Biomedical subjects

M Karin

Publications and source records attributed to M Karin.

At least 145 records · Page 8Linked to original sources

PubMed1994

JNK is involved in signal integration during costimulation of T lymphocytes.

T lymphocyte activation and interleukin-2 (IL-2) production require at least two signals, generated by phorbol ester (TPA) and Ca2+ ionophore or costimulation of the T cell receptor (TCR) and the CD28 auxiliary receptor. We investigated how these stimuli affect mitogen activated protein (MAP) kinases. Full activation of the MAP kinases that phosphorylate the Jun activation domain, JNK1 and JNK2, required costimulation of T cells with either TPA and Ca2+ ionophore or antibodies to TCR and CD28. Alone, each stimulus resulted in little or no activation. Similar to its effect on IL-2 induction, cyclosporin A (CsA) inhibited the synergistic activation of JNK, and a competitive inhibitor of Jun phosphorylation by JNK inhibited IL-2 promoter activation. By contrast, the MAP kinases ERK1 and ERK2 were fully activated by TPA or TCR stimulation and were not affected by Ca2+, CD28, or CsA. Hence, integration of signals that lead to T cell activation occurs at the level of JNK activation.

Animals↗

PubMed1994

The UV response involving the Ras signaling pathway and AP-1 transcription factors is conserved between yeast and mammals.

UV irradiation of mammalian cells activates AP-1 through a Ras-dependent pathway, independently of DNA damage. We show that the yeast S. cerevisiae has a remarkably similar UV response involving the AP-1 factor Gcn4, which is distinct from the DNA damage response. Transcriptional activation of HIS3 and HIS4 by Gcn4 is triggered by UV irradiation in a Ras-dependent fashion. Moreover, resistance of yeast to UV irradiation is correlated with the level of Ras activity and Gcn4 function. Like mammalian cells in which activated Ras leads to increased c-Jun synthesis and phosphorylation, the effects in yeast involve increased translation of GCN4 mRNA and a posttranslational event. However, this effect on GCN4 translation is different from the response to amino acid or purine starvation. Therefore, a UV signaling pathway involving Ras and AP-1 is an ancient and universal mechanism involved in protection against damage to cellular components other than DNA.

Amitrole↗

PubMed1994

JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.

The ultraviolet (UV) response of mammalian cells is characterized by a rapid and selective increase in gene expression mediated by AP-1 and NF-kappa B. The effect on AP-1 transcriptional activity results, in part, from enhanced phosphorylation of the c-Jun NH2-terminal activation domain. Here, we describe the molecular cloning and characterization of JNK1, a distant relative of the MAP kinase group that is activated by dual phosphorylation at Thr and Tyr during the UV response. Significantly, Ha-Ras partially activates JNK1 and potentiates the activation caused by UV. JNK1 binds to the c-Jun transactivation domain and phosphorylates it on Ser-63 and Ser-73. Thus, JNK1 is a component of a novel signal transduction pathway that is activated by oncoproteins and UV irradiation. These properties indicate that JNK1 activation may play an important role in tumor promotion.

Amino Acid Sequence↗

PubMed1994

Signal transduction from the cell surface to the nucleus through the phosphorylation of transcription factors.

The activation of several transcription factors in response to extracellular stimuli is fairly well understood. In all cases, activation is mediated through phosphorylation by signal responsive protein kinases. The specificity of transcription factor phosphorylation is likely to be ensured by physical interactions between the protein kinases and their substrates. Distinct biological responses are likely to be mediated through activation of different constellations of protein kinases, resulting in phosphorylation of different transcription factors.

Animals↗

PubMed1994

Isolation of a cDNA encoding a metal response element binding protein using a novel expression cloning procedure: the one hybrid system.

A new method for isolation of cDNA clones encoding sequence-specific DNA-binding proteins is described. This method, the one-hybrid system, is based on the use of reporter genes whose transcription can be activated through synthetic cis elements recognized by the sought-after DNA-binding protein. These reporter genes are used for in vivo screening of a library of cDNAs fused to a DNA fragment encoding the GAL4 activation domain. cDNA clones expressing the appropriate fusion proteins lead to activation of these reporter genes in transformed yeast cells. We have used this approach to isolate a mammalian cDNA clone encoding a sequence-specific DNA-binding protein that recognizes the metal response elements (MREs) of the metallothionein (MT) genes. The protein encoded by this cDNA, M96, shows similarity to the trithorax proteins. Expression of a functional DNA-binding form of M96 requires Zn2+ ions. The recombinant protein binds to several different MREs but fails to recognize nonfunctional mutant MREs. M96 may be involved in the activation of MT genes in response to heavy-metal ions.

