Molecular cell biology research communications : MCBRC最新文献

Raf-1, a key kinase in the Ras signaling pathway, plays critical roles in cell differentiation, proliferation, and tumorigenesis. However, knowledge of the Raf-1 in inflammation is limited. Using an inducible oncogenic Raf-1, we show that the Raf-1 orchestrates the discrete NF-κB activating pathways. While the Raf-1 activation induces a modest IκB degradation by enhancing the basal IκB kinase activity, it contradictorily suppresses the proinflammatory cytokine inducible IκB kinase complex, leading to an inhibition of TNF-α- and IL-1β-induced NF-κB activation. Despite considerable degrees of overlap, LPS signaling is not affected by Raf-1. By either conditionally reducing Raf-1 activity or completely disrupting the Raf-1 signaling by PD98059, a specific inhibitor of MEK1, the otherwise inhibited cytokine responses can be restored. Moreover, when the activity of Raf-1 is up-regulated during the cell cycle progression from the G₀ phase to the late G₁ phase, the enhanced Raf-1 activity suffices to shift the TNF-α response from the sensitive to the insensitive state. Together, these studies elucidate a mechanism by which signaling outputs are shaped by the intracellular Raf-1, thus explaining the “cellular context”-dependent cytokine response.

The treatment of thymocytes with protein phosphatase inhibitors such as calyculin A and okadaic acid resulted in apoptosis with a concomitant increase in phosphorylation of nuclear proteins. The phosphorylated protein in the thymocyte nuclei induced by protein phosphatase inhibitors was identified as histones by the use of two-dimensional polyacrylamide gel electrophoresis. These compounds accelerated the phosphorylation of histone H2A, H3, and H1. On the other hand, little phosphorylation of H2B and H4 by these compounds was observed. The effect of these compounds on the level of nuclear histones was also examined using high-performance capillary electrophoresis. No significant changes in the level of histones were seen in the nuclei of thymocytes treated with calyculin A and okadaic acid. Thus, the induction of thymocyte apoptosis is involved in the chemical modification of histones but not the change in their quantity. Moreover, the treatment of thymocytes with calyculin A increased the sensitivity toward endogenous DNase in the nuclei. These results suggest that phosphorylation of histones, especially H2A, H3, and H1, is an early step of triggering DNA fragmentation in thymocyte apoptosis.

Yeast strains containing a new temperature-sensitive allele of the RML2 gene, encoding a component of the large subunit of the mitochondrial ribosome, display normal growth on acetate, slowed growth on glycerol and an inability to grow on oleic acid. These cells, denoted rml2(fat21), have an apparent inability to induce peroxisomal function, as evidenced by a deficiency in oleic acid induction of beta-oxidation. However, the oleic acid regulation of genes encoding core enzymes of peroxisomal beta-oxidation is normal. In contrast, up-regulation of CTA1 (catalase) mRNA expression and enzyme activity is interrupted. Upon comparison of the induction requirements of catalase and the genes of beta-oxidation, we hypothesized that the rml2(fat21) mutation alters the activity of the transcription factor Adr1p. In support of this hypothesis, over-expression of ADR1 in rml2(fat21) cells restores CTA1 induction. Several assays of mitochondria from rml2(fat21) strains suggest normal mitochondrial function. Thus, the modulation of Adr1p-associated gene regulation is not due to overt mitochondrial dysfunction.

Receptor mediated stimulation of the G protein-α subunit leads to exchange of GDP for GTP, activating the protein. Spontaneous GDP release from Gα can also lead to the active state, if GTP in solution binds the nucleotide binding pocket. The purpose of this study is to evaluate the molecular determinants for maintaining the spontaneous GDP release rates between two Gα subunits. Gα_t has a low rate of nucleotide release, compared to Gα_i1. Gα_t/i1 chimeras were used to explore the molecular basis for this behavior. The C-terminal α4-helix, the N-terminal 56 residues and the Switch I/II regions of Gα_t were shown to affect the low spontaneous GDP release rate in Gα_t. A specific molecular contact between Asp26 and Asn191 was found in Gα_t that is not present in Gα_i1. In two chimeras disrupting this interaction produced an increased spontaneous GDP release; restoring the contact present in Gα_t into these chimeras decreased the GDP release rate by half as compared to the original chimeras. Similarly, introduction of this contact in wild-type Gα_i1 decreased the GDP release rate of Gα_i1 by half. Differences in GDP release rates may reflect physiological roles these proteins play in living systems.

