Cytokine-inducible SH2 protein (CIS3) and JAK2 binding protein (JAB) abolish prolactin receptor-mediated STAT5 signaling

In vivo microdialysis was used to measure release of endogenous l-glutamate and l-aspartate in the nucleus tractus solitarius of the anaesthetised rat evoked by baroreceptor loading. Aortic constriction, the method of loading, elicited a reproducible increase in extracellular levels of l-glutamate to 322+/-139% of basal levels, which could be attenuated by concomitant local administration of the nicotinic acetylcholine receptor antagonist mecamylamine (100 microM).     

Growth hormone stimulates tyrosine phosphorylation of JAK2 and STAT5, but not insulin receptor substrate-1 or SHC proteins in liver and skeletal muscle of normal rats in vivo

GH has been shown to stimulate tyrosine phosphorylation of JAK2, several STAT proteins, insulin receptor substrate-1 (IRS-1), and SHC proteins in cultured cells. The goal of this study was to determine GH effects on protein tyrosine phosphorylation in liver and skeletal muscle of normal rats in vivo. Nonfasted male Sprague-Dawley rats (225-250 g) were injected with GH iv, and tissues were obtained after 5, 15, 30, or 60 min. At a maximally effective GH dose (1.5 mg/kg body weight), phosphotyrosine antibody immunoblots demonstrated marked stimulation of the tyrosine phosphorylation of JAK2 (maximal at 5 min) and a 95,000 Mr protein (maximal at 15 min) in both liver and skeletal muscle. The 95,000 Mr protein was recognized and immunodepleted by STAT5 antibody, but not by other STAT protein antibodies. Although basal tyrosine phosphorylation of IRS-1 and SHC was evident, GH did not stimulate tyrosine phosphorylation of either of these proteins in liver or skeletal muscle. In conclusion, GH stimulates the tyrosine phosphorylation of JAK2 and STAT5, but not IRS-1, SHC, or other STAT proteins in liver and skeletal muscle of normal rats. These results differ from findings in cultured cells and support the concept that selectivity for tyrosine kinase substrates is an important determinant of postreceptor signaling specificity in vivo.     

Repression of c-fos and c-jun gene expression is not part of AT2 receptor coupled signal transduction

Recent world-wide publications were reviewed in order to determine the clinical characteristics and therapeutic relevance of the chlamydial respiratory tract infections in humans. It was emphasized that Chlamydia pneumoniae could initiate asthma and may be associated with acute asthma exacerbation. Laboratory procedures for identifying chlamydia and difficulties concerned with the diagnostics of this intracellular pathogen were also presented. In patients with evidence of chlamydial infection the casual treatment (macrolides, tetracyclines, fluoroquinolones) may induce major improvement or complete resolution of asthma.     

Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice

PRL is an anterior pituitary hormone that, along with GH and PLs, forms a family of hormones that probably resulted from the duplication of an ancestral gene. The PRLR is also a member of a larger family, known as the cytokine class-1 receptor superfamily, which currently has more than 20 different members. PRLRs or binding sites are widely distributed throughout the body. In fact, it is difficult to find a tissue that does not express any PRLR mRNA or protein. In agreement with this wide distribution of receptors is the fact that now more than 300 separate actions of PRL have been reported in various vertebrates, including effects on water and salt balance, growth and development, endocrinology and metabolism, brain and behavior, reproduction, and immune regulation and protection. Clearly, a large proportion of these actions are directly or indirectly associated with the process of reproduction, including many behavioral effects. PRL is also becoming well known as an important regulator of immune function. A number of disease states, including the growth of different forms of cancer as well as various autoimmune diseases, appear to be related to an overproduction of PRL, which may act in an endocrine, autocrine, or paracrine manner, or via an increased sensitivity to the hormone. The first step in the mechanism of action of PRL is the binding to a cell surface receptor. The ligand binds in a two-step process in which site 1 on PRL binds to one receptor molecule, after which a second receptor molecule binds to site 2 on the hormone, forming a homodimer consisting of one molecule of PRL and two molecules of receptor. The PRLR contains no intrinsic tyrosine kinase cytoplasmic domain but associates with a cytoplasmic tyrosine kinase, JAK2. Dimerization of the receptor induces tyrosine phosphorylation and activation of the JAK kinase followed by phosphorylation of the receptor. Other receptor-associated kinases of the Src family have also been shown to be activated by PRL. One major pathway of signaling involves phosphorylation of cytoplasmic State proteins, which themselves dimerize and translocate to nucleus and bind to specific promoter elements on PRL-responsive genes. In addition, the Ras/Raf/MAP kinase pathway is also activated by PRL and may be involved in the proliferative effects of the hormone. Finally, a number of other potential mediators have been identified, including IRS-1, PI-3 kinase, SHP-2, PLC gamma, PKC, and intracellular Ca2+. The technique of gene targeting in mice has been used to develop the first experimental model in which the effect of the complete absence of any lactogen or PRL-mediated effects can be studied. Heterozygous (+/-) females show almost complete failure to lactate after the first, but not subsequent, pregnancies. Homozygous (-/-) females are infertile due to multiple reproductive abnormalities, including ovulation of premeiotic oocytes, reduced fertilization of oocytes, reduced preimplantation oocyte development, lack of embryo implantation, and the absence of pseudopregnancy. Twenty per cent of the homozygous males showed delayed fertility. Other phenotypes, including effects on the immune system and bone, are currently being examined. It is clear that there are multiple actions associated with PRL. It will be important to correlate known effects with local production of PRL to differentiate classic endocrine from autocrine/paracrine effects. The fact that extrapituitary PRL can, under some circumstances, compensate for pituitary PRL raises the interesting possibility that there may be effects of PRL other than those originally observed in hypophysectomized rats. The PRLR knockout mouse model should be an interesting system by which to look for effects activated only by PRL or other lactogenic hormones. On the other hand, many of the effects reported in this review may be shared with other hormones, cytokines, or growth factors and thus will be more difficult to study. (ABSTRACT TRUNCATED)     

Prolactin stimulates phosphorylation of the human T-cell antigen receptor complex and ZAP-70 tyrosine kinase: a potential mechanism for its immunomodulation

Prolactin (PRL) is an immunomodulatory hormone which promotes T-cell activation and proliferation. However, the intracellular mechanisms of this action in normal lymphocytes are unknown. Because the PRL receptor (PRLR) activates several signals also activated by the T-cell antigen receptor (TCR)/CD3 complex, we evaluated whether signaling "cross-talk" occurs between these distinct receptors. Using human thymocytes, human peripheral blood lymphocytes and the rat Nb2 lymphoma T-cell, we found that PRL induced rapid phosphorylation of multiple, TCR/CD3 complex proteins, an event required for lymphocyte activation. Two of these phosphorylated proteins were identified to be CD3 epsilon and ZAP-70 tyrosine kinase, molecules essential for TCR function. Further, PRL induced tyrosyl phosphorylation of ZAP-70 in each population of T-lymphocytes tested, demonstrating for the first time that ZAP-70 is a target of PRL action. Taken together, our results suggest that the PRLR directly affects T-lymphocyte activation by means of signaling cross-talk with the TCR/CD3 complex.     

The chemokine monocyte chemotactic protein 1 triggers Janus kinase 2 activation and tyrosine phosphorylation of the CCR2B receptor

Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin did not affect the dissociation of actin-gelsolin (1:1) complexes induced by neutrophil stimulation with N-formyl-Met-Leu-Phe. Guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) indirectly promoted the dissociation of actin-gelsolin complexes in a cell-free system using neutrophil cytosol, and this effect was blocked by the GDP dissociation inhibitor for Rho (Rho-GDI). The GTPgammaS-loaded ialpha2 and the beta1gamma2 subunits of heterotrimeric G-proteins (Gialpha2 and Gbeta1gamma2) also triggered actin-gelsolin dissociation in a Rho-GDI-sensitive manner. GTP-loaded activated Rac, but not activated Rho, induced the dissociation of cytosolic actin-gelsolin complexes. The guanine nucleotide exchange on Rac was increased by addition of GTPgammaS-loaded Gialpha2 or Gbeta1gamma2 to neutrophil cytosol. These findings suggest that activation of Rac by G-protein-coupled receptors in neutrophils triggers uncapping of actin filaments, independently of PI 3-kinase.     

