Showing papers by "Michael Karin published in 1998"
TL;DR: In this paper, a large, cytokine-responsive IkappaB kinase (IKK) complex has been purified and the genes encoding two of its subunits have been cloned.
Abstract: Pro-inflammatory cytokines activate the transcription factor NF-kappaB by stimulating the activity of a protein kinase that phosphorylates IkappaB, an inhibitor of NF-kappaB, at sites that trigger its ubiquitination and degradation. This results in the nuclear translocation of freed NF-kappaB dimers and the activation of transcription of target genes. Many of these target genes code for immunoregulatory proteins. A large, cytokine-responsive IkappaB kinase (IKK) complex has been purified and the genes encoding two of its subunits have been cloned. These subunits, IKK-alpha and IKK-beta, are protein kinases whose function is needed for NF-kappaB activation by pro-inflammatory stimuli. Here, by using a monoclonal antibody against IKK-alpha, we purify the IKK complex to homogeneity from human cell lines. We find that IKK is composed of similar amounts of IKK-alpha, IKK-beta and two other polypeptides, for which we obtained partial sequences. These polypeptides are differentially processed forms of a third subunit, IKK-gamma. Molecular cloning and sequencing indicate that IKK-gamma is composed of several potential coiled-coil motifs. IKK-gamma interacts preferentially with IKK-beta and is required for the activation of the IKK complex. An IKK-gamma carboxy-terminal truncation mutant that still binds IKK-beta blocks the activation of IKK and NF-kappaB.
909 citations
TL;DR: Purified recombinant IKKα and IKKβ expressed in insect cells were used to demonstrate that each protein can directly phosphorylate IκB proteins, explaining how free IkkB can accumulate in cells in which IKK is still active and thus can contribute to the termination of NF-κB activation.
Abstract: A large protein complex mediates the phosphorylation of the inhibitor of κB (IκB), which results in the activation of nuclear factor κB (NF-κB) Two subunits of this complex, IκB kinase α (IKKα) and IκB kinase β (IKKβ), are required for NF-κB activation Purified recombinant IKKα and IKKβ expressed in insect cells were used to demonstrate that each protein can directly phosphorylate IκB proteins IKKα and IKKβ were found to form both homodimers and heterodimers Both IKKα and IKKβ phosphorylated IκB bound to NF-κB more efficiently than they phosphorylated free IκB This result explains how free IκB can accumulate in cells in which IKK is still active and thus can contribute to the termination of NF-κB activation
450 citations
TL;DR: It is found that gamma radiation led to activation of IKK, the protein kinase that phosphorylates IkappaBalpha at Ser-32 and Ser-36, whereas UV-C radiation did not, and expression of a catalytically inactive IKKbeta mutant prevented NF-kappaB activation by gamma radiation, but not byUV-C.
Abstract: We examined the mechanisms by which two different types of photonic radiation, short wavelength UV (UV-C) and γ radiation, activate transcription factor NF-κB. Exposure of mammalian cells to either form of radiation resulted in induction with similar kinetics of NF-κB DNA binding activity, nuclear translocation of its p65(RelA) subunit, and degradation of the major NF-κB inhibitor IκBα. In both cases, induction of NF-κB activity was attenuated by proteasome inhibitors and a mutation in ubiquitin-activating enzyme, suggesting that both UV-C and γ radiation induce degradation of IκBs by means of the ubiquitin/proteasome pathway. However, although the induction of IκBα degradation by γ rays was dependent on its phosphorylation at Ser-32 and Ser-36, UV-C-induced IκBα degradation was not dependent on phosphorylation of these residues. Even the “super repressor” IκBα mutant, which contains alanines at positions 32 and 36, was still susceptible to UV-C-induced degradation. Correspondingly, we found that γ radiation led to activation of IKK, the protein kinase that phosphorylates IκBα at Ser-32 and Ser-36, whereas UV-C radiation did not. Furthermore, expression of a catalytically inactive IKKβ mutant prevented NF-κB activation by γ radiation, but not by UV-C. These results indicate that γ radiation and UV-C activate NF-κB through two distinct mechanisms.
434 citations
TL;DR: Signaling pathways that stabilize interleukin-2 (IL-2) messenger RNA (mRNA) in activated T cells were examined and multiple elements within IL-2 mRNA modulate its stability in a combinatorial manner.
