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F Mercurio

Publications and source records attributed to F Mercurio.

28 records · Page 2Linked to original sources
PubMed1996

Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation.

Extracellular stimuli that activate the transcription factor NF-kappaB cause rapid phosphorylation of the IkappaBalpha inhibitor, which retains NF-kappaB in the cytoplasm of nonstimulated cells. Phosphorylation of IkappaBalpha is followed by its rapid degradation, the inhibition of which prevents NF-kappaB activation. To determine the relationship between these events, we mapped the inducible phosphorylation sites of IkappaBalpha. We found that two residues, serines 32 and 36, were phosphorylated in response to either tumor necrosis factor, interleukin-1, or phorbol ester. Substitution of either serine blocks or slows down induction of IkappaBalpha degradation. Substitutions of the homologous sites in IkappaBbeta, serines 19 and 23, also prevent inducible IkappaBbeta degradation. We suggest that activation of a single IkappaB kinas e or closely related IkappaB kinases is the first cr itical step in NF-kappaB activation. Once phosphorylated, IkappaB is ubiquitinated. Unlike wild-type IkappaBalpha, the phosphorylation-defective mutants do not undergo inducible polyubiquitination. As substitution of a conserved lysine residue slows down the ubiquitination and degradation of IkappaBalpha without affecting its phosphorylation, polyubiquitination is required for inducible IkappaB degradation.

3T3 Cells↗
PubMed1995

Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2.

One Ras-dependent protein kinase cascade leading from growth factor receptors to the ERK (extracellular signal-regulated kinases) subgroup of mitogen-activated protein kinases (MAPKs) is dependent on the protein kinase Raf-1, which activates the MEK (MAPK or ERK kinase) dual specificity kinases. A second protein kinase cascade leading to activation of the Jun kinases (JNKs) is dependent on MEKK (MEK kinase). A dual-specificity kinase that activates JNK, named JNKK, was identified that functions between MEKK and JNK. JNKK activated the JNKs but did not activate the ERKs and was unresponsive to Raf-1 in transfected HeLa cells. JNKK also activated another MAPK, p38 (Mpk2; the mammalian homolog of HOG1 from yeast), whose activity is regulated similarly to that of the JNKs.

Amino Acid Sequence↗
PubMed1995

Phosphorylation of I kappa B alpha precedes but is not sufficient for its dissociation from NF-kappa B.

NF-kappa B is an important activator of immune and inflammatory response genes. NF-kappa B is sequestered in the cytoplasm of nonstimulated cells through interaction with the I kappa B inhibitors. These inactive complexes are dissociated in response to a variety of extracellular signals, thereby allowing free NF-kappa B dimers to translocate to the nucleus and active transcription of specific target genes. The current dogma is that phosphorylation of the I kappa Bs is responsible for dissociation of the inactive complexes, an event that is rendered irreversible by rapid I kappa B degradation. Here, we show that inducers of NF-kappa B activity stimulate the hyperphosphorylation of one of the I kappa Bs, I kappa B alpha. However, contrary to the present dogma the hyperphosphorylated form of I kappa B alpha remains associated with NF-kappa B components such as RelA (p65). Thus, phosphorylation of I kappa B alpha is not sufficient to cause dissociation of the inactive NF-kappa B:I kappa B alpha complex. However, that complex is disrupted through the selective degradation of phosphorylated I kappa B alpha in response to extracellular signals. Using a variety of protease inhibitors, some of which have specificity towards the multicatalytic proteinase complex, we demonstrate that degradation of I kappa B alpha is required for NF-kappa B activation. The results of these experiments are more consistent with a new model according to which phosphorylation of I kappa B alpha associated with NF-kappa B marks it for proteolytic degradation. I kappa B alpha is degraded while bound to NF-kappa B. The selective degradation of I kappa B alpha releases active NF-kappa B dimers which can translocate to the nucleus to activate specific target genes.

Amino Acid Sequence↗
PubMed1993

p105 and p98 precursor proteins play an active role in NF-kappa B-mediated signal transduction.