Amino Acid Sequence↗

PubMed1994

c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases.

c-Jun transcriptional activity is stimulated by phosphorylation at two N-terminal sites: Ser-63 and -73. Phosphorylation of these sites is enhanced in response to a variety of extracellular stimuli, including growth factors, cytokines, and UV irradiation. New members of the mitogen-activated protein (MAP) kinase group of signal-transducing enzymes, termed JNKs, bind to the activation domain of c-Jun and specifically phosphorylate these sites. However, the N-terminal sites of c-Jun were also suggested to be phosphorylated by two other MAP kinases, ERK1 and ERK2. Despite these reports, we find that unlike the JNKs, ERK1 and ERK2 do not phosphorylate the N-terminal sites of c-Jun in vitro; instead they phosphorylate an inhibitory C-terminal site. Furthermore, the phosphorylation of c-Jun in vivo at the N-terminal sites correlates with activation of the JNKs but not the ERKs. The ERKs are probably involved in the induction of c-fos expression and thereby contribute to the stimulation of AP-1 activity. Our study suggests that two different branches of the MAP kinase group are involved in the stimulation of AP-1 activity through two different mechanisms.

Animals↗

PubMed1994

BCL3 encodes a nuclear protein which can alter the subcellular location of NF-kappa B proteins.

BCL3 is a candidate proto-oncogene involved in the recurring translocation t(14;19) found in some patients with chronic lymphocytic leukemia. BCL3 protein acts as an I kappa B in that it can specifically inhibit the DNA binding of NF-kappa B factors. Here, we demonstrate that BCL3 is predominantly a nuclear protein and provide evidence that its N terminus is necessary to direct the protein into the nucleus. In contrast to I kappa B alpha (MAD3), BCL3 does not cause NF-kappa B p50 to be retained in the cytoplasm; instead, in cotransfection assays, it alters the subnuclear localization of p50. The two proteins colocalize, suggesting that they interact in vivo. Further immunofluorescence experiments showed that a mutant p50, lacking a nuclear localization signal and restricted to the cytoplasm, is brought into the nucleus in the presence of BCL3. Correspondingly, a wild-type p50 directs into the nucleus a truncated BCL3, which, when transfected alone, is found in the cytoplasm. We tested whether BCL3 could overcome the cytoplasmic retention of p50 by I kappa B alpha. Results from triple cotransfection experiments with BCL3, I kappa B alpha, and p50 implied that BCL3 can successfully compete with I kappa B alpha and bring p50 into the nucleus; thus, localization of NF-kappa B factors may be affected by differential expression of I kappa B proteins. These novel properties of BCL3 protein further establish BCL3 as a distinctive member of the I kappa B family.

Animals↗

PubMed1994

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

Yeast strains in which the Ras-cyclic AMP (cAMP) pathway is constitutively active are sensitive to heat shock, whereas mutants in which the activity of this pathway is low are hyperresistant to heat shock. To determine the molecular basis for these differences, we examined the transcriptional induction of heat shock genes in various yeast strains. Activation of heat shock genes was attenuated in the strains in which the Ras-cAMP pathway is constitutively active. In contrast, in a strain deficient in cAMP production, several heat shock genes were induced by removal of cAMP from the medium. These results indicate that the Ras-cAMP pathway affects the induction of heat shock genes. In all of the mutants, heat shock transcription factor expression and activity were identical to those in wild-type cells. The response to heat shock in Ha-ras-transformed rat fibroblasts was also studied. While no induction of Hsp68 was observed in Ha-ras-transformed cells, proper regulation of heat shock transcription factor was found. Therefore, in mammals, as in Saccharomyces cerevisiae, the Ras pathway controls the transcription of heat shock genes via a mechanism not involving the heat shock transcription factor.

3T3 Cells↗

PubMed1994

Protein kinase A and C site-specific phosphorylations of LAP (NF-IL6) modulate its binding affinity to DNA recognition elements.

LAP (NF-IL6 or C/EBP beta), is a liver transcriptional activator protein that confers liver-specific gene expression. Because LAP has a characteristic phosphoacceptor sequence for cAMP-dependent protein kinase A (PKA), we tested if in vitro phosphorylation of LAP by PKA modulates its interaction with specific DNA sequences. The major PKA phosphorylation site of LAP was identified as Ser105, which is a predicted PKA site. As expected, this PKA phosphorylation site disappears after mutation of Ser105 to Ala. Kinetic studies with LAP and LAP Asp105 (which mimics a phosphoserine residue) demonstrated that phosphorylation of Ser105 itself has no effect on DNA binding. Phosphorylation of other sites by PKA, identified in the region between Ser173 and Ser223 and at Ser240, by analysis of truncated and mutated LAP peptides, resulted in an inhibition of DNA binding. LAP was also phosphorylated by purified protein kinase C in vitro, and the major phosphoacceptor was shown to be Ser240 within the DNA-binding domain of LAP. Phosphorylation of LAP at this residue or introduction of a Ser240 to Asp mutation resulted in marked decrease in its binding to DNA. These results suggest that site-specific phosphorylations of LAP modulate transactivation of its target genes.

