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R T Hay

Publications and source records attributed to R T Hay.

At least 37 records · Page 2Linked to original sources
PubMed1999

Control of NF-kappa B transcriptional activation by signal induced proteolysis of I kappa B alpha.

In unstimulated cells the transcription factor NF-kappa B is held in the cytoplasm in an inactive state by I kappa B inhibitor proteins. Ultimately activation of NF-kappa B is achieved by ubiquitination and proteasome-mediated degradation of I kappa B alpha and we have therefore investigated factors which control this proteolysis. Signal-induced degradation of I kappa B alpha exposes the nuclear localization signal of NF-kappa B, thus allowing it to translocate into the nucleus and activate transcription from responsive genes. An autoregulatory loop is established when NF-kappa B induces expression of the I kappa B alpha gene and newly synthesized I kappa B alpha accumulates in the nucleus where it negatively regulates NF-kappa B-dependent transcription. As part of this post-induction repression, the nuclear export signal on I kappa B alpha mediates transport of NF-kappa B-I kappa B alpha complexes from the nucleus to the cytoplasm. As nuclear export of I kappa B alpha is blocked by leptomycin B this drug was used to examine the effect of cellular location on susceptibility of I kappa B alpha to signal-induced degradation. In the presence of leptomycin B, I kappa B alpha is accumulated in the nucleus and in this compartment is resistant to signal-induced degradation. Thus signal-induced degradation of I kappa B alpha is mainly, if not exclusively a cytoplasmic process. An efficient nuclear export of I kappa B alpha is therefore essential for maintaining a low level of I kappa B alpha in the nucleus and allowing NF-kappa B to be transcriptionally active upon cell stimulation. We have detected a modified form of I kappa B alpha, conjugated to the small ubiquitin-like protein SUMO-1, which is resistant to signal-induced degradation. SUMO-1 modified I kappa B alpha remains associated with NF-kappa B and thus overexpression of SUMO-1 inhibits the signal-induced activation of NF-kappa B-dependent transcription. Reconstitution of the conjugation reaction with highly purified proteins demonstrated that in the presence of a novel E1 SUMO-1 activating enzyme, Ubch9 directly conjugated SUMO-1 to I kappa B alpha on residues K21 and K22, which are also used for ubiquitin modification. Thus, while ubiquitination targets proteins for rapid degradation, SUMO-1 modification acts antagonistically to generate proteins resistant to degradation.

Animals↗
PubMed1999

A complex containing betaTrCP recruits Cdc34 to catalyse ubiquitination of IkappaBalpha.

Activation of transcription factor NF-kappaB is accomplished by degradation of its inhibitor IkappaBalpha. Signal induced phosphorylation of IkappaBalpha on serine 32 and 36 targets the protein for ubiquitination on lysine 21 and 22. Here we use a phosphorylated peptide substrate representing residues 20-43 of IkappaBalpha to investigate requirements for ubiquitination of IkappaBalpha. Phosphorylation dependent polyubiquitination is carried out by a multiprotein complex containing betaTrCP, Skp1 and Cdc53 (Cull). In the presence of ubiquitin activating enzyme and the protein complex containing betaTrCP, polyubiquitination of IkappaBalpha peptide was dependent on the presence of Cdc34, while Ubc5 only stimulated mono- and di-ubiquitination.

Amino Acid Sequence↗
PubMed1999

Characterisation of the adenovirus preterminal protein and its interaction with the POU homeodomain of NFIII (Oct-1).

Formation of the preinitiation complex for adenovirus DNA replication involves the incoming preterminal protein-adenovirus DNA polymerase heterodimer being positioned at the origin of replication by protein-DNA and protein-protein interactions. Preterminal protein directly binds to the cellular transcription factor nuclear factor III (Oct-1), via the POU homeodomain. Co-precipitation of POU with individual domains of preterminal protein expressed by in vitro translation indicated that POU contacts multiple sites on preterminal protein. Partial proteolysis of preterminal protein in the presence or absence of POU homeodomain demonstrated that many sites accessible to proteases in free preterminal protein were resistant to cleavage in the presence of POU homeodomain. The accessibility of sites in free preterminal protein to cleavage by trypsin was strongly dependent on the ionic strength, suggesting that preterminal protein may undergo a sodium chloride-induced conformational change. It is therefore likely that the POU homeodomain contacts a number of sites on preterminal protein to induce a conformational change which may influence the initiation of adenovirus DNA replication.