The Rel/NF-kappa B family of transcription factors is composed of two distinct subgroups, proteins that undergo proteolytic processing and contain SWI6/ankyrin repeats in their carboxyl termini (p105, p98), and those without such repeats that do not require processing (p65, c-Rel, RelB, and Dorsal). We demonstrate that the p105 and p98 precursors share functional properties with the I kappa B proteins, which also contain SWI6/ankyrin repeats. Both p105 and p98 were found to form stable complexes with other Rel/NF-kappa B family members, including p65 and c-Rel. Association with the precursors is sufficient for cytoplasmic retention of either p65 or c-Rel, both of which are otherwise nuclear. These complexes undergo stimulus-responsive processing to produce active p50/c-Rel and p55/c-Rel complexes. These observations suggest a second pathway leading to NF-kappa B induction, in which processing of the precursors rather than phosphorylation of I kappa B plays a major role.

Antigen-Antibody Complex↗
PubMed1992

Molecular cloning and characterization of a novel Rel/NF-kappa B family member displaying structural and functional homology to NF-kappa B p50/p105.

The NF-kappa B transcription factor has been implicated in the inducible expression of many genes, including inflammatory, immune, and acute-phase response genes. NF-kappa B consists of two subunits, 50K and 65K polypeptides. The genes encoding p50 and p65 have sequence similarities with the c-rel proto-oncogene and the Drosophila maternal effect gene dorsal. We describe the cloning and characterization of a novel rel-related gene encoding a 98K product that shares extensive homology with the p105 precursor of the NF-kappa B p50 protein, containing both a Rel homology and SWI6/ankyrin repeat domain. We demonstrate that p98 is proteolytically processed in vivo to generate a 55K polypeptide, which binds to kappa B sites. p55 is capable of forming heterocomplexes with other Rel/NF-kappa B family members, which can bind to kappa B motifs in vitro, and stimulate transcription of reporter genes containing these cis-elements in vivo. The identification of a homolog for NF-kappa B p50/p105, termed p55/p98, gives further support to the idea that NF-kappa B is a collection of structurally related complexes of which contribute to the pleiotropic regulatory processes originally assigned to NF-kappa B.

Amino Acid Sequence↗
PubMed1989

Ubiquitous and lymphocyte-specific factors are involved in the induction of the mouse interleukin 2 gene in T lymphocytes.

The immediate upstream region of the mouse interleukin 2 (Il-2) gene harbors a strong transcriptional enhancer. This enhancer contains most, if not all of the sequence elements necessary for the T cell specific induction of the Il-2 gene by the phorbol ester TPA and the plant lectin Concanavalin A. DNase I footprinting studies with fractionated extracts obtained from induced and uninduced E14 T cells revealed numerous recognition sites for potential trans-acting factors. Five of these sites are also recognized by the TPA-activated HeLa cell factors AP-1 and AP-3. Other sites including two TATA-boxes, two purine-rich sequence motifs and two copies of the GGGPuTTTCAA motif are recognized by lymphocyte specific factors. The latter motif is highly conserved between several lymphokine genes and is therefore designated as a T cell element (TCE). In E14 T cells, pentamers of the distal TCEd confer an activity similar to that of the entire Il-2 enhancer, whereas in B and HeLa cells, the TCEd-pentamer is inactive as is the Il-2 enhancer. These data indicate the involvement of the TCEd and its recognition factor(s) in the cell type specific induction of the Il-2 gene during T cell activation.

Animals↗
PubMed1989

Transcription factors AP-3 and AP-2 interact with the SV40 enhancer in a mutually exclusive manner.

The 'core' sequence is critical for efficient transcriptional activity of the SV40 enhancer. Moreover, the core was shown to be involved in a signal transduction pathway elicited by treatment of cells with phorbol ester tumor promoters. We report here the identification and characterization of activator protein-3 (AP-3), which recognizes the core element. AP-3 was purified to near homogeneity and identified as a 48K polypeptide. The purified protein is an efficient transcriptional activator in vitro. In addition, we show that AP-3 and a second factor that recognizes the SV40 enhancer, AP-2, interact in a mutually exclusive manner. These studies should facilitate understanding of the mechanism by which the SV40 enhancer achieves its characteristic broad cell-type specificity.

