The control and execution of programmed cell death: an update.
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Animals↗
Biomedical subjects
C S Duckett
Publications and source records attributed to C S Duckett.
Induction of nuclear factor kappaB by the CD30 receptor is mediated by TRAF1 and TRAF2.
CD30 is a lymphoid cell-specific surface receptor which was originally identified as an antigen expressed on Hodgkin's lymphoma cells. Activation of CD30 induces the nuclear factor kappaB (NF-kappaB) transcription factor. In this study, we define the domains in CD30 which are required for NF-kappaB activation. Two separate elements of the cytoplasmic domain which were capable of inducing NF-kappaB independently of one another were identified. The first domain (domain 1) mapped to a approximately 120-amino-acid sequence in the membrane-proximal region of the CD30 cytoplasmic tail, between residues 410 and 531. A second, more carboxy-terminal region (domain 2) was identified between residues 553 and 595. Domain 2 contains two 5- to 10-amino-acid elements which can mediate the binding of CD30 to members of the tumor necrosis factor receptor-associated factor (TRAF) family of signal transducing proteins. Coexpression of CD30 with TRAF1 or TRAF2 but not TRAF3 augmented NF-kappaB activation through domain 2 but not domain 1. NF-kappaB induction through domain 2 was inhibited by coexpression of either full-length TRAF3 or dominant negative forms of TRAF1 or TRAF2. In contrast, NF-kappaB induction by domain 1 was not affected by alterations in TRAF protein levels. Together, these data support a model in which CD30 can induce NF-kappaB by both TRAF-dependent and -independent mechanisms. TRAF-dependent induction of NF-kappaB appears to be regulated by the relative levels of individual TRAF proteins in the cell.
Animals↗
A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors.
The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. Here we show that iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, we have isolated Drosophila and human genes which encode IAP-like proteins (dILP and hILP). Like IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis.
Amino Acid Sequence↗
CD30 contains two binding sites with different specificities for members of the tumor necrosis factor receptor-associated factor family of signal transducing proteins.
CD30 is a member of the tumor necrosis factor (TNF) receptor family of proteins. CD30 can regulate proliferation of lymphocytes and may also play an important role in human immunodeficiency virus replication. However, little is known about CD30 signal transduction. We performed a yeast two-hybrid library screen with the cytoplasmic domain of CD30 and isolated multiple independent cDNAs encoding human tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, and CRAF1 (TRAF3). The ability of TRAF1, TRAF2, and CRAF1 to associate with CD30 was confirmed using an in vitro coprecipitation assay, further demonstrating that the interaction was specific and direct. The TRAF-binding domain of CD30 was mapped to the COOH-terminal 36 amino acid residues, which contained two independent binding sites. CRAF1 bound only a single site, which contained the sequence PEQET, whereas TRAF1 and TRAF2 were capable of binding to either the PEQET site or an additional downstream domain. These data indicate that the TRAF protein binding pattern of CD30 differs from other TNF receptor family members and suggest that signaling specificity through TNF receptor family proteins may be achieved through differences in their abilities to bind TRAF proteins.
Base Sequence↗
Cytokine induction of nuclear factor kappa B in cycling and growth-arrested cells. Evidence for cell cycle-independent activation.
Nuclear factor kappa B (NF-kappa B) is a pleiotropic transcription factor which regulates the expression of a large number of cellular and viral genes. Induction of NF-kappa B has been shown previously to occur during cell cycle transition from G0 to G1, but the relationship of cytokine induction of this transcription factor to cell cycling has not been directly addressed. Here we examine the inductions of NF-kappa B in serum-deprived and cycling cells in response to tumor necrosis factor-alpha (TNF-alpha). In 3T3 fibroblasts deprived of serum, and in the temperature-sensitive G2 phase mutant carcinoma line FT210, we find that NF-kappa B DNA binding activity is rapidly induced upon addition of TNF-alpha. In addition, NF-kappa B induction in cycling cells occurs without a significant change in cell cycle distribution. These data reveal that NF-kappa B is rapidly induced by TNF-alpha in both proliferating and arrested cells and suggest that distinct activation pathways can lead to cell cycle-dependent or -independent induction of NF-kappa B.
3T3 Cells↗
Regulation of human retroviral latency by the NF-kappa B/I kappa B family: inhibition of human immunodeficiency virus replication by I kappa B through a Rev-dependent mechanism.
The cellular transcription factor NF-kappa B stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional initiation, but its role in the retroviral life cycle has not been fully defined. In this report, we show that I kappa B alpha acts as a cellular inhibitor of human retroviral replication through a discrete mechanism, independent of its effect on HIV transcription. I kappa B alpha inhibited HIV replication and gp160 expression by negatively regulating Rev function, most likely acting through a cellular factor involved in Rev transactivation. A similar effect was observed with human T leukemia virus I, in which I kappa B alpha inhibited Rex function. In contrast, no effect was observed on the replication of a DNA virus, adenovirus type 5. The NF-kappa B/I kappa B regulatory pathway therefore modulates human retroviral replication by regulating a program of cellular gene expression required for several steps in the viral life cycle, including not only viral transcription but also RNA export. This interaction between cellular and viral gene products suggests that NF-kappa B plays a broader role in the regulation of human retroviral replication, providing a previously unrecognized link between two important regulators of HIV gene expression and common NF-kappa B-dependent programs of gene expression used by human retroviruses.