Abstract: Signaling pathways that stabilize interleukin-2 (IL-2) messenger RNA (mRNA) in activated T cells were examined. IL-2 mRNA contains at least two cis elements that mediated its stabilization in response to different signals, including activation of c-Jun amino-terminal kinase (JNK). This response was mediated through a cis element encompassing the 5' untranslated region (UTR) and the beginning of the coding region. IL-2 transcripts lacking this 5' element no longer responded to JNK activation but were still responsive to other signals generated during T cell activation, which were probably sensed through the 3' UTR. Thus, multiple elements within IL-2 mRNA modulate its stability in a combinatorial manner, and the JNK pathway controls turnover as well as synthesis of IL-2 mRNA.
360 citations
TL;DR: As GRdim/dim mice appear relatively normal and healthy under standard laboratory conditions, the most striking outcome of these experiments is that GRE-mediated gene activation is not necessary for development and survival.
341 citations
TL;DR: It is demonstrated that lasting c-Jun S73 phosphorylation and JNK activity are part of neuronal stress response after neurodegenerative disorders in the adult mammalian brain with Fas-ligand as a putative apoptotic effector.
Abstract: Transcription factor c-Jun is proposed to control neuronal cell death and survival, but its activation by N-terminal phosphorylation and the underlying activity of the c-Jun N-terminal kinases (JNKs) remain to be elucidated in the adult mammalian brain. We generated a polyclonal antiserum that specifically recognizes c-Jun phosphorylated at its serine 73 (S73) residue after UV irradiation of 3T3 cells. Disruption of the c-jun locus in 3T3 cells abolished this reaction, and retransfection of the human c-jun at the c-jun-/- background restored it. The phospho-c-Jun antiserum was used to visualize N-terminally phosphorylated c-Jun in the adult rat brain with cellular resolution. Prolonged c-Jun S73 phosphorylation was detected in affected neurons up to 5 d after transient occlusion of medial cerebral artery or up to 50 d after transection of central nerve fiber tracts. After cerebral ischemia-reperfusion, phosphorylation of c-Jun was linked with induced expression of Fas-ligand (APO-1, CD95-ligand), whose gene is a putative c-Jun/AP-1 target, and with terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactivity, a marker for apoptosis. After nerve fiber transection, however, lasting c-Jun phosphorylation occurred in axotomized neurons negative for Fas-ligand or TUNEL and regardless of degeneration or survival. In contrast to these lasting phosphorylation patterns, transient seizure activity by pentylenetetrazole provoked only a brief c-Jun phosphorylation and JNK activation. In extracts from ischemic or axotomized brain compartments, c-Jun phosphorylation correlated with enhanced long-term JNK activity, and in-gel kinase assays visualized proteins with sizes corresponding to JNK isoforms as the only c-Jun N-terminally phosphorylating enzymes. These results demonstrate that lasting c-Jun S73 phosphorylation and JNK activity are part of neuronal stress response after neurodegenerative disorders in the adult mammalian brain with Fas-ligand as a putative apoptotic effector.
334 citations
TL;DR: MAPK cascades were recently found to be activated by a variety of stresses including ischemia reperfusion, neuronal injury, osmotic shock, and exposure to UV irradiation, suggesting they are likely to be important regulatory elements in a range of stress responses.
Abstract: Mitogen-activated protein kinase (MAPK) cascades play an important role in transducting environmental stimuli to the transcriptional machinery in the nucleus by virtue of their ability to phosphorylate and regulate the activity of various transcription factors. Originally found to be activated in response to occupancy of cell surface receptors for polypeptide hormones, cytokines, and growth factors, MAPK cascades were recently found to be activated by a variety of stresses including ischemia reperfusion, neuronal injury, osmotic shock, and exposure to UV irradiation. Therefore, MAPK cascades are likely to be important regulatory elements in a variety of stress responses.
328 citations
TL;DR: This study found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs and are suggested to participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specificExocytosis in clostidial NT-resistant pathways.
Abstract: The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been ...
311 citations
TL;DR: The results indicate that PKC isozymes have distinct biological functions and suggest that synergistic JNK activation is an important function for PKC‐θ in T‐cell activation.
