Brandi L. Williams

Sequential Involvement of Lck and SHP-1 with MHC-Recognizing Receptors on NK Cells Inhibits FcR-Initiated Tyrosine Kinase Activation

Recognition of major histocompatibility (MHC) class I complexes on target cells by killer cell inhibitory receptors (KIR) blocks natural killer (NK) and T cell cytotoxic function. The inhibitory effect of KIR ligation requires the phosphotyrosine-dependent association of KIR with the cytoplasmic SH2-containing protein tyrosine phosphatase SHP-1. Using a somatic genetic model, we first define a requirement for the Src family protein tyrosine kinase (PTK) Lck in mediating KIR tyrosine phosphorylation. We then investigate how KIR ligation interrupts PTK-dependent NK cell activation signals. Specifically, we show that KIR ligation inhibits the Fc receptor (FcR)-induced tyrosine phosphorylation of the FcR-associated zeta signaling chain, the PTK ZAP-70, and phospholipase C gamma. Overexpression of catalytically inactive SHP-1 (acting as a dominant negative) restores the tyrosine phosphorylation of these signaling events and reverses KIR-mediated inhibition of NK cell cytotoxic function. These results suggest sequential roles for Lck and SHP-1 in the inhibition of PTK following MHC recognition by NK cells.

Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.

ABSTRACT T-cell antigen receptor (TCR) engagement activates multiple protein tyrosine kinases (PTKs), including the Src family member, Lck, and the Syk-related PTK, ZAP-70. Studies in ZAP-70-deficient humans have demonstrated that ZAP-70 plays crucial roles in T-cell activation and development. However, progress toward a detailed understanding of the regulation and function of ZAP-70 during TCR signaling has been hampered by the lack of a suitable T-cell model for biochemical and genetic analyses. In this report, we describe the isolation and phenotypic characterization of a Syk- and ZAP-70-negative somatic mutant derived from the Jurkat T-cell line. The P116 cell line displays severe defects in TCR-induced signaling functions, including protein tyrosine phosphorylation, intracellular Ca2+ mobilization, and interleukin-2 promoter-driven transcription. These signaling defects were fully reversed by reintroduction of catalytically active versions of either Syk or ZAP-70 into the P116 cells. However, in contrast to ZAP-70 expression, Syk expression triggered a significant degree of cellular activation in the absence of TCR ligation. Transfection experiments with ZAP-70–Syk chimeric proteins indicated that both the amino-terminal regulatory regions and the carboxy-terminal catalytic domains of Syk and ZAP-70 contribute to the distinctive functional properties of these PTKs. These studies underscore the crucial role of ZAP-70 in TCR signaling and offer a powerful genetic model for further analyses of ZAP-70 regulation and function in T cells.

Phosphorylation of Tyr319 in ZAP‐70 is required for T‐cell antigen receptor‐dependent phospholipase C‐γ1 and Ras activation

Accumulating evidence indicates that the interdomain B regions of ZAP‐70 and Syk play pivotal roles in the coupling of T‐cell antigen receptor (TCR) stimulation to the activation of downstream signaling pathways. The interdomain B region of ZAP‐70 contains at least three candidate sites of tyrosine phosphorylation. In this report, we identify Tyr319 as a functionally important phosphorylation site in the ZAP‐70 interdomain B region. TCR crosslinkage triggered a rapid increase in the phosphorylation of Tyr319 in Jurkat T cells. Although mutation of Tyr319 to Phe had no effect on the tyrosine kinase activity of ZAP‐70, the resulting ZAP(Y319→F) mutant failed to reconstitute TCR‐dependent Ca2+ mobilization, Ras activation, CD69 expression and NFAT‐dependent transcription in ZAP‐70‐deficient Jurkat cells. These defects were correlated with reduced tyrosine phosphorylation of phospholipase C (PLC)‐γ1 and the LAT adapter protein in the ZAP(Y319→F)‐expressing cells. On the other hand, ZAP(Y319→F)‐expressing cells displayed normal increases in SLP‐76 phosphorylation and ERK activation during TCR stimulation. Phosphorylation of Tyr319 promoted the association of ZAP‐70 with the SH2 domains of two key signaling molecules, Lck and PLC‐γ1. These studies suggest that Tyr319 phosphorylation is required for the assembly of a ZAP‐70‐containing signaling complex that leads to the activation of the PLC‐γ1‐ and Ras‐dependent signaling cascades in antigen‐stimulated T cells.

Antiproliferative action of interferon-alpha requires components of T-cell-receptor signalling

Signal transduction through both cytokine and lymphocyte antigen receptors shares some common pathways by which they initiate cellular responses, such as activation of mitogen-activated protein kinase(s),. However, other signalling components appear to be uniquely coupled to each receptor. For example, the interferon receptors transduce regulatory signals through the JAK/STAT pathway, resulting in an inhibition of growth and of antiviral effects, whereas this pathway apparently plays no role in T-cell-receptor (TCR)-dependent gene expression,. Conversely, signal transduction through the TCR requires the tyrosine kinases Lck and ZAP-70 and the tyrosine phosphatase CD45 (ref. 5). Here we show that, unexpectedly, transmission of growth-inhibitory signals by interferon-α (IFN-α) in T cells requires the expression and association of CD45, Lck and ZAP-70 with the IFN-α-receptor signalling complex.