Base Sequence↗
iPABP, an inducible poly(A)-binding protein detected in activated human T cells.
The poly(A)-binding protein (PABP) binds to the poly(A) tail present at the 3' ends of most eukaryotic mRNAs. PABP is thought to play a role in both translation and mRNA stability. Here we describe the molecular cloning and characterization of an inducible PABP, iPABP, from a cDNA library prepared from activated T cells. iPABP shows 79% sequence identity to PABP at the amino acid level. The RNA binding domains of iPABP and PABP are nearly identical, while their C termini are more divergent. Like PABP, iPABP is primarily localized to the cytoplasm. iPABP is expressed at low levels in resting normal human T cells; following T-cell activation, however, iPABP mRNA levels are rapidly up-regulated. In contrast, PABP is constitutively expressed in both resting and activated T cells. iPABP mRNA was also expressed at much higher levels than PABP mRNA in heart and skeletal muscle tissue. These data suggest that the regulation of cytoplasmic poly(A)-binding activity is more complex than previously believed. In most tissues, poly(A)-binding activity is likely to be the result of the combined effects of constitutively expressed PABP and iPABP, whose expression is subject to more complex regulation.
Amino Acid Sequence↗
Transcription factor AP-2 regulates human immunodeficiency virus type 1 gene expression.
Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by an enhancer region composed of multiple potential cis-acting regulatory sites. Here, we describe binding sites for the transcription factor AP-2 in the HIV-1 long terminal repeat which modulate HIV enhancer function. One site is embedded within the two previously described kappa B elements, and a second site is detected further downstream. DNase I footprinting and electrophoretic mobility shift assay experiments demonstrated that AP-2 binds to the site between the kappa B elements. Interestingly, AP-2 and NF-kappa B bind to this region in a mutually exclusive manner. Mutations which disrupt this AP-2-binding site lower basal levels of transcription but do not affect NF-kappa B-mediated induction by tumor necrosis factor alpha in Jurkat T leukemia cells.
Base Sequence↗
A cooperative interaction between NF-kappa B and Sp1 is required for HIV-1 enhancer activation.
The human immunodeficiency virus (HIV-1) long terminal repeat (LTR) contains two binding sites for NF-kappa B in close proximity to three binding sites for the constitutive transcription factor, Sp1. Previously, stimulation of the HIV enhancer in response to mitogens has been attributed to the binding of NF-kappa B to the viral enhancer. In this report, we show that the binding of NF-kappa B is not by itself sufficient to induce HIV gene expression. Instead, a protein-protein interaction must occur between NF-kappa B and Sp1 bound to an adjacent site. Cooperativity both in DNA binding and in transcriptional activation of NF-kappa B and Sp1 was confirmed by electrophoretic mobility shift gel analysis, DNase footprinting, chemical cross-linking and transfection studies in vivo. With a heterologous promoter, we find that the interaction of NF-kappa B with Sp1 is dependent on orientation and position, and is not observed with other elements, including GATA, CCAAT or octamer. An increase in the spacing between the kappa B and Sp1 elements virtually abolishes this functional interaction, which is not restored when these sites are brought back into the same helical position. Several other promoters regulated by NF-kappa B also contain kappa B in proximity to Sp1 binding sites. These findings suggest that an interaction between NF-kappa B and Sp1 is required for inducible HIV-1 gene expression and may serve as a regulatory mechanism to activate specific viral and cellular genes.
Avian Sarcoma Viruses↗
Differential regulation of vascular cell adhesion molecule 1 gene expression by specific NF-kappa B subunits in endothelial and epithelial cells.
Vascular cell adhesion molecule 1 (VCAM-1) is expressed in both endothelial and epithelial cell types, where it contributes to lymphocyte migration to sites of inflammation. Its expression is regulated by cytokines, in part through two kappa B-like regulatory elements. Because NF-kappa B can be composed of multiple alternative subunits with differential effects on gene expression, the role of different specific NF-kappa B family members subunits in VCAM-1 regulation is unknown. In this report, we define the contribution of different NF-kappa B family members to VCAM-1 gene regulation. We show that both kappa B sites in the VCAM-1 enhancer are required to optimally stimulate gene expression, but the enhancer is differentially regulated by specific combinations of NF-kappa B subunits. At low concentrations, RelA(p65) acted in concert with the approximately 50-kDa product of p105 NF-kappa B, NF-kappa B1(p50), to stimulate transcription, and at high concentrations, RelA(p65) alone stimulated the VCAM-1 promoter. In contrast, NF-kappa B2 inhibited functional activation of the VCAM reporter by p65. Consistent with this finding, an additional binding complex was detected by using recombinant NF-kappa B2(p49)/RelA(p65) with radiolabeled VCAM kappa B site probes. Interestingly, the human immunodeficiency virus enhancer responded differently to stimulation by NF-kappa B subunits, with optimal response to p49(100)/p65. Analysis of NF-kappa B mRNA in human umbilical vein endothelial cells revealed that nfkb1, nfkb2, and relA NF-kappa B but not c-rel were induced by tumor necrosis factor alpha and lipopolysaccharide, which also induce VCAM-1. These data suggest that specific subunits of NF-kappa B regulate VCAM-1 and differentially activate other genes in these cells.
