of PKC kinase activity requires ZAP-70. PKC was immunoprecipitated from lysates containing P116 and P116.c39 T cells that had been either left unstimulated or stimulated with anti-CD3 (1:100 OKT3 ascites) and anti-CD28 (2 µg/ml 9.3) mAbs for 10 min. PKC immunoprecipitates were subjected to an in vitro PKC kinase assay as described in Materials and Methods.

A potential site of action for ZAP-70 in regulating the pathway leading to NF-B activation that would also be consistent with a role for PKC would be at the level of Vav-1 tyrosine phosphorylation and activation. ZAP-70 has been reported to be able to phosphorylate Vav-1 in heterologous overexpression systems (15, 41, 42), and deficient Vav-1 phosphorylation has been reported in T cells that overexpress a dominant negative ZAP-70 (43) or that fail to express ZAP-70 (10). Therefore, Vav-1 tyrosine phosphorylation was examined over a 20-min time period under the stimulatory conditions used for assessing IB degradation and NF-B band-shift (Fig. 6, top). Unexpectedly, under these conditions of stimulation, only minor differences in Vav-1 tyrosine phosphorylation were observed between the ZAP-70-negative and -replete T cell lines. Equal amounts of Vav-1 were precipitated from each cell line. Examination of whole-cell lysate samples from these same cells for tyrosine phosphorylated proteins showed interesting differences both in the region between 50 and 60 kDa and around 70 kDa. The former is consistent with the region where the Src-family PTKs Lck, Fyn, and Yes migrate, and the later is consistent with where ZAP-70, SLP-76, and Itk migrate. The P116 cells lack any signal in the 70 kDa range, consistent with the absence of ZAP-70, yet they exhibit a much stronger signal in the region where the Src-family proteins migrate, perhaps suggesting a compensatory increase in Src-family PTK activity, which could account for the persistence of Vav-1 tyrosine phosphorylation.

View larger version (41K): [in this window] [in a new window]   FIGURE 6. Tyrosine phosphorylation of Vav-1 in response to CD3/CD28 costimulation is ZAP-70 independent. Whole-cell lysates were prepared from serum-starved P116 and P116.c39 T cells that were incubated in the presence of Abs to CD3 (1:100 OKT3 ascites) and CD28 (2 µg/ml 9.3) for 0, 5, 10, or 20 min. Vav-1 immunoprecipitated from the lysates was immunoblotted for phosphotyrosine (4G10), stripped, and blotted for Vav-1. Whole-cell lysates were blotted for phosphotyrosine (4G10). Arrowhead indicates position of ZAP-70. Small arrow indicates position of Src family kinases.

Although there was no apparent requirement for ZAP-70 in supporting phosphorylation of Vav-1 in the P116 cells in response to CD3/CD28 stimulation, it remained possible that ZAP-70 could be affecting Vav-1 function indirectly. One possibility that was investigated was whether ZAP-70 expression was required to support the association of Vav-1 with SLP-76. Although the role of SLP-76 in regulating PKC and NF-B activation has not been tested, SLP-76 and Vav-1 functionally interact in other pathways, such as the activation of NF-AT, Rac-1, and Cdc42 activation (51, 53, 54) and physically interact via an association involving the SH2 domain of Vav-1 and specific tyrosine residues in SLP-76 that are phosphorylated by ZAP-70 (15, 51, 52). To test this possibility, Vav-1 was immunoprecipitated from the lysates of either P116 or P116.c39 cells that were stimulated for 0, 3, 5, 10, or 30 min at 37°C with mAb to CD3 and CD28 (Fig. 7A). In the P116.c39 cells, there was a rapid association of SLP-76 with Vav-1 that could be detected as early as 3 min after stimulation. The association persisted for 10 min, dropping to baseline by 30 min. No stimulation-induced increase in SLP-76 association with Vav-1 could be detected in the P116 cells. Comparable amounts of Vav-1 were brought down in each immunoprecipitate.

