Positive and negative regulation of T-cell activation by adaptor proteins.

Adaptor proteins, molecules that mediate intermolecular interactions, are now known to be as crucial for lymphocyte activation as are receptors and effectors. Extensive work from numerous laboratories has identified and characterized many of these adaptors, demonstrating their roles as both positive and negative regulators. Studies into the molecular basis for the actions of these molecules shows that they function in various ways, including: recruitment of positive or negative regulators into signalling networks, modulation of effector function by allosteric regulation of enzymatic activity, and by targeting other proteins for degradation. This review will focus on a number of adaptors that are important for lymphocyte function and emphasize the various ways in which these proteins carry out their essential roles.

Separation of Notch1 promoted lineage commitment and expansion/transformation in developing T cells.

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4(+)CD8(+) double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2(-/)-) or Src homology 2 domain--containing leukocyte protein of 76 kD (SLP-76)(-/)- mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2(-/)- progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3 epsilon and pre-T alpha mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2(-/)- mice with a TCR beta transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell-specific signals associated with development of DP thymocytes.

Coupling of the TCR to integrin activation by Slap-130/Fyb.

SLAP-130/Fyb (SLP-76-associated phosphoprotein or Fyn-binding protein; also known as Fyb/Slap) is a hematopoietic-specific adapter, which associates with and modulates function of SH2-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76). T cells from mice lacking SLAP-130/Fyb show markedly impaired proliferation following CD3 engagement. In addition, the T cell receptor (TCR) in SLAP-130/Fyb mutant cells fails to enhance integrin-dependent adhesion. Although TCR-induced actin polymerization is normal, TCR-stimulated clustering of the integrin LFA-1 is defective in SLAP-130/Fyb-deficient cells. These data indicate that SLAP-130/Fyb is important for coupling TCR-mediated actin cytoskeletal rearrangement with activation of integrin function, and for T cells to respond fully to activating signals.

Protective effect of oxidative stress in HaCaT keratinocytes expressing E7 oncogene.

In a previous study, we established a stable cell line which constitutively expresses E7 in HaCaT human keratinocyte cell line and identified various relevant factors including oxygen modulators affected by the E7 oncogene. E7-expressing HaCaT cells (HaCaT/E7) appeared to be more resistant to H2O2-induced cell death. Here, we demonstrate how E7 oncogene would modulate oxidative stress-induced cell death. In addition, we verified the increased expression of catalase in the HaCaT/E7 by Western blot analysis. The results suggest that the E7 oncogene would induce higher resistance to ROS-induced cell injury in the E7-infected cells via the upregulation of catalase. To investigate these paradoxical effects of high concentrations of H2O2 (500 microM-1 mM), we examined their effects on receptor mediated apoptosis, cell death via the mitochondrial pathway and modulation of apoptosis related factors. Our results revealed that HaCaT keratinocytes infected with HPV 16 E7 oncogene modulated expressions of catalase, Bcl-xL, IL-18, Fas, Bad, and cytochrome c as well as NF-kappaB, resulting in the resistance to oxidative stress-induced cell death.

Adapter proteins in lymphocyte antigen-receptor signaling.

Adapter molecules contain discrete modular domains that direct specific intermolecular interactions to orchestrate assembly of signaling complexes. A number of adapter proteins play critical roles in both positive and negative regulation of antigen-receptor signaling, influencing lymphocyte development and function.

Conversion of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase by GPI-PLD.

Enzymatic conversion of brain glycosylphosphatidylinositol-linked alkaline phosphatase (GPI-AP), amphiphilic, was examined. When GPI-AP was incubated with glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), a negligible conversion of GPI-AP to hydrophilic form was observed. The inclusion of monoacylglycerols enhanced the enzymatic conversion, although the action of monoacylglycerols differed greatly according to the size of acyl group; the enzymatic conversion was enhanced considerably in the presence of monoacylglycerols possessing acyl group of longer chain length (C10-C18), while monoacylglycerols with acyl moiety of shorter length (C4-C8) did fail to augment the enzymatic conversion. Noteworthy, monooleoylglycerol was much more effective than the other monoacylglycerols in promoting the enzymatic conversion, indicating a beneficial role of the unsaturation in acyl chain. Meanwhile, ionic amphiphiles such as monohexadecyllysophosphatidylcholine and palmitoyl-carnitine decreased the enzymatic conversion of GPI-AP in a concentration-dependent manner, with monohexadecyllysophosphatidylcholine being more inhibitory than palmitoylcarnitine. Separately, when GPI-AP was exposed to various oxidants prior to the incubation with GPI-PLD, a remarkable decrease of the enzymatic conversion was observed with hypochlorite and peroxynitrite generators, but not H2O2. In further study, hypochlorite was found to inactivate GPI-PLD at low concentrations (3 to approximately 100 microM). From these results, it is suggested that the enzymatic conversion of GPI-AP by GPI-PLD may be regulated in vivo system.

G1 to S phase transition protein 1 induces apoptosis signal-regulating kinase 1 activation by dissociating 14-3-3 from ASK1.