Adenoviridae↗
PubMed1999

Mutations in the IkBa gene in Hodgkin's disease suggest a tumour suppressor role for IkappaBalpha.

The NF-kappaB/Rel family of transcription factors regulates a wide variety of genes whose products play a fundamental role in inflammatory and immune responses. The implication of NF-kappaB/Rel proteins and their IkappaB regulatory subunits in the control of cellular growth and oncogenesis, was suggested by the induction of fatal lymphomas in birds by the v-rel oncoprotein, and the rearrangement and amplification of several genes encoding the NF-kappaB/Rel/IkappaB signal transduction factors in human malignancies, primarily of lymphoid origin. Hodgkin's disease (HD) is a lymphoma characterized by a low frequency of malignant Hodgkin and Reed-Sternberg (H/RS) cells in a reactive background of non-neoplastic cells. The peculiar activated phenotype of Hodgkin and Reed-Sternberg cells and their pattern of cytokine secretion are believed to be a consequence of constitutive activation of the NF-kappaB transcription factor. Here, we report the detection of mutations of the IkBa gene, in two HD-derived cell lines and in two out of eight biopsy samples from patients with relapsed Hodgkin's disease. The presence of defective IkappaBalpha is thus likely to explain the constitutive activation of NF-kappaB in these cells and suggests that IkappaBalpha is a tumour suppressor controlling the oncogenic activation of NF-kappaB in Hodgkin and Reed-Sternberg cells.

Alleles↗
PubMed1999

Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1.

The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IkappaBalpha, and PML. SUMO-1-modified proteins display altered subcellular targeting and/or stability. We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitin-activating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub-1p. In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IkappaBalpha. As SAE1/SAE2, Ubch9, SUMO-1, and IkappaBalpha are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IkappaBalpha in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.

Adenosine Triphosphate↗
PubMed1999

Nuclear retention of IkappaBalpha protects it from signal-induced degradation and inhibits nuclear factor kappaB transcriptional activation.

Transcriptional activation of nuclear factor kappaB (NF-kappaB) is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. However, NF-kappaB activation induces rapid resynthesis of IkappaBalpha, which is responsible for post-induction repression of transcription. Newly synthesized IkappaBalpha translocates to the nucleus, where it dissociates NF-kappaB from DNA and transports NF-kappaB from the nucleus to the cytoplasm in a nuclear export sequence-dependent process that is sensitive to leptomycin B (LMB). In the present study, LMB was used as a tool to inhibit nuclear export sequence-mediated nuclear protein export and evaluate the consequences for regulation of NF-kappaB-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-kappaB-dependent transcriptional activation mediated by interleukin 1beta or tumor necrosis factor alpha. This is a consequence of the inhibition of signal-induced degradation of IkappaBalpha. Although LMB treatment does not affect the signal transduction pathway leading to IkappaBalpha degradation, it blocks IkappaBalpha nuclear export. IkappaBalpha is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IkappaBalpha is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IkappaBalpha is therefore essential for maintaining a low level of IkappaBalpha in the nucleus and allowing NF-kappaB to be transcriptionally active upon cell stimulation.

Biological Transport↗
PubMed1999

Characterization of IkappaBalpha nuclear import pathway.

IkappaBalpha controls the transcriptional activity of nuclear factor (NF)-kappaB by retaining it in the cytoplasm; but, when expressed in the nucleus, it can also inhibit the interaction of NF-kappaB with DNA and promote the export of NF-kappaB from the nucleus to the cytoplasm. Here, we report that IkappaBalpha, when not bound to NF-kappaB, is constitutively transported to the nucleus, and we confirm that the interaction of IkappaBalpha with NF-kappaB retains IkappaBalpha in the cytoplasm. Nuclear import of IkappaBalpha does not result from passive diffusion but from a specific energy-dependent transport process that requires the ankyrin repeats of IkappaBalpha. Nuclear accumulation of IkappaBalpha is dependent on importins alpha and beta as well as the small GTPase Ran, which are also responsible for the nuclear import mediated by basic nuclear localization sequences (NLS). However, these proteins are not sufficient to promote IkappaBalpha nuclear translocation. Factor(s) can be removed selectively from cell extracts with ankyrin repeats of IkappaBalpha which strongly reduce import of IkappaBalpha but not of proteins containing basic NLS. These findings indicate that IkappaBalpha is imported in the nucleus by a piggy-back mechanism that involves additional protein(s) containing a basic NLS and able to interact with ankyrin repeats of IkappaBalpha.