View larger version (57K): [in this window] [in a new window]   FIGURE 7. CD3/CD28-stimulated association of SLP-76 with Vav-1 requires ZAP-70 expression. A, P116 and P116.c39 T cells were stimulated with mAb to anti-CD3 (1:500 OKT3 ascites) and anti-CD28 (2 µg/ml 9.3) mAbs for 0, 3, 5, 10, or 30 min. Vav-1 was immunoprecipitated from 1 x 107 cell equivalents and was immunoblotted for SLP-76 and Vav-1. B, Vav-SH2 affinity precipitates were prepared from 2 x 107 Jurkat and P116 T cells after stimulation with anti-CD3 (1:100 OKT3) for 0, 0.5, 2, 8, 15, and 30 min. Samples were precleared with 20 µg of GST bound on glutathione beads before affinity precipitation. The affinity precipitates and a whole-cell lysate sample from 2-min OKT3-stimulated Jurkat T cells (last lane) underwent SDS-PAGE on a 6% Tris-glycine gel and were blotted with an Ab for phosphotyrosine (4G10).

SLP-76 is required for IB degradation and NF-B activity in response to CD3 and CD28 costimulation

Given that SLP-76 is a substrate of ZAP-70 (14, 15) and that there is a correlation in the CD3/CD28-stimulated P116 cells between failure to form a complex between SLP-76 and Vav-1 and the failure to activate NF-B, we next examined whether or not SLP-76 is required for NF-B activation in response to these stimuli. SLP-76-replete (J14.76.11) and -negative (J14.v.29) Jurkat T cell lines were used for this analysis. Surface expression levels of CD3 and CD28 were found to be comparable for the two cell lines (Fig. 1), as was the expression level of ZAP-70 (Fig. 8A, bottom). After stimulation with PMA and calcium ionophore, which would be expected to exert their effects downstream of SLP-76, IB degradation, as measured by western blot (Fig. 8A, top), and NF-B activation, as measured by EMSA (Fig. 8B), were readily detected at 40 min in both the SLP-76-replete and -negative cell lines. However, when these cells were costimulated with anti-CD3 and anti-CD28 mAbs, only the cells that are replete for SLP-76 underwent IB degradation (Fig. 8A, top) and NF-B DNA-binding (Fig. 8B). As expected, SLP-76 was only detected in the SLP-76 reconstituted cells and not in the vector control cells (Fig. 8A, middle). Inhibition and supershift studies demonstrated that the shifted band contains an NF-B DNA-binding complex made up of p50 and p65 subunits (data not shown). The SLP-76 dependence of NF-B activation noted here necessitated that we measure the relative levels of SLP-76 expression in Jurkat, P116, and P116.c39 cells. These three cell lines were found to have equivalent levels of SLP-76 in their cellular lysates (Fig. 2, bottom), ruling out differences in SLP-76 expression as contributing toward the NF-B signaling deficiency of the P116 cell line.

View larger version (60K): [in this window] [in a new window]   FIGURE 8. SLP-76 is required for CD3/CD28-stimulated IB degradation and NF-B activation. A, Whole cell lysates were prepared from J14.76.11 (SLP-76-replete) and J14.v.29 (SLP-76-negative) Jurkat T cells that were incubated in the presence of PMA (50 ng/ml) and A23187 (500 ng/ml) or Abs to CD3 (1:100 OKT3 ascites) and CD28 (2 µg/ml 9.3), for 0, 10, 40, or 120 min. Lysates were loaded at 10 µg/lane onto 4–12% NuPAGE gels in MOPS buffer, transferred to nitrocellulose, and immunoblotted with the indicated Ab. Membranes were blotted for IB, stripped, and blotted for SLP-76 and then stripped and blotted for ZAP-70 B, Nuclear extracts were collected from J14.76.11 and J14.v.29 cells that were incubated as in A and analyzed as in Fig. 2C.

SLP-76 is required for PKC activation in response to CD3 and CD28 costimulation

Because SLP-76 expression is clearly required for NF-B activation, the role of SLP-76 in PKC activation also was assessed. PKC was immunoprecipitated from the cellular lysates of J14-v-29 and J14-76-11 Jurkat T cells, and the intrinsic kinase activity measured. There was little to no CD3/CD28-stimulated increase in kinase activity in PKC isolated from J14-v-29 cells, whereas the kinase was strongly stimulated in the CD3/CD28-activated J14-76-11 cells (Fig. 9A). This correlates well with the NF-B activation results observed with these cells.