Abstract: Costimulation of the T cell receptor (TCR) and CD28 is required for optimal interleukin-2 (IL-2) induction. These signals, which can be replaced by the pharmacological agents phorbol ester (PMA) and Ca2+ ionophore, synergistically activate the mitogen-activated protein kinase (MAPK) JNK. Cyclosporin A, an inhibitor of the Ca2+-dependent phosphatase calcineurin which blocks IL-2 induction, abrogates Ca2+-triggered synergistic JNK activation. As protein kinase C (PKC) downregulation inhibits PMA+ionophore-induced JNK activation, we examined whether a particular PKC isoform is preferentially involved in this response. We found that PKC-theta but neither PKC-alpha nor PKC-epsilon participates in JNK activation, whereas all three PKCs lead to ERK MAPK activation. PKC-theta specifically cooperates with calcineurin, and together their signals converge on (or upstream of) Rac leading to potent JNK activation. Similarly, calcineurin and PKC-theta specifically synergize to induce transcription of reporters driven by the c-jun and IL-2 promoters. PKC-theta and calcineurin are also partially responsible for the synergistic activation of JNK following TCR and CD28 ligation. Preferential cooperation between PKC-theta and calcineurin is observed in Jurkat T cells but not in HeLa cells. These results indicate that PKC isozymes have distinct biological functions and suggest that synergistic JNK activation is an important function for PKC-theta in T-cell activation.
273 citations
TL;DR: When different mammalian MAPKKs were examined in vitro or in vivo for phosphorylation and activation by MEKK1, a MAPKK called JNKK1 (MKK4 or SEK1), whose function is JNK (and p38 MAPK) activation was found to be the preferred MeKK1 substrate, thus providing a clear biochemical explanation for the marked pro-JNK bias of MEK1.
Abstract: MEKK1 (MEK kinase 1) is a mammalian serine/threonine kinase in the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) group (1). Being the first mammalian homolog of STE11, a MAPKKK that activates the pheromone responsive MAPK cascade of budding yeast, MEKK1 as its name indicates was thought to be an activator of the MAPK kinase (MAPKK) MEK1/2 and thus an activator of the ERK MAPK cascade. It therefore was rather surprising that titration experiments (2) or analysis of cells engineered to express MEKK1 from an inducible promoter (3) revealed that it is a far more potent activator of the JNK MAPK cascade. These observations made by using either the catalytic domain of MEKK1 (MEKK1Δ) or a 672-aa C-terminal fragment recently were confirmed by using full-length human MEKK1 (Y. Xia, Z. Wu, B. Su, B. Murray, and M.K., unpublished work). Most importantly, when different mammalian MAPKKs were examined in vitro or in vivo for phosphorylation and activation by MEKK1, a MAPKK called JNKK1 (MKK4 or SEK1), whose function is JNK (and p38 MAPK) activation (4) was found to be the preferred MEKK1 substrate (Y. Xia, Z. Wu, B. Su, B. Murray, and M.K., unpublished work). Based on specificity constants, MEKK1 phosphorylates JNKK1 45-fold more efficiently than it phosphorylates MEK1/2 (Y. Xia, Z. Wu, B. Su, B. Murray, and M.K., unpublished work), thus providing a clear biochemical explanation for the marked pro-JNK bias of MEKK1. Targets for JNK include transcription factors c-Jun and ATF2, which are components of the AP-1 dimer that are involved in induction of the c-jun protooncogene (5). JNK-mediated phosphorylation enhances the transcriptional activity of both c-Jun and ATF2 (6, 7). Correspondingly, MEKK1 expression plasmids are potent activators of a chimeric c-Jun-GAL4 transcription factor, in which the c-Jun activation domain is fused to the GAL4 DNA binding domain (8 …
214 citations
TL;DR: A role for the amino-terminal extension of MAPKKs in determination of response specificity is underscored, as JNK and MEKK1 compete for binding to JnKK1 and activation of JNKK1 prevents its binding to MEKK2.