Characterization of the role of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit in the activation of JAK2 and STAT5

The high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) consists of an alpha (GMRalpha) and a common beta (betac) subunit. The intracellular domain of betac has been extensively characterized and has been shown to be critical for the activation of both the JAK/STAT and MAP kinase pathways. The function of the intracellular domain of GMRalpha, however, is not as well characterized. To determine the role of this domain in GMR signaling, an extensive structure-function analysis was performed. Truncation mutants alpha362, alpha371, and alpha375 were generated, as well as the site-directed mutants alphaVQVQ and alphaVVVV. Although alpha375beta, alphaVQNQbeta, and alphaVVVVbeta stimulated proliferation in response to human GM-CSF, the truncation mutants alpha362beta and alpha371beta were incapable of transducing a proliferative signal. In addition, both alpha371 and alphaVVVV were expressed at markedly reduced levels, indicating the importance of residues 372 to 374 for proper protein expression. More importantly, we show that GMRalpha plays a direct role in the activation of the JAK/STAT pathway, and electrophoretic mobility shift assays (EMSA) indicate that both GMRalpha and betac play a role in determining the STAT5 DNA binding complex activated by the GMR. Thus, the intracellular domain of the human GMRalpha is important for activation of the JAK/STAT pathway and protein stabilization.     

Cholesterol efflux-mediated signal transduction in mammalian sperm. beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation

Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin and OH-propyl-beta-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these beta-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different beta-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the beta-cyclodextrins with cholesterol-SO4- to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The beta-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The beta-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, beta-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release.     

Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival

Interleukin-5 (IL-5) regulates the growth and function of eosinophils. It induces rapid tyrosine phosphorylation of Lyn and Jak2 tyrosine kinases. The role of tyrosine phosphatases in IL-5 signal transduction has not been investigated. In this study, we provide first evidence that SH2 protein tyrosine phosphatase 2 (SHPTP2) phosphotyrosine phosphatase plays a key role in prevention of eosinophil death by IL-5. We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min. The tyrosine phosphorylated SHPTP2 was complexed with the adapter protein Grb2 in IL-5-stimulated eosinophils. Furthermore, SHPTP2 appeared to physically associate with beta common (betac) chain of the IL-5 receptor (IL-5betacR). The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612. The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2. Only SHPTP2 antisense oligonucleotides, but not sense SHPTP2, could inhibit tyrosine phosphorylation of microtubule-associated protein kinase, and reverse the eosinophil survival advantage provided by IL-5. Therefore, we conclude that the physical association of SHPTP2 with the phosphorylated betac receptor and Grb2 and its early activation are required for the coupling of the receptor to the Ras signaling pathway and for prevention of eosinophil death by IL-5.     

Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling

Some forms of G protein-coupled receptor signaling, such as activation of mitogen-activated protein kinase cascade as well as resensitization of receptors after hormone-induced desensitization, require receptor internalization via dynamin-dependent clathrin-coated pit mechanisms. Here we demonstrate that activation of beta2-adrenergic receptors (beta2-ARs) leads to c-Src-mediated tyrosine phosphorylation of dynamin, which is required for receptor internalization. Two tyrosine residues, Tyr231 and Tyr597, are identified as the major phosphorylation sites. Mutation of these residues to phenylalanine dramatically decreases the c-Src-mediated phosphorylation of dynamin following beta2-AR stimulation. Moreover, expression of Y231F/Y597F dynamin inhibits beta2-AR internalization and the isoproterenol-stimulated mitogen-activated protein kinase activation. Thus, agonist-induced, c-Src-mediated tyrosine phosphorylation of dynamin is essential for its function in clathrin mediated G protein-coupled receptor endocytosis.     