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, plays a critical role in mediating apoptosis signals initiated by a variety of death stimuli such as hydrogen peroxide and tumor necrosis factor-alpha. Owing to its critical role in inducing apoptosis, the activity of ASK1 is tightly regulated by various mechanisms such as post-translational modifications and protein-protein interactions. Here we describe the identification of G(1) to S phase transition protein 1 (GSPT1), which is associated with protein translation, as a regulator of ASK1. GSPT1 interacts with ASK1 and enhances ASK1-induced apoptotic activity through the activation of caspase-3. In vitro kinase assay data show that GSPT1 enhances ASK1 autophosphorylation and its kinase activity. Cell cycle-dependent GSPT1 induction and small interfering RNA analyses show that ASK1 autophosphorylation is dependent on the expression level of endogenous GSPT1 in cells. GSPT1 inhibits the binding of ASK1 to the 14-3-3 protein, an ASK1 inhibitor, while GSPT1 has no effect on the interaction between ASK1 and TRAF2, a C-terminal-binding activator of ASK1. Thus, our results reveal a novel role of GSPT1 in the regulation of ASK1-mediated apoptosis.

Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity.

The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser133 of CREB is essential for its activation by CaMKIV. Because both CaMKII and CaMKIV can phosphorylate CREB, we pursued further the mechanism by which CaMKII and CaMKIV differentially regulate CREB activity. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser133 only, whereas CaMKII phosphorylates CREB at Ser133 and a second site, Ser142. Transient transfection studies revealed that phosphorylation of Ser142 by CaMKII blocks the activation of CREB that would otherwise occur when Ser133 is phosphorylated. When Ser142 was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser142 to alanine enhanced the ability of Ca2+ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser142 in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca2+ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca2+ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca2+/calmodulin-dependent protein kinases.

Interaction of divalent metal ions with Zn(2+)-glycerophosphocholine cholinephosphodiesterase from ox brain.

The effect of divalent metal ions on the activity of glycerophosphocholine cholinephosphodiesterse from ox brain was examined. Zn(2+)- and Co(2+)-glycerophosphocholine cholinephosphodiesterases were prepared from the exposure of apoenzyme to Zn2+ and Co2+, respectively, and the properties of two metallo-phosphodiesterases were compared to those of native phosphodiesterase. Although two metallo-enzymes were similar in expressing Km value, optimum pH or sensitivity to Cu2+, they differed in the susceptibility to the inhibition by thiocholine or tellurite; while Co(2+)-phosphodiesterase was more sensitive to tellurites, Zn(2+)-phosphodiesterase was more susceptible to inhibition by thiocholine. In addition, Zn(2+)-phosphodiesterase was more thermo-stable than Co2+ enzyme. Separately, when properties of native phosphodiesterase were compared to those of each metallo-phosphodiesterase, native phosphodiesterase was found to be quite similar to Zn(2+)-phosphodiesterase in many respects. Even in thermo-stability, native enzyme resembled Zn(2+)-phosphodiesterase rather than Co(2+)-enzyme. Consistent with this, the stability of native phosphodiesterase was maintained in the presence of Zn2+, but not Co2+, Mn2+ was also as effective as Zn2+ in the stabilization of the enzyme. Noteworthy, the native enzyme was found to be inhibited competitively by Cu2+ with a Ki value of 20 microM, and its inhibitory action was antagonized effectively by Zn2+ or Co2+. Also, choline, another competitive inhibitor of the enzyme, appeared to antagonize the inhibitory action of Cu2+. Taken together, it is suggested that there may be multiple binding sites for divalent metal ions in the molecule of glycerophosphocholine cholinephosphodiesterase.

Differential regulation of the IL-12 p40 promoter and of p40 secretion by CpG DNA and lipopolysaccharide.

Challenge of macrophages with DNA containing an internal CpG motif results in IL-12 p40 secretion. In the presence of IFN-gamma, CpG DNA induces more p40 secretion than does LPS. In the RAW 264 macrophage cell line, both CpG DNA and LPS activate a p40 promoter-reporter construct, and the promoter response to either agent is augmented 2- to 5-fold by IFN-gamma. While either LPS or CpG DNA induces p40 promoter activity, only CpG DNA induces an increase in p40 mRNA or protein secretion. Even though IFN-gamma augmented LPS-driven p40 promoter activity in RAW 264 cells, the combination of IFN-gamma and LPS induced less p40 mRNA or protein secretion than the combination of IFN-gamma and CpG DNA. The ability of IFN-gamma to augment LPS or CpG DNA-induced p40 promoter activation was observed with truncation mutants of the IL-12 promoter containing as few as 250 bp 5' of the TATA box. Although LPS alone is a poor inducer of p40 transcription, both LPS and CpG DNA induce similar nuclear translocation of NF-kappaB. This binding is not augmented by costimulation with IFN-gamma. Thus, CpG DNA induces p40 transcription by a mechanism that includes NF-kappaB translocation; however, CpG DNA appears to induce other factor(s) necessary for p40 transcription. These results illustrate fundamental differences between CpG DNA and LPS with respect to activation of IL-12 p40 secretion.