The cytokine tumor necrosis factor (TNF) elicits a wide range of biological responses, including inflammation, cell proliferation, differentiation, and apoptosis. Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and death is still unknown. This review will focus on the crosstalk that exists between proteins of the TNF receptor (TNF-R) signalosome, and which are involved in the initiation of nuclear factor kappa B (NF-κB) activation or apoptosis. At least four different mechanisms of regulation can be distinguished: (i) NF-κB-mediated induction of proteins of the TNF-R complex; (ii) NF-κB-independent protection against apoptosis by the TNF-R-associating factor 2 (TRAF2)-mediated recruitment of antiapoptotic proteins; (iii) dual activation of apoptosis and NF-κB by a single molecule; and (iv) amplification of the death signal by proteolytic inactivation of signaling proteins that are involved in NF-κB activation or cell survival.

Rel/NF-κB proteins are eukaryotic transcription factors that control the expression of genes involved in a large variety of cellular processes. Rel proteins share a highly conserved DNA-binding/dimerization domain called the Rel Homology (RH) domain. We have constructed and characterized a composite cDNA encoding most of the chicken RelB transcription factor. The predicted chicken RelB protein has a high degree of sequence similarity to other vertebrate RelB proteins within the RH domain, but is much less conserved outside this domain. Chicken RelB does not bind DNA as a homodimer, but forms DNA-binding heterodimers with NF-κB p50 or p52. Overexpressed chicken RelB localizes to the nucleus in chicken embryo fibroblasts, and the nonconserved C-terminal sequences of chicken RelB contain a transactivation domain that functions in chicken and mouse fibroblasts. Thus, chicken RelB has functional properties similar to other vertebrate RelB proteins. However, Western blotting of diverse chicken tissues indicates that chicken RelB is more widely expressed than mammalian RelB.

To elucidate the apoptotic signaling pathway, we have generated a cell culture model: S2 cells stably transfected with a Drosophila cell death gene, reaper (rpr). Following rpr overexpression, caspase activation-mediated apoptotic cell death was induced in the cells. Apoptosis triggered by rpr required intracellular Ca²⁺ ions and calmodulin. Furthermore, protein kinase inhibitors H-7 (a PKC, PKA, PKG, MLCK, and CKI inhibitor), calphostin C (a PKC inhibitor), or H-89 (a PKA and PKG inhibitor) completely blocked apoptosis induced by rpr, suggesting that some kind of serine/threonine protein kinase(s) act upstream of caspase in apoptotic pathway induced by rpr in S2 cells.

Dlxin-1, a unique member of the necdin/melanoma associated antigen gene (MAGE) family, is a novel protein that binds Dlxin-5 and regulates its transcriptional function. We have cloned the homology region between Dlxin-1 and necdin from mouse melanoma cells. Here we report the expression cloning, characterization, and detailed tissue-specific expression studies of Dlxin-1. A unique expression pattern of Dlxin-1 emerged from the work wherein strong expression of a 3.2-Kb transcript was observed in mouse brain and embryos. Amongst the representative established cell lines of different tumor categories studied the presence of transcript was detected only in sarcomas and neuroectodermal tumors. Characteristically, lymphomas, leukaemias, adenocarcinomas, and carcinomas did not express Dlxin-1. Also, we observed a growth suppression on ectopic expression of this cDNA possibly due to the close homology shared with necdin, a neuron-specific growth suppressor. The extensive homology of our Dlxin-1 clone to necdin makes it an attractive system to understand the importance of the necdin/MAGE family of molecules in cell cycle regulation.

We previously identified the hydrophilic residues that are essential for ligand binding in the C-type lectin-like domain (CTLD) of human lectin-like oxidized LDL receptor (hLOX-1). To provide a more detailed understanding of ligand binding, we selected in the present study 13 conserved hydrophobic residues in the CTLD of hLOX-1 for mutagenesis analysis. The selected residues were replaced either by Ser (drastic mutation) or by size- and structure-based alternative hydrophobic residues (conserved mutation). Mutation targeted at F228, Y238, and G232 deprived hLOX-1 of ligand binding without alteration of protein expression and localization. In contrast, drastic mutation introduced into positions W203, W215, and W217 resulted in mislocalization, whereas conserved mutation at the same sites resulted in clones with similar cell surface localization and ligand binding to native hLOX-1. Our results indicate that F228, Y238, and G232 are essential for ligand binding, while W203, W215, W217, and L206 play a structural role.