Growth hormone and prolactin stimulate tyrosine phosphorylation of insulin receptor substrate-1, -2, and -3, their association with p85 phosphatidylinositol 3-kinase (PI3-kinase), and concomitantly PI3-kinase activation via JAK2 kinase

Growth hormone (GH) and prolactin (PRL) binding to their receptors, which belong to the cytokine receptor superfamily, activate Janus kinase (JAK) 2 tyrosine kinase, thereby leading to their biological actions. We recently showed that GH mainly stimulated tyrosine phosphorylation of epidermal growth factor receptor and its association with Grb2, and concomitantly stimulated mitogen-activated protein kinase activity in liver, a major target tissue. Using specific antibodies, we now show that GH was also able to induce tyrosine phosphorylation of insulin receptor substrate (IRS)-1/IRS-2 in liver. In addition, the major tyrosine-phosphorylated protein in anti-p85 phosphatidylinositol 3-kinase (PI3-kinase) immunoprecipitate from liver of wild-type mice was IRS-1, and IRS-2 in IRS-1 deficient mice, but not epidermal growth factor receptor. These data suggest that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in physiological target organ of GH, liver. We also show that PRL was able to induce tyrosine phosphorylation of both IRS-1 and IRS-2 in COS cells transiently transfected with PRLR and in CHO-PRLR cells. Moreover, we show that tyrosine phosphorylation of IRS-3 was induced by both GH and PRL in COS cells transiently transfected with IRS-3 and their cognate receptors. By using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, we show that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3. Finally, a specific PI3-kinase inhibitor, wortmannin, completely blocked the anti-lipolytic effect of GH in 3T3 L1 adipocytes. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings appears to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase and its downstream biological effects.     

Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase

Formyl peptide receptor activation of three mitogen-activated protein kinase (MAPK) cascades, extracellular signal-regulated kinases (ERKs), N-terminal kinases (JNKs), and p38 MAPK was examined in differentiated HL-60 granulocytes. FMLP stimulated a concentration- and time-dependent increase in ERK, JNK, and p38 MAPK activities, all of which were dependent on a pertussis toxin-sensitive G protein. Pharmacologic inhibitors were used to examine the roles of tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C. FMLP-stimulated ERK activity was dependent on tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C; p38 MAPK activation was dependent on phosphatidylinositol 3-kinase and phospholipase C; while JNK activation was independent of all of these signaling components. The mitogen-activated protein kinase/ERK kinase inhibitor PD098059 reduced ERK activation by 90%, while an inhibitor of p38 MAPK, SB203580, inhibited p38 MAPK activation by 80%. Both PD098059 and SB203580 inhibited FMLP-stimulated superoxide release, as did inhibitors directed against protein kinase C, tyrosine kinases, and phosphatidylinositol 3-kinase. We conclude that formyl peptide receptors are coupled to three MAPK cascades by Gi proteins. ERKs, p38 MAPK, and JNKs are each activated by distinct proximal signal transduction pathways. Activation of p38 MAPK is necessary for FMLP stimulation of respiratory burst activity; however, a second signal that may involve ERK is also required for this activity.     

The low-molecular-weight phosphotyrosine protein phosphatase, when overexpressed, reduces the mitogenic response to macrophage colony-stimulating factor and tyrosine phosphorylation of its receptor

Inert gases at raised pressure exert anaesthetic effects. It is assumed that anaesthesia by the inert gases is fundamentally similar to anaesthesia produced by general anaesthetics. However, do general anaesthetics bind directly to proteins or influence activity by indirectly perturbing membrane lipids still remains a major question. Although the pressure required to achieve anaesthesia with inert gases has been suggested to exert potentially some pressure antagonism per se, this has not been studied yet to our knowledge. We investigated this possibility using nitrogen, argon, and nitrous oxide. Whatever the narcotic agent used, our results showed that the pressure of narcotic required to induce anaesthetic effects increased, as compression rate increased, in a sigmoid fashion rather than in a linear fashion. Evidence for sigmo?dal responses vs. linear responses depended of the narcotic potency of the anaesthetic agent used (nitrogen: r2=0.973 vs. r2=0.941; argon: r2=0. 971 vs. r2=0.866; nitrous oxide: r2=0.995 vs. r2=0.879). Since a linear antagonism is predicted by lipid theories, we think it likely that these findings indicate that inert gases bind to a modulatory site of a protein receptor and act as allosteric modulators. Since other workers provided evidence for binding processes using volatile anaesthetics, the present findings could indicate that all classes of general anaesthetics, including inert gases, could act by binding directly to proteins rather than by dissolving in some lipids of the cellular membrane.     