Biological Transport↗
PubMed1999

Role of the conserved lysine 80 in stabilisation of NF-kappaB p50 DNA binding.

The transcriptional rate of a variety of genes involved in acute-phase response, inflammation, lymphocytic activation, and cell growth or differentiation, is regulated by the DNA binding activity of the inducible transcription factor NF-kappaB. NF-kappaB p50 homodimers bind specifically to DNA, via base and backbone contacts mediated by residues in the flexible loops which link secondary structure elements in both of its two distinct domains. However, it has been suggested that additional contacts which stabilise DNA binding are made by lysine residues located in the C-terminus of the flexible loop which connects A and B beta-sheets of the N-terminal domain of p50. To determine the importance of each of the lysine residues in this region (K77, K79, K80), a series of mutated p50 proteins were generated in which the lysines were changed to alanines. The DNA binding properties of these mutants were analysed by gel electrophoresis DNA binding assays and surface plasmon resonance. This study revealed that the C-terminus of AB loop interacts with DNA through an additional lysine-phosphate backbone ionic bond which makes a significant contribution to the binding energy, thus stabilising the complex. The lysine residue responsible for this interaction is K80 which is conserved in all NF-kappaB/Rel/Dorsal molecules.

Amino Acid Sequence↗
PubMed1999

Persistent activation of nuclear factor-kappaB in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IkappaB family proteins.

Rat hepatic stellate cells (HSC) cultured in serum-containing medium underwent a rapid (3-hour) classical induction of p50:p65 and p65:p65 nuclear factor-kappaB (NF-kappaB) dimers. Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high-mobility NF-kappaB DNA binding complex consisting of potentially novel Rel-like protein(s). Formation of the latter complex was competed for by specific double-stranded oligonucleotides, was up-regulated by treatment of HSCs with tumor necrosis factor alpha (TNF-alpha), and was maintained at basal levels of expression by a soluble HSC-derived factor. An NF-kappaB-responsive CAT reporter gene was highly active in early cultured HSCs but was also trans-activated at a lower but significant level in longer-term cultured cells and could be completely suppressed by expression of dominant negative IkappaB-alpha. Physiological significance of the lower persistent NF-kappaB activities was also demonstrated by the ability of long-term cultured HSCs to support the activity of the NF-kappaB-dependent human intercellular adhesion molecule-1 (ICAM-1) promoter. Freshly isolated HSCs expressed high levels of IkappaB-alpha and IkappaB-beta. Culture activation was accompanied by a long-term reduction in levels of IkappaB-alpha with no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus. IkappaB-beta expression was transiently reduced and, upon replenishment, was associated with appearance of a lower-mobility IkappaB-beta antibody-reactive species. Bcl3 expression was absent in freshly isolated HSC but was induced during culturing and became a persistent feature of the activated HSC. Inhibition of NF-kappaB DNA binding activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF-kappaB in cultured HSC is required for expression of specific genes associated with the activated phenotype such as ICAM-1 and may be antiapoptotic for rat HSCs.

Adipocytes↗
PubMed1999

p75-mediated NF-kappaB activation enhances the survival response of developing sensory neurons to nerve growth factor.

We have investigated whether the transcription factor NF-kappaB plays a role in regulating neuronal survival by manipulating NF-kappaB activation in the nerve growth factor (NGF)-dependent sensory neurons of the embryonic mouse trigeminal ganglion. Overexpression of either the p65 or the p50 NF-kappaB subunits resulted in NF-kappaB activation and promoted in vitro survival as effectively as NGF. Expression of a superrepressor IkappaB-alpha protein prevented NF-kappaB activation in p65/p50-overexpressing neurons and caused the neurons to die as rapidly as NGF-deprived neurons. NGF treatment also activated NF-kappaB, and preventing this activation with superrepressor IkappaB-alpha reduced the NGF survival response. Antibodies that block binding of NGF to the p75 receptor prevented NGF-induced NF-kappaB activation and reduced the NGF survival response to the same extent as superrepressor IkappaB-alpha. Trigeminal neurons cultured from p65(-/-) embryos showed a reduced survival response to NGF compared with neurons from wild-type embryos and there was increased apoptosis of neurons in the trigeminal ganglia of p65(-/-) embryos in vivo. However, as with p75-deficient sensory neurons, p65-deficient sensory neurons showed a normal survival response to BDNF. These results reveal a role for NF-kappaB in regulating neuronal survival during embryonic development and suggest that in addition to the well-established Trk receptor tyrosine kinase signaling cascade, NGF enhances neuronal survival by signaling via a p75-mediated pathway.

