In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.

The initiation of biochemical signal transduction following ligation of surface receptors with intrinsic cytoplasmic tyrosine kinase activity is common for many cell types. T lymphocytes also require activation of tyrosine kinases following T cell receptor (TCR) ligation for maximal stimulation. However, the TCR has no intrinsic tyrosine kinase activity. Instead, the TCR must rely on cytoplasmic tyrosine kinases that localize to the TCR complex and initiate TCR-mediated signaling events. Although much has been learned regarding how these cytosolic tyrosine kinases are activated and recruited to the TCR complex, relatively little is understood about how these initial events are translated into transcriptional activation of genes that regulate cytokine production, cell proliferation, and cell death. Recently, it has become clear that the class of intracellular molecules known collectively as adapter proteins, molecules with modular domains capable of recruiting additional proteins but that exhibit no intrinsic enzymatic activity, serve to couple proximal biochemical events initiated by TCR ligation with more distal signaling pathways.

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have a consensus binding site for the SH2 domain of Vav (DYESP) (2-5) and are heavily tyrosine-phosphorylated following TCR engagement, whereas Tyr 145 , which falls in the sequence DYEPP, is phosphorylated to a lesser extent (6). SLP-76 also contains a central proline-rich region that mediates the association with Grb2 (7) and a carboxyl-terminal SH2 domain that binds to at least two tyrosine-phosphorylated proteins, SLAP-130 (SLP-76-associated phosphoprotein of 130 kDa) (8), a 62-kDa protein, and an uncharacterized serine/threonine kinase after TCR engagement (7). SLP-76 is believed to be an essential adapter protein in T cells. Overexpression of SLP-76 results in an enhancement of TCR-mediated induction of nuclear factor of activated T cell and interleukin-2 promoter activity (3, 5-7, 9, 10). More recently, lack of expression of SLP-76 in Jurkat cells demonstrated that SLP-76 is necessary for tyrosine phosphorylation of phospholipase C-␥ 1 (PLC-␥ 1 ) and activation of the Ras pathway (11). Moreover, SLP-76 is required for normal thymocyte development, since SLP-76 knockout mice lack peripheral T cells (12, 13).The three tyrosine phosphorylation sites, the proline-rich region, and the SH2 domain of SLP-76 have all been shown to be important for the regulation of T cell interleukin 2 production (10). The inducible tyrosine phosphorylation of SLP-76 is mediated by ZAP-70 or Syk in COS cells (9) and rat basophilic leukemia cells (14), respectively. The mechanism by which SLP-76 is phosphorylated by ZAP-70 or Syk is not known.We have previously reported the association of tyrosinephosphorylated SLP-76 with the SH3 domain of Grb2 in platelets in response to stimulation of the low affinity IgG immunoreceptor Fc␥RIIA (15). Increasing evidence suggests that the collagen receptor underlying the major increase in tyrosine phosphorylation in platelets also signals like an immune receptor. The collagen receptor is believed to comprise a multimeric structure, containing the glycoprotein VI (GPVI), and the Fc receptor (FcR) ␥-chain (16 -19). Binding of collagen to GPVI induces tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of FcR ␥-chain (18), leading to tyrosine phosphorylation of Syk and

Recent studies indicate that reactive oxygen intermediates (ROI) serve as second messengers in cell signaling. ROI have been implicated in the activation of NF-kappaB as well as c-Jun N-terminal kinase (JNK) in response to IL-1 and TNF-alpha stimulation. In this report we examine whether intracellular ROI are involved in CD40 receptor signaling. We show that CD40 engagement on resting splenic B lymphocytes and murine B lymphoma WEHI 231 cells generates ROI. Blocking ROI production by preincubation with the antioxidant N-acetyl-L-cysteine inhibits JNK activation, NF-kappaB-driven luciferase activity, and IL-6 secretion following CD40 ligation, suggesting a role for ROI in CD40-mediated signaling events. Furthermore, transfection of WEHI 231 cells with a plasmid encoding Mn-superoxide dismutase interferes with CD40-induced NF-kappaB activation, providing further support for ROI involvement in this pathway. Collectively, these data demonstrate that ROI may serve as second messengers linking CD40 engagement on B cells to important downstream activation events.