Regulation of brain glycosylphosphatidylinositol-specific phospholipase D by natural amphiphiles.

Brain glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD)-catalyzed conversion of amphiphilic form of Zn2+ -glycerophosphocholine cholinephosphodiesterase (Amp-GPC PDE) into hydrophilic form was investigated in the presence of natural amphiphiles. Monoacylglycerols enhanced considerably the conversion by GPI-PLD of Amp-GPC PDE to hydrophilic form, with the enhancing effect of monoacylglycerols being dependent on the size of acyl group (C8-C18). Whereas the maximal enhancement of GPI-PLD action was the greatest with monodecanoylglycerol, the concentration (EC50) required to achieve 50% maximal effect was the smallest for monomyristoyl- or monopalmitoylglycerol. In addition, monolaurylglycerol or its alkyl analogue, monododecylglycerol, showed a remarkable decrease in enhancing effect at high concentrations (>1 mM). Presence of double bond in acyl chain, as exemplified by monooleoylglycerol or mono-11-eicosenoin, further enhanced the conversion by GPI-PLD. Meanwhile, lysophosphatidylcholine (IC50, 25 microM) and phosphatidic acid (IC50, >100 microM), ionic amphiphiles, inhibited the GPI-PLD activity, which was determined in the presence of monooleoylglycerol as a detergent. From these results, it is suggested that the activity of GPI-PLD in vivo system may be regulated by natural amphiphiles.

Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through alpha IIb beta 3 and collagen receptors.

Mice deficient in the hematopoietic cell-specific adapter protein SLP-76 demonstrate a failure of T cell development and fetal hemorrhage. Although SLP-76-deficient platelets manifest defective collagen receptor signaling, this alone may not explain the observed bleeding diathesis. Because alpha IIb beta 3, the platelet fibrinogen receptor, is required for normal hemostasis, we explored a potential role for SLP-76 in alpha IIb beta 3 signaling. Interaction of soluble or immobilized fibrinogen with normal human or murine platelets triggers rapid tyrosine phosphorylation of SLP-76. Moreover, platelet adhesion to fibrinogen stimulates actin rearrangements, filopodial and lamellipodial extension, and localization of tyrosine phosphorylated proteins to the cell periphery. In contrast, SLP-76-deficient murine platelets bind fibrinogen normally, but spread poorly and exhibit reduced levels of phosphotyrosine. The in vivo bleeding diathesis as well as the defects in platelet responses to fibrinogen and collagen are reversed by retroviral transduction of SLP-76 into bone marrow derived from SLP-76-deficient mice. These studies establish that SLP-76 functions downstream of alpha IIb beta 3 and collagen receptors in platelets. Furthermore, expression of SLP-76 in hematopoietic cells, including platelets, plays a necessary role in hemostasis.

In vitro and in vivo macrophage function can occur independently of SLP-76.

Expression of SH2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), a hematopoietic cell-specific adapter protein, is required to couple Syk family tyrosine kinase activation to downstream mediators such as phospholipase C (PLC)-gamma following TCR, platelet collagen receptor and mast cell Fc epsilon R stimulation. In addition to T cells, mast cells and platelets, SLP-76 is expressed in monocytes and macrophages. To determine the role of SLP-76 in Fc gamma R-stimulated signaling pathways in macrophages, we examined cultured bone marrow-derived macrophages (BMM) from SLP-76(-/-) and wild-type mice. In this study, we show that Fc gamma R cross-linking rapidly induces tyrosine phosphorylation of SLP-76 in wild-type BMM. Surprisingly, however, BMM from SLP-76(-/-) mice activate ERK2 and phosphorylate PLC-gamma 2 following Fc gamma R ligation. Furthermore, SLP-76(-/-) BMM display normal Fc gamma R-dependent phagocytic function and reactive oxygen intermediate production. SLP-76(-/-) and SLP-76(+/+) BMM secrete comparable levels of IL-12 in response to lipopolysaccharide and IFN-gamma. To examine macrophage function in vivo, SLP-76(-/-) mice were challenged i.v. with Listeria monocytogenes. SLP-76(-/-) mice survive and efficiently contain the acute phase of infection similar to wild-type mice but exhibit a stable chronic infection attributed to the lack of mature T cells. These data show that, although SLP-76 is required to couple Syk family PTK activity to downstream mediators and effector functions in Fc gamma R-induced pathways in some cell types, activation of Fc gamma R-dependent pathways occurs independently of SLP-76 in BM

Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength.

Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.

Differential requirement for SLP-76 domains in T cell development and function.

The hematopoietic cell-specific adaptor protein, SLP-76, is critical for T cell development and mature T cell receptor (TCR) signaling; however, the structural requirements of SLP-76 for mediating thymopoiesis and mature T cell function remain largely unknown. In this study, transgenic mice were generated to examine the requirements for specific domains of SLP-76 in thymocytes and peripheral T cells in vivo. Examination of mice expressing various mutants of SLP-76 on the null background demonstrates a differential requirement for specific domains of SLP-76 in thymocytes and T cells and provides new insight into the molecular mechanisms underlying SLP-76 function.