Albumins↗

PubMed1994

Negative transcriptional regulation by nuclear receptors.

Steroid and thyroid hormones, and vitamins A and D bind to nuclear receptors, which act as ligand-modulated transcription factors. In many cases, ligand-activated nuclear receptor binds to positively acting hormone response elements (p-HREs) to induce gene transcription. However, ligand-activated receptors also repress transcription of specific genes and several mechanisms that account for negative regulation have recently emerged. One major form of negative regulation is based on transcriptional interference between nuclear receptors and other transcription factors, such as AP-1. In this case, the liganded receptor prevents AP-1 or other positively acting transcription factors from fruitful interaction with the transcription initiation complex. A second form of negative regulation is based on binding of nuclear receptors to specialized negative HREs (n-HREs). Binding of unliganded receptor to such an element results in constitutive activation, which is terminated by the binding of ligand. While transcriptional interference with AP-1 has been described for many members of the nuclear receptor family, negative regulation through n-HREs so far has been shown only for one of the thyroid hormone receptors. However, this type of negative regulation is likely to be widespread.

Animals↗

PubMed1993

A novel cis element mediating ligand-independent activation by c-ErbA: implications for hormonal regulation.

A novel type of hormone-responsive element (HRE) is described. Unlike classical HREs, this element, RSV-T3RE (found in Rous sarcoma virus-long terminal repeat), mediates strong activation by the c-ErbA alpha thyroid hormone (T3) receptor in the absence of T3, and addition of T3 reverses this response. Whereas both c-ErbA alpha and v-ErbA are potent ligand-independent activators through the RSV-T3RE, c-ErbA beta is not. The RSV-T3RE is recognized and activated by either c-ErbA alpha homodimers or c-ErbA alpha/retinoid X receptor (RXR) heterodimers. Ligand-independent activation by c-ErbA alpha depends on a unique N-terminal activation domain, while the C-terminal activation domain is not absolutely required. Ligand-dependent activation, on the other hand, requires the C-terminal but not the N-terminal activation domain. Upon binding to the RSV-T3RE, c-ErbA alpha assumes a different conformation than when bound to a classical T3RE. c-ErbA alpha is therefore capable of selective deployment of activation domains, dictated both by the HRE with which it interacts and by T3 binding.

Alkylation↗

PubMed1993

NF-kappa B activation by ultraviolet light not dependent on a nuclear signal.

Exposure of mammalian cells to radiation triggers the ultraviolet (UV) response, which includes activation of activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B). This was postulated to occur by induction of a nuclear signaling cascade by damaged DNA. Recently, induction of AP-1 by UV was shown to be mediated by a pathway involving Src tyrosine kinases and the Ha-Ras small guanosine triphosphate-binding protein, proteins located at the plasma membrane. It is demonstrated here that the same pathway mediates induction of NF-kappa B by UV. Because inactive NF-kappa B is stored in the cytosol, analysis of its activation directly tests the involvement of a nuclear-initiated signaling cascade. Enucleated cells are fully responsive to UV both in NF-kappa B induction and in activation of another key signaling event. Therefore, the UV response does not require a signal generated in the nucleus and is likely to be initiated at or near the plasma membrane.

Alleles↗

PubMed1993

Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain.

One of the members of the bZIP family of transcriptional activators is NF-IL6/LAP (IL-6 DBP, C/EBP beta, CRP2). NF-IL6/LAP protein is highly expressed in liver nuclei, where it has been implicated as a master regulator of the acute-phase response, induced by interleukin-6 (IL-6) and other inflammatory mediators. Also, NF-IL6/LAP is involved in the activation of the IL-6 promoter in response to IL-1 and bacterial lipopolysaccharide. The control of NF-IL6/LAP expression and activity is complex and poorly understood. Under some conditions the NF-IL6/LAP gene is transcriptionally activated by IL-1 and lipopolysaccharide, whereas in other instances, its binding to cognate DNA sequences is enhanced by cytokines. Additionally, the ability of constitutively expressed NF-IL6/LAP to activate transcription is strongly augmented by IL-6, through an unknown signalling pathway. We now show that stimulation of the protein kinase C pathway increases the phosphorylation of Ser 105 within the activation domain of NF-IL6/LAP, and enhances its transcriptional efficacy.

Animals↗

PubMed1993

Various modes of gene regulation by nuclear receptors for steroid and thyroid hormones.