View larger version (13K): [in this window] [in a new window]   FIGURE 9. CD3/CD28-stimulated activation of PKC kinase activity requires SLP-76. A, PKC was immunoprecipitated from J14-76-11 and J14-v-29 T cells that had been either left unstimulated or stimulated with anti-CD3 (1:100 OKT3 ascites) and anti-CD28 (2 µg/ml 9.3) mAb for 10 min. PKC immunoprecipitates were subjected to an in vitro PKC kinase assay as in Fig. 5. Results are expressed as percent increases in kinase activity between stimulated and unstimulated cells. B, An NF-B luciferase reporter gene (10 µg) and -galactosidase gene (5 µg) were cotransfected with 20 µg of empty vector, wild-type SLP-76, or SLP-76Y3F into 1.2 x 107 J14-v-29 cells. The transfected cells were cultured at 37°C for 15 h and then washed and resuspended to 2 x 106 cells/ml in complete medium. Cells were either left unstimulated, stimulated with Abs to CD3 (OKT3 ascites 1:100) and CD28 (2 µg/ml 9.3), or stimulated with PMA (50 ng/ml) and A23187 (500 ng/ml) for 6 h. Lysates were prepared and analyzed for luciferase and -gal activities. The results are expressed in terms of their percentage of the maximum response elicited with PMA and A23187.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
NF-B is a transcription factor the activity of which is critical for T cell proliferation. Although events proximal to NF-B activation are increasingly well understood, the early T cell signaling events emanating from TCR and CD28 engagement that combine to lead to NF-B activation are less clear. Given the demonstrated importance of ZAP-70 and SLP-76 in supporting T cell activation, we sought to determine whether these proteins play a role in NF-B activation after engagement of the TCR and the costimulatory molecule, CD28. With Jurkat T cell clones lacking ZAP-70 or SLP-76, we found a requirement for both ZAP-70 and SLP-76 in signaling IB degradation and the subsequent activation of NF-B. That the inability to activate NF-B in these cells was in fact attributable to the absence of ZAP-70 or SLP-76 is indicated by two separate lines of evidence. First, stimuli such as PMA and A23187 that bypass early signaling events were capable of inducing IB degradation and nuclear translocation of NF-B to the same extent in both the parental and ZAP-70- or SLP-76-deficient cell lines. TNF-, which acts via a ZAP-70-independent pathway, could also induce IB degradation and nuclear translocation of NF-B equally well in the parental and ZAP-70-negative cells. Second, and more importantly, reintroduction of normal expression levels of ZAP-70 and SLP-76 by stable transfection restored the ability of these cells to activate NF-B in response to stimulation through CD3 and CD28. Our findings are consistent with those of Ouellet et al. who recently reported that Lck, ZAP-70, and SLP-76 are required for NF-B activation in response to bisperoxovanadium, a phosphotyrosyl phosphatase inhibitor that mimics many aspects of TCR signaling (65). However, to the best of our knowledge, this is the first demonstration that ZAP-70 and SLP-76 are required for PKC and NF-B activation in T cells stimulated through TCR and costimulatory receptor engagement.

ZAP-70 is one of the major mediators of the signal delivered to the T cell in response to engagement of the TCR, so it is perhaps not unexpected that ZAP-70 would play an important role in TCR-stimulated NF-B activation. However, it is interesting to note that while other ZAP-70-mediated signaling events, such as increases in intracellular [Ca²⁺], and the activation of NF-AT and extracellular signal-related kinase (Erk), can all be maximally stimulated through TCR engagement alone, ZAP-70-mediated activation of NF-B does not occur efficiently unless the accessory signal delivered by CD28 also is present (Fig. 1B and Ref. 66). This has generally been interpreted as indicating that some element in the signal transduction pathway leading to NF-B activation in T cells requires a signal from CD28 in addition to the TCR to become activated, whereas other signaling pathways do not have such a requirement. Alternatively, it has been proposed that CD28 may act principally, and perhaps solely, by augmenting the TCR signal (24, 25). Under this model, NF-B activation would simply require a greater signal through the TCR, rather than a unique signal originating from CD28.

