Structure-activity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants.
(65/568)
Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells. Clustering of the intracellular domain of the receptor, the so-called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli. Circular dichroism, steady-state and time-resolved fluorescence spectroscopy were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas-associating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3-dimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution. This prompted a homology modeling of the TNFR DD 3-D structure using FADD as a template. In vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations. Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55 DD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference. (+info)
The atypical protein kinase C-interacting protein p62 is a scaffold for NF-kappaB activation by nerve growth factor.
(66/568)
Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor kappaB (NF-kappaB). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-kappaB pathway, which mediates NGF survival and differentiation responses. (+info)
14-3-3 is involved in p75 neurotrophin receptor-mediated signal transduction.
(67/568)
The low affinity neurotrophin receptor (p75NTR) has been shown to mediate the apoptosis signaling to neural cells. However, the specific mechanisms of intracellular signal transduction of this process are largely unknown. To understand p75NTR-mediated signal transduction, we previously identified a protein that interacts with the intracellular domain of p75NTR, and we named it p75NTR-associated cell death executor (NADE). To elucidate further the signaling mechanisms utilized by p75NTR and NADE, we screened for NADE-binding protein(s) with the yeast two-hybrid method, and we identified 14-3-3epsilon as a NADE-binding protein in vivo. To examine whether 14-3-3epsilon affects the induction of p75NTR-mediated apoptosis, wild type or various deletion mutant forms of 14-3-3epsilon were co-expressed in HEK293, PC12nnr5, and oligodendrocytes. Interestingly, transient expression of the mutant form of 14-3-3epsilon lacking the 208-255 amino acid region blocked nerve growth factor-dependent p75NTR/NADE-mediated apoptosis, although this mutant form of 14-3-3epsilon continued to associate with NADE. These results suggest that 14-3-3epsilon plays an important role in the modulation of nerve growth factor-dependent p75NTR/NADE-mediated apoptosis. (+info)
Preemptive control of graft-versus-host disease in a murine allogeneic transplant model using retrovirally transduced murine suicidal lymphocytes.
(68/568)
Suicidal lymphocytes could greatly expand the role of allogeneic transplantation by reducing graft-versus-host disease (GVHD) as a barrier to transplantation, but optimization of their use is hindered by the lack of adequate animal models. To develop an animal model that used retrovirally transduced suicidal lymphocytes in a GVHD setting, a well-characterized MHC-matched murine transplant model (B10.BR-->AKR/J) was adapted. B10.BR splenic lymphocytes stimulated with concanavalin A and interleukin 2 were infected with a retrovirus containing the low-affinity nerve growth factor receptor (LNGFR) and the HSV-TK gene and immunomagnetically selected; these LNGFR+/TK+ allogeneic lymphocytes were then cotransplanted with 1 x 10(7) bone marrow cells into lethally irradiated AKR/J recipients. The LNGFR+/TK+ donor lymphocytes persisted in the peripheral circulation for 6 months in both syngeneic and allogeneic settings. Doses of 2 x 10(6) TK+ allogeneic lymphocytes produced GVHD with a severity and time course similar to that induced by naive lymphocytes. Survival of TK+ allogeneic lymphocyte-bearing mice was significantly improved (P = 0.01) when ganciclovir (GCV; 2 mg/day) was administered on days 7-13 post transplant by i.p. injection, demonstrating that GVHD could be prevented. Fluorescence-activated cell sorting analysis demonstrated 4-fold reduction but persistent circulation of LNGFR+ lymphocytes in mice treated with GCV at various time points 1-3 months after transplantation, demonstrating selective killing of GVHD-reactive cells. We conclude that retrovirally transduced LNGFR+/TK+ murine lymphocytes can be produced, persist after transplant, remain alloreactive, and can be killed by GCV administration, resulting in reduced GVHD. (+info)
The p75 neurotrophin receptor activates Akt (protein kinase B) through a phosphatidylinositol 3-kinase-dependent pathway.
(69/568)
The Akt kinase plays a crucial role in supporting Trk-dependent cell survival, whereas the p75 neurotrophin receptor (p75NTR) facilitates cellular apoptosis. The precise mechanism that p75NTR uses to promote cell death is not certain, but one possibility is that p75NTR-dependent ceramide accumulation inhibits phosphatidylinositol 3-kinase-mediated Akt activation. To test this hypothesis, we developed a system for examining p75NTR-dependent apoptosis and determined the effect of p75NTR on Akt activation. Surprisingly, p75NTR increased, rather than decreased, Akt phosphorylation in a variety of cell types, including human Niemann-Pick fibroblasts, which lack acidic sphingomyelinase activity. The p75NTR expression level required to elicit Akt phosphorylation was much lower than that required to activate the JNK pathway or to mediate apoptosis. We show that p75NTR-dependent Akt phosphorylation was independent of TrkA signaling, required active phosphatidylinositol 3-kinase, and was associated with increased tyrosine phosphorylation of p85 and Shc and with reduced cytosolic tyrosine phosphatase activity. Finally, we show that p75NTR expression increased survival in cells exposed to staurosporine or subjected to serum withdrawal. These findings indicate that p75NTR facilitates cell survival through novel signaling cascades that result in Akt activation. (+info)
Endogenous brain-derived neurotrophic factor and neurotrophin-3 antagonistically regulate survival of axotomized corticospinal neurons in vivo.