SLP-76 Is a Direct Substrate of SHP-1 Recruited to Killer Cell Inhibitory Receptors

Activation of immune system cells via antigen-, Fc-, or natural killer cell-triggering-receptor stimulation is aborted by co-engagement of inhibitory receptors. Negative signaling by killer cell inhibitory receptors and related receptors depends on the Src homology 2 (SH2)-containing protein tyrosine phosphatase SHP-1. Using a combination of direct binding and functional assays, we demonstrated that the SH2 domain-containing leukocyte protein 76 (SLP-76) is a specific target for dephosphorylation by SHP-1 in T cells and natural killer cells. Furthermore, we showed that tyrosine-phosphorylated SLP-76 is required for optimal activation of cytotoxic lymphocytes, suggesting that the targeted dephosphorylation of SLP-76 by SHP-1 is an important mechanism for the negative regulation of immune cell activation by inhibitory receptors.

Pleiotropic Contributions of Phospholipase C-γ1 (PLC-γ1) to T-Cell Antigen Receptor-Mediated Signaling: Reconstitution Studies of a PLC-γ1-Deficient Jurkat T-Cell Line

ABSTRACT Phospholipase C-γ1 (PLC-γ1) plays a crucial role in the coupling of T-cell antigen receptor (TCR) ligation to interleukin-2 (IL-2) gene expression in activated T lymphocytes. In this study, we have isolated and characterized two novel, PLC-γ1-deficient sublines derived from the Jurkat T-leukemic cell line. The P98 subline displays a >90% reduction in PLC-γ1 expression, while the J.gamma1 subline contains no detectable PLC-γ1 protein. The lack of PLC-γ1 expression in J.gamma1 cells caused profound defects in TCR-dependent Ca2+ mobilization and NFAT activation. In contrast, both of these responses occurred at normal levels in PLC-γ1-deficient P98 cells. Unexpectedly, the P98 cells displayed significant and selective defects in the activation of both the composite CD28 response element (RE/AP) and the full-length IL-2 promoter following costimulation with anti-TCR antibodies and phorbol ester. These transcriptional defects were reversed by transfection of P98 cells with a wild-type PLC-γ1 expression vector but not by expression of mutated PLC-γ1 constructs that lacked a functional, carboxyl-terminal SH2 [SH2(C)] domain or the major Tyr783 phosphorylation site. On the other hand, the amino-terminal SH2 [SH2(N)] domain was not essential for reconstitution of RE/AP- or IL-2 promoter-dependent transcription but was required for the association of PLC-γ1 with LAT, as well as the tyrosine phosphorylation of PLC-γ1 itself, in activated P98 cells. These studies demonstrate that the PLC-γ1 SH2(N) and SH2(C) domains play functionally distinct roles during TCR-mediated signaling and identify a non-Ca2+-related signaling function linked to the SH2(C) domain, which couples TCR plus phorbol ester-CD28 costimulation to the activation of the IL-2 promoter in T lymphocytes.

EDD Mediates DNA Damage-induced Activation of CHK2

EDD, the human orthologue of Drosophila melanogaster “hyperplastic discs,” is overexpressed or mutated in a number of common human cancers. Although EDD has been implicated in DNA damage signaling, a definitive role has yet to be demonstrated. Here we report a novel interaction between EDD and the DNA damage checkpoint kinase CHK2. EDD and CHK2 associate through a phospho-dependent interaction involving the CHK2 Forkhead-associated domain and a region of EDD spanning a number of putative Forkhead-associated domain-binding threonines. Using RNA interference, we demonstrate a critical role for EDD upstream of CHK2 in the DNA damage signaling pathway. EDD is necessary for the efficient activating phosphorylation of CHK2 in response to DNA damage following exposure to ionizing radiation or the radiomimetic, phleomycin. Cells depleted of EDD display impaired CHK2 kinase activity and an inability to respond to DNA damage. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasize the potential importance of EDD in cancer.

Interdomain B in ZAP-70 Regulates but Is Not Required for ZAP-70 Signaling Function in Lymphocytes

ABSTRACT The protein tyrosine kinase ZAP-70 plays an important role in T-cell activation and development. After T-cell receptor stimulation, ZAP-70 associates with the receptor and is phosphorylated on many tyrosines, including Y292, Y315, and Y319 within interdomain B. Previously, we demonstrated that Y292 negatively regulates ZAP-70 function and that Y315 positively regulates ZAP-70 function by interacting with Vav. Recent studies have suggested that Y319 also positively regulate ZAP-70 function. Paradoxically, removal of interdomain B (to create the construct designated Δ), containing the Y292, Y315, and Y319 sites, did not eliminate the ability of ZAP-70 to induce multiple gene reporters in Syk-deficient DT-40 B cells and ZAP-70/Syk-deficient Jurkat cells. Here we show that Δ still utilizes the same pathways as wild-type ZAP-70 to mediate NF-AT induction. This is manifested by the ability of Δ to restore induction of calcium fluxes and mitogen-activated protein kinase activation and by the ability of dominant negative Ras and FK506 to block the induction of NF-AT activity mediated by Δ. Biochemically we show that the stimulated tyrosine phosphorylation of Vav, Shc, and ZAP-70 itself is diminished, whereas that of Slp-76 is increased in cells reconstituted with Δ. Deletion of interdomain B did not affect the ability of ZAP-70 to bind to the receptor. The in vitro kinase activity of ZAP-70 lacking interdomain B was markedly reduced, but the kinase activity was still required for the protein’s in vivo activity. Based on these data, we concluded that interdomain B regulates but is not required for ZAP-70 signaling function leading to cellular responses.