Abstract: MAP kinase (MAPK) cascades are composed of a MAPK, MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Despite the existence of numerous components and ample opportunities for crosstalk, most MAPKs are specifically and distinctly activated. We investigated the basis for specific activation of the JNK subgroup of MAPKs. The specificity of JNK activation is determined by the MAPKK JNKK1, which interacts with the MAPKKK MEKK1 and JNK through its amino-terminal extension. Inactive JNKK1 mutants can disrupt JNK activation by MEKK1 or tumor necrosis factor (TNF) in intact cells only if they contain an intact amino-terminal extension. Mutations in this region interfere with the ability of JNKK1 to respond to TNF but do not affect its activation by physical stressors. As JNK and MEKK1 compete for binding to JNKK1 and activation of JNKK1 prevents its binding to MEKK1, activation of this module is likely to occur through sequential MEKK1:JNKK1 and JNKK1:JNK interactions. These results underscore a role for the amino-terminal extension of MAPKKs in determination of response specificity.
TL;DR: It is demonstrated that Tax triggers activation of cellular protein kinases, IκB kinase α (IKKα) and IKKβ, which phosphorylate the NF-κB inhibitory protein IKKBα, resulting in its degradation and NF-σκB activation.
Journal Article•
TL;DR: Signals generated by PTKs are functionally distinct and need to be integrated to induce transcriptional responses and, unlike Syk, the PTK Lck activates JNK but does not cooperate with Rac, resulting in weak AP-1 and NF-AT activation.
TL;DR: It is shown that other microorganisms (Candida albicans, Schizosaccharomyces pombe, and Escherichia coli) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms.
Abstract: Stalk formation is a novel pattern of multicellular organization Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (05 to 30 cm) and thin (05 to 4 mm in diameter) multicellular structures We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces We show that other microorganisms (Candida albicans, Schizosaccharomyces pombe, and Escherichia coli) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores
Patent•
23 Feb 1998TL;DR: In this article, isolated nucleic acid molecules encoding IκB kinase (IKK) catalytic subunit polypeptides were associated with an IKK serine protein kinase that phosphorylates a protein (IκB) that inhibits the activity of the NF-κB transcription factor.
Abstract: The present invention provides an isolated nucleic acid molecules encoding IκB kinase (IKK) catalytic subunit polypeptides, which are associated with an IKK serine protein kinase that phosphorylates a protein (IκB) that inhibits the activity of the NF-κB transcription factor, vectors comprising such nucleic acid molecules and host cells containing such vectors. In addition, the invention provides nucleotide sequences that can bind to a nucleic acid molecule of the invention, such nucleotide sequences being useful as probes or as antisense molecules. The invention also provides isolated IKK catalytic subunits, which can phosphorylate an IκB protein, and peptide portions of such IKK subunit. In addition, the invention provides anti-IKK antibodies, which specifically bind to an IKK complex or an IKK catalytic subunit, and IKK-binding fragments of such antibodies. The invention further provides methods of substantially purifying an IKK complex, methods of identifying an agent that can alter the association of an IKK complex or an IKK catalytic subunit with a second protein, and methods of identifying proteins that can interact with an IKK complex or an IKK catalytic subunit.
01 Jan 1998
TL;DR: The SAPKs were identified by virtue of their ability to phosphorylate the N-terminal sites of c-Jun, which is encoded by the c-jun proto-oncogene.
Abstract: Publisher Summary Mammalian cells respond to extracellular stimuli by activating signaling cascades that lead to long-term changes in gene expression. These signal transduction cascades are mediated by serine/threonine kinases of the mitogen-activated protein (MAP) kinase family. Several subgroups of MAP kinases have been identified in mammalian cells, which differ in their substrate specificities and regulation. Understanding the mechanisms by which signal transduction pathways transmit information to the nucleus is important in order to develop a better understanding of how normal cells respond to extracellular stimuli. The JNKs were identified by virtue of their ability to phosphorylate the N-terminal sites of c-Jun, which is encoded by the c-jun proto-oncogene. The JNKs were also independently identified as cyclohexamide-activated protein kinases and were named SAPKs. Of the different Juns, only c-Jun is an efficient JNK substrate. Like other MAP kinases, JNK binding is stimulated in response to a signal transduction cascade that is triggered when cells are exposed to extracellular stimuli. The JNKs are activated by a variety of different types of extracellular stimuli including growth factors, cytokines, and cellular stresses, such as heat shock, hyperosmolarity, and UV irradiation.