Animals↗
PubMed1999

SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation.

PML is a nuclear phosphoprotein that was first identified as part of a translocated chromosomal fusion product associated with acute promyelocytic leukaemia (APL). PML localises to distinct nuclear multi-protein complexes termed ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs), which are disrupted in APL and are the targets for immediate early viral proteins, although little is known about their function. In a yeast two-hybrid screen, we first identified a ubiquitin-like protein named PIC1 (now known as SUMO-1), which interacts and co-localises with PML in vivo. More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and IkappaBalpha and is proposed to play a role in either targeting modified proteins and/or inhibiting their degradation. The precise molecular role for the SUMO-1 modification of PML is unclear, and the specific lysine residues within PML that are targeted for modification and the PML sub-domains necessary for mediating the modification in vivo are unknown. Here we show that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain. Using site-specific mutagenesis, we have identified the primary PML-SUMO-1 modification site as being part of the nuclear localisation signal (Lys487 or Lys490). However SUMO-1 modification is not essential for PML nuclear localisation as only nuclear PML is modified. The sequence of the modification site fits into a consensus sequence for SUMO-1 modification and we have identified several other nuclear proteins which could also be targets for SUMO-1. We show that SUMO-1 modification appears to be dependant on the correct subcellular compartmentalisation of target proteins. We also find that the APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RARA. Our results provide significant insights into the role of SUMO-1 modification of PML in both normal cells and the APL disease state.

Consensus Sequence↗
PubMed1998

Defective IkappaBalpha in Hodgkin cell lines with constitutively active NF-kappaB.

The molecular mechanisms underlying Hodgkin's disease remain obscure, but it has been recognized that the neoplastic cells display high levels of constitutively active nuclear NF-kappaB. Here we demonstrate that although nuclear NF-kappaB is transcriptionally active, the Hodgkin cells fail to activate NF-kappaB dependent transcription in response to CD40 ligand. In three Hodgkin cell lines examined each had abnormalities in expression of IkappaBalpha which could account for the deregulated NF-kappaB. Although all three cell lines had greater than normal levels of IkappaBalpha mRNA no IkappaBalpha protein could be detected in the KM-H2 cells, while the L428 cell line contains a C-terminally truncated IkappaBalpha species that fails to associate with NF-kappaB. The HDLM-2 cell line contains a more slowly migrating form of IkappaBalpha that can associate with NF-kappaB, but increasing the level of this protein within the cell fails to inhibit nuclear NF-kappaB. Addition of recombinant IkappaBalpha to nuclear extracts from all three cell lines resulted in complete inhibition of NF-kappaB DNA binding activity and introduction of a plasmid expressing IkappaBalpha into the cells inhibited the transcriptional activity of an NF-kappaB dependent reporter plasmid. Thus the constitutive expression of NF-kappaB in Hodgkin cells is a direct consequence of the abnormal expression of IkappaBalpha rather than changes in NF-kappaB that render it refractory to inhibition by IkappaB proteins. These changes could, at least in part, account for the characteristic activated phenotype of Hodgkin cells and their pattern of cytokine secretion, which determine the pathological appearance and clinical manifestations of Hodgkin's disease.

CD40 Ligand↗
PubMed1998

Adenovirus DNA polymerase: domain organisation and interaction with preterminal protein.

Adenovirus DNA polymerase is one of three viral proteins and two cellular proteins required for replication of the adenovirus genome. During initiation of viral DNA synthesis the viral DNA polymerase transfers dCMP onto the adenovirus preterminal protein, to which it is tightly bound. The domain structure of the 140 kDa DNA polymerase has been probed by partial proteolysis and the sites of proteolytic cleavage determined by N-terminal sequencing. At least four domains can be recognised within the DNA polymerase. Adenovirus preterminal protein interacts with three of the four proteolytically derived domains. This was confirmed by cloning and expression of each of the individual domains. These data indicate that, like other members of the pol alpha family of DNA polymerases, the adenovirus DNA polymerase has a multidomain structure and that interaction with preterminal protein takes place with non-contiguous regions of the polypeptide chain over a large surface area of the viral DNA polymerase.

Adenoviridae↗
PubMed1998

SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation.