Given that ZAP-70 appears to represent a signaling bottleneck in TCR signaling, and given our results, which establish that ZAP-70 is required for NF-B activation in response to CD3/CD28 coengagement, one would predict that if in signaling to NF-B CD28 were merely augmenting the signal arising from the TCR, then CD28 cross-linking would necessarily lead to an augmentation of ZAP-70 activation in our system. However, this was not observed when the effect of CD28 cross-linking on submaximal anti-CD3-induced ZAP-70 phosphorylation was measured. No augmentation of ZAP-70 phosphorylation by CD28 engagement could be detected. These results argue against the only role of CD28 as being an enhancer of TCR signaling and indicate that CD28 provides a unique, as yet unidentified, signal that is required for NF-B activation in these cells. However, we cannot rule out the possibility that CD28 is augmenting a ZAP-70-independent, TCR-initiated signal that is required for NF-B activation rather than contributing a unique signal. It should be noted that the failure of CD28 engagement to augment anti-CD3 mAb-induced ZAP-70 tyrosine phosphorylation reported here is in conflict with the report of Tuosto and coworkers (24). The reason for the disparity may arise from the different methods used for stimulation. Although both studies used Jurkat T cells, the present study used mAbs to CD3 and CD28, whereas Tuosto and colleagues used superantigen-pulsed L cells in the presence or absence of CTLA4-Ig, to block CD28 engagement (24).

In considering possible sites of action for ZAP-70 in regulating NF-B activation, Vav-1 seemed a likely candidate, as Vav-1 has been shown to be required for NF-B activation in CD3/CD28 costimulated CD4⁺ T cells (39). Furthermore, studies with overexpression of kinase-dead, TCR-proximal kinases support a role for ZAP-70 in Vav-1 tyrosine phosphorylation and activation (43), as do several other studies (10, 41, 42). However, we unexpectedly found that Vav-1 could be phosphorylated to comparable levels in both the ZAP-70-negative and ZAP-70-replete Jurkat T cells when activated via CD3 and CD28. This finding was unexpected, because Salojin and colleagues (10) have reported defective Vav-1 tyrosine phosphorylation in P116 T cells, the same cell line that was used in the present study. The explanation for the different findings is not yet clear but may stem from differences in the way that the cells were stimulated in the two studies. In the study by Salojin and colleagues, CD3 and CD28 were co-cross-linked to one another with secondary Ab. Whereas no secondary Ab cross-linking of CD3 to CD28 was used in the current study. The more potent stimulus provided by co-cross-linking may accentuate the differences in signaling capacity of the ZAP-70-negative and ZAP-70-replete Jurkat T cells. Notably, in a few experiments Vav-1 phosphorylation was reduced in the P116 T cells, but even in these experiments, the fold increase in phosphorylation level with stimulation was always equal to or greater than that observed in ZAP-70 replete Jurkat T cells. These data argue against Vav-1 tyrosine phosphorylation as the point of insertion for ZAP-70 into the NF-B activation pathway. However, without actually mapping out which sites in Vav-1 become phosphorylated on stimulation in the two cell lines, we cannot rule out the possibility that a different pattern of sites is being phosphorylated in the presence or absence of ZAP-70, with possible functional consequences for Vav-1.

Like Vav-1, PKC has been reported to play a T cell-specific role in NF-B activation (27, 28, 29, 30, 40). The possibility that PKC or events upstream of its activation could represent a site of action of ZAP-70 in supporting NF-B was considered, and PKC activation in response to CD3/CD28 stimulation was found to be greatly deficient in the absence of ZAP-70. Precisely how ZAP-70 acts to regulate PKC activity remains to be determined. The role of ZAP-70 in supporting activation of PLC and the consequent production of PKC-activating DAG presents one possibility (7, 9, 67). PKC also has been reported to be subject to stimulation-induced tyrosine phosphorylation (68, 69); however, the role of ZAP-70 in this process, if any, has yet to be assessed. Our preliminary data (not shown) show no clear differences between the ZAP-70-replete and -deficient cells in terms of CD3/CD28-stimulated PKC tyrosine phosphorylation.