(70/568)
Neuronal growth factors regulate the survival of neurons by their survival and death-promoting activity on distinct populations of neurons. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) promote neuronal survival via tyrosine kinase (Trk) receptors, whereas NGF and BDNF can also induce apoptosis in developing neurons through p75(NTR) receptors in the absence of their respective Trk receptors. Using mutant mice and inactivation of neurotrophins and their receptors with antibodies in rats, we show that endogenous NT-3 induces death of adult BDNF-dependent, axotomized corticospinal neurons (CSNs). When NT-3 is neutralized, the neurons survive even without BDNF, suggesting complete antagonism. Whereas virtually all unlesioned and axotomized CSNs express both trkB and trkC mRNA, p75 is barely detectable in unlesioned CSNs but strongly upregulated in axotomized CSNs by day 3 after lesion, the time point when cell death occurs. Blocking either cortical TrkC or p75(NTR) receptors alone prevents death, indicating that the opposing actions of NT-3 and BDNF require their respective Trk receptors, but induction of death depends on p75(NTR) cosignaling. The results show that neuronal survival can be regulated antagonistically by neurotrophins and that neurotrophins can induce neuronal death in the adult mammalian CNS. We further present evidence that signaling of tyrosine kinase receptors of the trk family can be crucially involved in the promotion of neuronal death in vivo. (+info)
cdc42 regulates the exit of apical and basolateral proteins from the trans-Golgi network.
(71/568)
It is well established that Rho-GTPases regulate vesicle fusion and fission events at the plasma membrane through their modulatory role on the cortical actin cytoskeleton. In contrast, their effects on intracellular transport processes and actin pools are less clear. It was recently shown that cdc42 associates with the Golgi apparatus in an ARF-dependent manner, similarly to coat proteins involved in vesicle formation and to several actin-binding proteins. We report here that mutants of cdc42 inhibited the exit of basolateral proteins from the trans-Golgi network (TGN), while stimulating the exit of an apical marker, in two different transport assays. This regulation may result from modulation of the actin cytoskeleton, as GTPase-deficient cdc42 depleted a perinuclear actin pool that rapidly exchanges with exogenous fluorescent actin. (+info)
HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction.
(72/568)
A recent report [Gil, Chaib-Oukadour, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182] describes activation of signal transduction pathways by tetanus toxin (TeTx), a Zn(2+)-dependent endopeptidase synthesized by the Clostridium tetani bacillus, which is responsible for tetanus disease. In the present work, specific activation of protein kinase C (PKC) isoforms and of intracellular signal-transduction pathways, which include nerve-growth-factor (NGF) receptor trkA, phospholipase C(PLC)gamma-1 and extracellular regulated kinases (ERKs) 1 and 2, by the recombinant C-terminal portion of the TeTx heavy chain (H(C)-TeTx) is reported. The activation of PKC isoforms was assessed through their translocation from the soluble (cytosolic) compartment to the membranous compartment, showing that clear translocation of PKC-alpha, -beta, -gamma and -delta isoforms exists, whereas PKC-epsilon showed a slight decrease in its soluble fraction immunoreactivity. The PKC-zeta isoform showed no consistent response. Using immunoprecipitation assays against phosphotyrosine residues, time- and dose-dependent increases in tyrosine phosphorylation were observed in the trkA receptor, PLCgamma-1 and ERK-1/2. The effects shown by the H(C)-TeTx fragment on tyrosine phosphorylation were compared with the effects produced by NGF. The trkA and ERK-1/2 activation were corroborated using phospho-specific antibodies against trkA phosphorylated on Tyr(490), and antibodies against Thr/Tyr phosphorylated ERK-1/2. Moreover, PLCgamma-1 phosphorylation was supported by its H(C)-TeTx-induced translocation to the membranous compartment, an event related to PLCgamma-1 activation. Since H(C)-TeTx is the domain responsible for membrane binding and lacks catalytic activity, the activations described here must be exclusively triggered by the interaction of TeTx with a membrane component. (+info)