Differential regulation of insulin receptor substrate-2 and mitogen-activated protein kinase tyrosine phosphorylation by phosphatidylinositol 3-kinase inhibitors in SH-SY5Y human neuroblastoma cells

Insulin-like growth factor I (IGF-I) is a potent neurotropic factor promoting the differentiation and survival of neuronal cells. SH-SY5Y human neuroblastoma cells are a well characterized in vitro model of nervous system growth. We report here that IGF-I stimulated the tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and insulin receptor substrate-2 (IRS-2) in a time- and concentration-dependent manner. These cells lacked IRS-1. After being tyrosine phosphorylated, IRS-2 associated transiently with downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-K) and Grb2. Treatment of the cells with PI 3-K inhibitors (wortmannin and LY294002) increased IGF-I-induced tyrosine phosphorylation of IRS-2. We also observed a concomitant increase in the mobility of IRS-2, suggesting that PI 3-K mediates or is required for IRS-2 serine/threonine phosphorylation, and that this phosphorylation inhibits IRS-2 tyrosine phosphorylation. Treatment with PI 3-K inhibitors induced an increased association of IRS-2 with Grb2, probably as a result of the increased IRS-2 tyrosine phosphorylation. However, even though the PI 3-K inhibitors enhanced the association of Grb2 with IRS-2, these compounds suppressed IGF-I-induced mitogen-activated protein kinase activation and neurite outgrowth. Together, these results indicate that although PI 3-K participates in a negative regulation of IRS-2 tyrosine phosphorylation, its activity is required for IGF-IR-mediated mitogen-activated protein kinase activation and neurite outgrowth.     

Calcium-regulated protein tyrosine phosphorylation is required for endothelin-1 to induce prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis in mesangial cells

The role of endothelin (ET)-1-mediated cytosolic calcium ([Ca2+]i) elevation in regulating ET-1-induced prostaglandin endoperoxide synthase, prostaglandin G/H synthase (PGHS)-2 mRNA expression and protein synthesis was investigated in mesangial cells (MC). Ionomycin, a calcium ionophore, and thapsigargin, an inhibitor of calcium ATPase, mimicked the ET-1-stimulated PGHS-2 mRNA and protein induction. Inhibition of [Ca2+]i increases with (2-?C2-bis-(carboxymethyl)-amino-5 methylphenoxy]methyl?-6-methoxy-8-bis-(carboxymethyl)-aminoquinoline tetra-(acetoxymethyl)ester (Quin/AM), a calcium chelator, or with the combined presence of [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl] (TMB), an inhibitor of intracellular calcium stores release, and ethyleneglycol-bis-(beta-aminoethyl)- N,N,N',N'-tetra-acetic acid (EGTA) suppressed ET-1, as well as ionomycin and thapsigargin-mediated PGHS-2 mRNA and protein formation. Also, the ET-1-, ionomycin-, and thapsigargin-induced PGHS-2 mRNA expression and protein formation was inhibited in MC pretreated with inhibitors of calcium calmodulin kinase. In contrast, these conditions did not inhibit interleukin (IL)-1-induced PGHS-2 mRNA expression and protein synthesis. Pretreatment with tyrosine kinase inhibitors abolished the ET-1-, ionomycin-, thapsigargin-, and IL-1-mediated PGHS-2 mRNA and protein induction. ET-1-, ionomycin-, and thapsigargin- induced protein tyrosine phosphorylation, but not IL-1-induced protein tyrosine phosphorylation, was suppressed by inhibiting either [Ca2+]i elevation or calcium calmodulin kinase activation. It was concluded that elevation of [Ca2+]i and activation of calcium calmodulin kinases are upstream mediators of ET-1-induced PGHS-2 gene expression through activation of non-receptor-linked protein tyrosine kinase in MC.     

Activation of OX40 signal transduction pathways leads to tumor necrosis factor receptor-associated factor (TRAF) 2- and TRAF5-mediated NF-kappaB activation

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-kappaB in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathione S-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256-263 (GGSFRTPI) was required for the association with TRAFs and NF-kappaB activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-kappaB activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-kappaB activation. These results indicate that the NF-kappaB activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3.