Animals↗
Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3).
Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.
Animals↗
Distinct combinations of NF-kappa B subunits determine the specificity of transcriptional activation.
The nuclear factor that binds to the kappa light-chain enhancer of B cells (NF-kappa B) is a transcription factor that regulates the expression of a variety of cellular and viral genes. NF-kappa B is composed of distinct subunits, and at least four independent genes (p105, p100, p65, and c-rel) have been isolated that encode related proteins that bind kappa B sites. Because it is possible that specific interactions of different subunits can allow selective gene activation, we have characterized the specificity of transcriptional activation by various combinations of these subunits. When tested alone, an approximately 49-kDa form (p49) of the p100 protein bound weakly to kappa B, but p49 associated with p65 to bind efficiently to this site. Furthermore, p49 acted in combination with either p65 or a Rel/VP16 fusion protein to activate kappa B-dependent transcription in Jurkat T leukemia cells. The p49/p65 or p49/Rel combination stimulated transcription mediated by the canonical kappa B site but did not stimulate reporter genes containing interleukin 2 receptor alpha or major histocompatibility complex kappa B elements, despite its ability to bind to these sites. Transactivation mediated by the p49/p100 and p65 NF-kappa B proteins is therefore sensitive to minor changes in the sequence of the kappa B site. Specificity determined by the association of NF-kappa B subunits provides a mechanism to selectively regulate variant kappa B sites associated with different cellular and viral genes.
Base Sequence↗
The proto-oncogene bcl-3 encodes an I kappa B protein.
The bcl-3 gene product, overexpressed in chronic lymphocytic leukemia (CLL) patients with the translocation t(14;19), is a member of the I kappa B family. The bcl-3 protein is able to inhibit the DNA binding and trans-activation of authentic NF-kappa B heterodimers p50-p65 and p49-p65, as well as p50 and p49 homodimers. The bcl-3 protein does not inhibit either the DNA-binding activity of the Rel protein or its ability to trans-activate genes linked to the kappa B site. A human 37-kD protein (I kappa B alpha), identified previously as a member of the I kappa B family, is also unable to inhibit DNA-binding activity of the Rel protein. However, unlike bcl-3, the 37-kD (I kappa B alpha) protein has no effect on the DNA-binding activity of p50 or p49 homodimers. Two dimensional phosphotryptic peptide maps of the human bcl-3 and the human 37-kD (I kappa B alpha) proteins reveal that the phosphopeptides from the 37-kD (I kappa B alpha) protein are nested within the bcl-3 protein. Furthermore, bcl-3 antisera immunoprecipitates an in vitro-radiolabeled 37-kD (I kappa B alpha) protein. Proteins of 56 and 38 kD can be identified in HeLa cells stimulated with PMA and immunoprecipitated with bcl-3 antisera. Comparison of tryptic peptide maps of the bcl-3 protein synthesized in vitro, and p56 and p38 from HeLa cells, shows that they are all structurally related. Removal of the amino-terminal sequences of the bcl-3 protein generates a protein that inhibits the DNA binding of the p50-p65 heterodimer but, like the 37-kD (I kappa B alpha) protein, is no longer able to inhibit the binding of the p50 and p49 homodimers with kappa B DNA. We propose that the bcl-3 and 37-kD (I kappa B alpha) proteins are related and are members of the I kappa B family.
3T3 Cells↗
Cloning of an NF-kappa B subunit which stimulates HIV transcription in synergy with p65.
The transcription factor NF-kappa B is a protein complex which comprises a DNA-binding subunit and an associated transactivation protein (of relative molecular masses 50,000 (50K) and 65K, respectively). Both the 50K and 65K subunits have similarity with the rel oncogene and the Drosophila maternal effect gene dorsal. The 50K DNA-binding subunit was previously thought to be a unique protein, derived from the 105K gene product (p105). We now report the isolation of a complementary DNA that encodes an alternative DNA-binding subunit of NF-kappa B. It is more similar to p105 NF-kappa B than other family members and defines a new subset of rel-related genes. It is synthesized as approximately 100K protein (p100) that is expressed in different cell types, contains cell cycle motifs and, like p105, must be processed to generate a 50K form. A 49K product (p49) can be generated independently from an alternatively spliced transcript; it has specific kappa B DNA-binding activity and can form heterodimers with other rel proteins. In contrast to the approximately 50K protein derived from p105, p49 acts in synergy with p65 to stimulate the human immunodeficiency virus (HIV) enhancer in transiently transfected Jurkat cells. p49/p100 NF-kappa B could therefore be important in the regulation of HIV and other kappa B-containing genes.
Amino Acid Sequence↗