AP-1 is a transcriptional activator composed of homo- and heterodimers of Jun and Fos proteins. It is involved in activation of genes, such as collagenase, stromelysin, IL-2 and TGF beta 1, by tumour promoters, growth factors and cytokines. AP-1 activity is also elevated in response to transforming oncogenes and is required for cell proliferation. AP-1 activity is subject to complex regulation both transcriptionally and post-transcriptionally. Transcriptional control of jun and fos gene expression determines the amount and composition of the AP-1 complex. The jun and fos genes are regulated both positively and negatively and are highly inducible in response to extracellular stimuli. Post translational control is also important. Both cJun and cFos are subject to regulated phosphorylation. In the case of cJun, phosphorylation of sites near the DNA-binding domain inhibits DNA-binding, while dephosphorylation reverses this inhibition. Phosphorylation of cJun on sites within the N-terminal activation domain increases its ability to activate transcription. The protein kinase phosphorylating these sites is stimulated by cytokines and growth factors. Another mechanism modulating AP-1 activity is transcriptional interference by members of the nuclear receptor family and is relevant for the pathophysiology of rheumatoid arthritis (RA). In RA, chronic inflammation leads to increased AP-1 activity in T cells,macrophages and synoviocytes as a response to secretion of cytokines such as IL-1 and TNF alpha. While the IL-2 gene plays a major role in T cell activation, another AP-1 target gene encodes an enzyme, collagenase, responsible for destruction of bone and tendon.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗

PubMed1993

Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain.

The activity of c-Jun is regulated by phosphorylation. Various stimuli including transforming oncogenes and UV light, induce phosphorylation of serines 63 and 73 in the amino-terminal activation domain of c-Jun and thereby potentiate its trans-activation function. We identified a serine/threonine kinase whose activity is stimulated by the same signals that stimulate the amino-terminal phosphorylation of c-Jun. This novel c-Jun amino-terminal kinase (JNK), whose major form is 46 kD, binds to a specific region within the c-Jun trans-activation domain and phosphorylates serines 63 and 73. Phosphorylation results in dissociation of the c-Jun-JNK complex. Mutations that disrupt the kinase-binding site attenuate the response of c-Jun to Ha-Ras and UV. Therefore the binding of JNK to c-Jun is of regulatory importance and suggests a mechanism through which protein kinase cascades can specifically modulate the activity of distinct nuclear targets.

3T3 Cells↗

PubMed1993

JunB differs from c-Jun in its DNA-binding and dimerization domains, and represses c-Jun by formation of inactive heterodimers.

JunB differs considerably from c-Jun in its ability to activate AP-1-responsive genes and induce oncogenic transformation. We demonstrate that the decreased ability of JunB to activate gene expression is the result of a small number of amino acid changes between its DNA-binding and dimerization motifs and the corresponding regions of c-Jun. These changes lead to a 10-fold decrease in the DNA-binding activity of JunB. JunB can be converted into a c-Jun-like activator by substituting four amino acids in its DNA-binding and dimerization motifs with the corresponding c-Jun sequences. JunB can also attenuate trans-activation by c-Jun, an activity mediated by its leucine zipper. This ability depends on two glycine residues that decrease the stability of the JunB leucine zipper, resulting in decreased homodimerization and increased heterodimerization. These results illustrate how small changes in primary structure, including chemically conservative changes, can result in functional divergence of two highly related transcriptional regulators.

Amino Acid Sequence↗

PubMed1993

GHF-1-promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice.

During pituitary development, the homeo domain protein GHF-1 is required for generation of somatotropes and lactotropes and for growth hormone (GH) and prolactin (PRL) gene expression. GHF-1 mRNA is detectable several days before the emergence of GH- or PRL-expressing cells, suggesting the existence of a somatotropic progenitor cell in which GHF-1 transcription is first activated. We have immortalized this cell type by using the GHF-1 regulatory region to target SV40 T-antigen (Tag) tumorigenesis in transgenic mice. The GHF-Tag transgene caused developmental entrapment of somatotropic progenitor cells that express GHF-1 but not GH or PRL, resulting in dwarfism. Immortalized cell lines derived from a transgenic pituitary tumor maintain the characteristics of the somato/lactotropic progenitor in that they express GHF-1 mRNA and protein yet fail to activate GH or PRL transcription. Using these cells, we identified an enhancer that activates GHF-1 transcription at this early stage of development yet is inactive in cells representing later developmental stages of the somatotropic lineage or in other cell types. These experiments not only demonstrate the potential for immortalization of developmental progenitor cells using the regulatory regions from cell type-specific transcription factor genes but illustrate the power of such model systems in the study of developmental control.

Animals↗

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↗