Base Sequence↗
PubMed1989

A carcinogenesis- and tumorigenesis-associated rat fetal protein: an immuno-histochemical and immuno-biochemical study utilizing a new monoclonal antibody, MOFP.

An oncofetal protein (OFP), which is a potential marker for carcinogenesis and tumorigenesis, was evaluated with monoclonal antibodies shown to be specific for the antigen. Treatment of partially hepatectomized rats with a single non-necrogenic dose of diethylnitrosamine induced OFP in the liver. Its concentration, as measured by a dual immuno/bioassay, increased steadily over a 5-week period of observation before reaching a constant level. Immunohistochemical localization of OFP in liver sections from rats treated with N-nitroso-N-diethyl-nitrosamine showed that the factor was primarily localized to the cell cytoplasm in cells of most of the altered hepatic foci although some of this shedding antigen was also extracellular. Monoclonal antibody 17-1A specific for 17-1A antigen, an established surface marker for adenocarcinomas of the gastrointestinal tract, showed a similar distribution in liver from the carcinogen-treated rats, but localized to the cell membrane and cytoplasm. Scattered cells surrounding the altered hepatic foci were also positive for both monoclonal antibodies. Immunolocalization studies showed fetal rat liver and hepatoma were positive for OFP but adult normal or regenerating liver was negative. It was not detected in cells which morphologically could be classified as oval cells. As assessed by immuno/bioassay, the OFP released to the peripheral blood (plasma) of hepato-carcinogen-treated rats increased for 3 weeks, before undergoing a transitory decrease. Circulating antibodies specific for the factor were detected in the blood around 3-5 weeks post-treatment. Development of Western blots of the OFP with antiphosphotyrosine IgG indicates that the marker protein contains phosphotyrosine.

Animals↗
PubMedDate not supplied

Mechanisms of ubiquitin-mediated, limited processing of the NF-kappaB1 precursor protein p105.

In most cases, target proteins of the ubiquitin system are completely degraded. In several exceptions, such as the first step in the activation of the transcriptional regulator NF-kappaB, the substrate, the precursor protein p105, is processed in a limited manner to yield the active subunit p50. p50 is derived from the N-terminal domain of p105, whereas the C-terminal domain is degraded. The mechanisms involved in this unique process have remained elusive. We have shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is one important processing signal and that it interferes with processing of the ubiquitinated precursor by the 26S proteasome. Also, amino acid residues 441-454 are important for processing under non-stimulated conditions. Lys 441 and 442 serve as ubiquitination targets, whereas residues 446-454 may serve as a ligase recognition motif. Following IkappaB kinase (IKK)-mediated phosphorylation, the C-terminal domain of p105, residues 918-934, recruits the SCF(beta-TrCP) ubiquitin ligase, and ubiquitination by this complex leads to accelerated processing. The two sites appear to be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. We have shown that ubiquitin conjugation and processing of a series of precursors of p105 that lack the C-terminal IKK phosphorylation/TrCP binding domain, is progressively inhibited with increasing number of ankyrin repeats. Inhibition is due to docking of active NF-kappaB subunits to the ankyrin repeat domain in the C-terminal half of p105 (IkappaBgamma). Inhibition is alleviated by phosphorylation of the C-terminal domain that leads to ubiquitin-mediated degradation of the ankyrin repeat domain and release of the anchored subunits. We propose a model that may explain the requirement for two sites: a) a basal site that may be involved in co-translational processing prior to the synthesis of the ankyrin repeat domain; and b) a signal-induced site that is involved in processing/degradation of the complete molecule following cell activation, with rapid release of stored, transcriptionally active subunits.

Amino Acid Motifs↗