The finding that the ZAP-70 substrate SLP-76 also is required for PKC activation offers another possible mechanism by which ZAP-70 could be regulating PKC activity but raises the question of what role SLP-76 is playing in PKC activation. Previous studies with this SLP-76-negative cell line found that SLP-76 is required for Ras, Erk, and NF-AT activation, as well as Ca²⁺ mobilization, but its role in PKC and NF-B activation was not assessed (20). The signaling element that is likely to be common to each of these signaling pathways, as well as to PKC activation, is the activation of PLC1, which supports NF-AT activation via increases in intracellular Ca²⁺ and supports the activation of Ras-GRP (and consequently Ras and Erk) and PKC via DAG production. In the absence of SLP-76 expression, PLC1 fails to become tyrosine phosphorylated, and there is no IP3 production in response to TCR stimulation (20). This action of SLP-76 seems to require tyrosine phosphorylation of specific tyrosine residues (Y113 and Y128), as mutation of these residues to phenylalanine strongly impairs NF-AT activation (70). Interestingly, whereas SLP-76 can be phosphorylated in vitro by the Src family kinases, Lck, and Fyn, only ZAP-70 can phosphorylate SLP-76 on Y113 and Y128 and support the ability of SLP-76 to bind to the SH2 domain of Vav-1 (14, 15, 54). Whether the reduced signaling capacity of Y113F/Y128F- SLP-76 reported above (70), and the reduced capacity of Y113F/Y128F/Y145F-SLP-76 to support NF-B activation (this report) is a consequence of a requirement for the formation of a SLP-76/Vav-1 complex or some other intermolecular interaction remains to be determined. However, given that we find that SLP-76 and ZAP-70 are both required for CD3/CD28-mediated NF-B activation, that ZAP-70 phosphorylates the N-terminal tyrosines of SLP-76 (14, 15, 54), and that mutation of the tyrosines in SLP-76 that get phosphorylated by ZAP-70 leads to defective SLP-76 signaling (54, 71), it is probable that failure to phosphorylate SLP-76 (and support required protein-protein interactions) is a contributing factor in the signaling failure observed in CD3/CD28-stimulated ZAP-70-negative P116 T cells.

Another ZAP-70 substrate, Lat, also represents a likely candidate for playing a role in coupling ZAP-70 to NF-B activation. Through the study of Lat-negative Jurkat mutant T cell lines, Lat expression has been shown to be required for Ca²⁺ mobilization, Ras, Erk, and NF-AT activation, as well as activation of IL-2 promoter-driven transcription (72, 73). Additionally, Lat can form a complex with both Vav-1 and SLP-76, and Lat expression is required for efficient TCR-stimulated tyrosine phosphorylation of these proteins. However, we were unable to assess the role of Lat in mediating ZAP-70 signaling to NF-B in these studies, because the Lat-negative cells are also negative for CD28 expression (72).

In summary, we report that ZAP-70 and SLP-76 are both required for activation of NF-B in Jurkat T cells stimulated by coengagement of TCR and CD28. The dependency of this process on these signaling proteins stems from their requirement for the activation of PKC. Because SLP-76 is a substrate of ZAP-70, it is likely that the failure to activate NF-B in both the ZAP-70-negative and SLP-76-negative cell lines is a consequence of a failure to signal to effector molecules downstream of SLP-76, principally PLC1. To our knowledge, this is the first study to demonstrate a role for these signaling molecules in PKC or NF-B activation in response to CD3/CD28 costimulation. This study further highlights the importance of these signaling molecules in regulating T cell function.

	Acknowledgments

 
We thank Drs. Robert Abraham, Paul Findell, Gary Koretzky, Carl June, and Arthur Weiss for their gift of reagents, Drs. Kip Hartman, Dan McVicar, and Henry Wong for helpful discussions, Francis J. Chrest, Christa M. Morris, and Dr. Robert Wersto of the Gerontology Research Center Flow Cytometry Laboratory, and Drs. Jason Wood and Maria-Cristina Seminario for their critical evaluation of the manuscript.

	Footnotes

 
¹ Current address: Howard Hughes Medical Institute, Johns Hopkins School of Medicine, Baltimore, MD 21205.

² Address correspondence and reprint requests to Dr. Ronald L. Wange, National Institute on Aging, Gerontology Research Center, Box 12, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825.

³ Abbreviations used in this paper: ZAP-70, chain associated protein of 70 kDa; PTK, protein tyrosine kinase; MAPK, mitogen-acivated protein kinase; Lat, linker for activation of T cells; SLP-76, SH2 domain containing leukocyte phosphoprotein of 76 kDa; Vav, p95^vav; PLC1, phospholipase C1; PKC, protein kinase C; IKK, IB kinase; SH2, Src homology domain 2; Ras, p21^ras oncoprotein; Erk, extracellular signal-regulated kinase; Itk, IL-2-inducible T cell kinase.

Received for publication January 28, 2000. Accepted for publication February 16, 2001.

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