Activation of NF-kappaB is achieved by ubiquitination and proteasome-mediated degradation of IkappaBalpha. We have detected modified IkappaBalpha, conjugated to the small ubiquitin-like protein SUMO-1, which is resistant to signal-induced degradation. In the presence of an E1 SUMO-1-activating enzyme, Ubch9 conjugated SUMO-1 to IkappaBalpha primarily on K21, which is also utilized for ubiquitin modification. Thus, SUMO-1-modified IkappaBalpha cannot be ubiquitinated and is resistant to proteasome-mediated degradation. As a result, overexpression of SUMO-1 inhibits signal-induced activation of NF-kappaB-dependent transcription. Unlike ubiquitin modification, which requires phosphorylation of S32 and S36, SUMO-1 modification of IkappaBalpha is inhibited by phosphorylation. Thus, while ubiquitination targets proteins for rapid degradation, SUMO-1 modification acts antagonistically to generate proteins resistant to degradation.

Amino Acid Sequence↗
PubMed1998

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) interacts with Lys-tRNA synthetase: implications for priming of HIV-1 reverse transcription.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 96-amino-acid 14-kDa protein (viral protein R [Vpr]), which is produced late in the viral life cycle and is incorporated into the virion. Although Vpr is not required for viral replication in transformed cell lines and primary T lymphocytes, it is essential for productive infection of macrophages and monocytes and appears to be important for pathogenesis in vivo. To establish the role of Vpr in HIV-1 replication and pathogenesis, we have isolated cellular proteins with which Vpr interacts. By using the yeast two-hybrid system, Lys-tRNA synthetase (LysRS) was identified as a Vpr-interacting protein. The interaction between Vpr and LysRS was characterized both in vitro and in vivo, and the domains of Vpr required for the interaction were defined. In the presence of Vpr, LysRS-mediated amino-acylation of tRNA(Lys) is inhibited. Since tRNA(Lys) is the primer for reverse transcription of the HIV-1 genome, this suggests that the interaction between Vpr and LysRS may influence the initiation of HIV-1 reverse transcription.

Acylation↗
PubMed1997

Ubch9 conjugates SUMO but not ubiquitin.

Ubiquitin conjugating enzymes participate in the thioester cascade that leads to protein ubiquitination. Although Ubc9 is homologous to E2 ubiquitin conjugating enzymes we have shown that it is unable to form a thioester with ubiquitin, but can form a thioester with the small ubiquitin-like protein SUMO. Thus Ubc9 is a SUMO conjugating enzyme rather than a ubiquitin conjugating enzyme. Transacetylation of Ubc9 by SUMO is not mediated by the E1 ubiquitin activating enzyme, but by a distinct enzymatic activity. SUMO conjugation to target proteins is mediated by a different, but parallel pathway to ubiquitination.

Acetylation↗
PubMed1997

The carboxy-terminus of I kappaB alpha determines susceptibility to degradation by the catalytic core of the proteasome.

The Rel/NF-kappaB family of transcription factors controls the expression of a wide variety of genes that are implicated in immune and inflammatory responses and cellular proliferation. Disregulation of NF-kappaB is associated with cellular transformation and the maintenance of a high anti-apoptotic threshold in transformed cells. NF-kappaB activity is in turn regulated by its sequestration in the cytoplasm by the inhibitor I kappaB. I kappaB alpha, the most abundant and well-characterized member of the I kappaB multiprotein family, is rapidly degraded in response to multiple physiologic stimuli. In the present study we show that not only the amino-terminus, but also the carboxy-terminus of I kappaB alpha contain transferable signals that must be simultaneously present in an unrelated protein to render it susceptible to activation-induced, proteasome-mediated degradation. We show here that I kappaB alpha amino-terminal modifications occur independently of the carboxy-terminus. Moreover, we present evidence indicating a critical role for the carboxy-terminal region in facilitating proteolysis by the catalytic core of the proteasome. When incubated with 20S proteasome extracted from rat liver, I kappaB alpha was quickly degraded while a deletion mutant lacking the carboxy-terminus was resistant to proteolysis. Likewise, chimeric proteins of beta-galactosidase with the I kappaB alpha carboxy-terminus were degraded in vitro independently of the presence of the I kappaB alpha amino-terminus, whereas chimeric proteins lacking the I kappaB alpha carboxy-terminus were stable. Our results identify the carboxy-terminus of I kappaB alpha as a domain critical for degradation through interaction with an as yet unidentified component of the proteasome.

Animals↗