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Biomedical subjects

M Karin

Publications and source records attributed to M Karin.

At least 163 records · Page 9Linked to original sources
PubMed1993

Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements.

The involvement of serine/threonine protein phosphatases in signaling pathways which modulate the activity of the transcription factor AP-1 was examined. Purified protein phosphatase types 1 (PP1) and 2A (PP2A) were microinjected into cell lines containing stably transfected lacZ marker genes under the control of an enhancer recognized by AP-1. Microinjection of PP2A potentiated serum-stimulated beta-galactosidase expression from the AP-1-regulated promoter. Similarly, transient expression of the PP2A catalytic subunit with c-Jun resulted in a synergistic transactivation of an AP-1-regulated reporter gene. PP2A, but not PP1, potentiated serum-induced c-Jun expression, which has been previously shown to be autoregulated by AP-1 itself. Consistent with these results, PP2A dephosphorylated c-Jun on negative regulatory sites in vitro, suggesting one possible direct mechanism for the effects of PP2A on AP-1 activity. Microinjection of PP2A had no effect on cyclic AMP (cAMP)-induced expression of a reporter gene containing a cAMP-regulated promoter, while PP1 injection abolished cAMP-induced gene expression. Taken together, these results suggest a specific role for PP2A in signal transduction pathways that regulate AP-1 activity and c-Jun expression.

Animals↗
PubMed1993

A conserved C-terminal sequence that is deleted in v-ErbA is essential for the biological activities of c-ErbA (the thyroid hormone receptor).

The thyroid hormone (T3) receptor type alpha, the c-ErbA alpha proto-oncoprotein, stimulates transcription of T3-dependent promoters, interferes with AP-1 activity, and induces erythroid differentiation in a ligand-dependent manner. The v-ErbA oncoprotein does not bind hormone and has lost all of these activities. Using c-ErbA/v-ErbA chimeras, we found that a deletion of 9 amino acids, conserved among many members of the nuclear receptor superfamily, which are located at the extreme carboxy terminus of c-ErbA alpha is responsible for loss of both transactivation and transcriptional interference activities. Single, double, and triple amino acid substitutions within this region completely abolished T3-dependent transcriptional activation, interference with AP-1 activity, and decreased T3 binding by c-ErbA alpha. However, the lower T3 binding by these mutants does not fully account for the loss of transactivation and transcriptional interference, since a c-ErbA/v-ErbA chimera which was similarly reduced in T3 binding activity has retained both of these functions. Deletion of homologous residues in the retinoic acid receptor alpha (RAR alpha) resulted in a similar loss of transactivation and transcriptional interference activities. The ability of c-ErbA alpha to induce differentiation of transformed erythroblasts is also impaired by all of the mutations introduced into the conserved carboxy-terminal sequence. We conclude that this 9-amino-acid conserved region is essential for normal biological function of c-ErbA alpha and RAR alpha and possibly other T3 and RA receptors.

Amino Acid Sequence↗
PubMed1993

Activation of activating protein 1 during hepatic acute phase response.

During an acute phase response following inflammatory stimuli, specific changes occur in the synthesis and secretion of many hepatic proteins. Because the expression of differentiated function requires the coordinated regulation of many genes, we investigated the activity of general and tissue-specific transcription factors using a rat liver model of the acute phase response induced by Freund's adjuvant. Nuclear extracts and RNAs were prepared throughout a 48-h posttreatment period. Mobility shift assays revealed increased binding activity by nuclear factor-kappa B, interleukin-6 (IL-6) responsive element binding protein, and activating protein 1 (AP-1). Two AP-1 complexes were induced during the acute phase response, and correlation between their presence and transcription activity was demonstrated by transfection studies. Elevated binding activity of AP-1 also correlated with elevated levels of c-jun, junD, junB, and c-fos mRNAs. Western blots showed elevated hepatic levels of c-Jun but not c-Fos proteins during the acute phase response. In addition, IL-6, tumor necrosis factor-alpha, and IL-1 beta, cytokine regulators of the acute phase response, stimulated expression of an AP-1 responsive reporter gene introduced by DNA-mediated transfection into adult rat hepatocytes in primary culture. These findings demonstrate the complexity of AP-1 hepatic transcription factor responses to humoral regulators with direct hepatocellular effects.

Acute-Phase Reaction↗
PubMed1992

The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases.

Exposure of mammalian cells to DNA-damaging agents induces the ultraviolet (UV) response, involving transcription factor AP-1, composed of Jun and Fos proteins. We investigated the mechanism by which UV irradiation induces the c-jun gene. The earliest detectable step was activation of Src tyrosine kinases, followed by activation of Ha-Ras and Raf-1. The response to UV was blocked by tyrosine kinase inhibitors and dominant negative mutants of v-src, Ha-ras, and raf-1. This signaling cascade leads to increased phosphorylation of c-Jun on two serine residues that potentiate its activity. These results strongly suggest that the UV response is initiated at or near the plasma membrane rather than the nucleus. The response may be elicited by oxidative stress, because it is inhibited by elevation of intracellular glutathione. Using tyrosine kinase inhibitors, we demonstrate that the UV response has a protective function.

Cell Membrane↗
PubMed1992

Construction and expression of a monomeric c-Jun protein that binds and activates transcription of AP-1-responsive genes.

c-Jun is a typical member of the bZIP (basic zipper) family of dimeric transcriptional activators. These proteins contain a basic region responsible for DNA sequence recognition and a leucine zipper that mediates dimerization. bZIP proteins regulate a large number of important physiological functions and, therefore, present an interesting target for molecular interference and mimicry. As a step toward the development of peptide and nonpeptide analogs of such proteins, we constructed a derivative of c-Jun that binds DNA as a monomer. This construction was done by connecting a second basic region to the natural basic region of c-Jun by means of a short peptide loop. Although the polypeptide backbone of the second basic region has an inverted polarity relative to that of the natural basic region, the monomeric c-Jun protein binds DNA with reasonably high affinity and indistinguishable specificity from the wild-type, dimeric c-Jun protein. Furthermore, the monomeric c-Jun protein can activate transcription in vivo. These findings indicate that the polypeptide backbone of the basic region contributes little to sequence recognition and that the leucine zipper is not directly involved in transcriptional activation.

Amino Acid Sequence↗
PubMed1992

Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity.

c-Jun, a major component of the inducible transcription factor AP-1, is a phosphoprotein. In nonstimulated fibroblasts and epithelial cells, c-Jun is phosphorylated on a cluster of two to three sites abutting its DNA-binding domain. Phosphorylation of these sites inhibits DNA binding, and their dephosphorylation correlates with increased AP-1 activity. We show that two of these sites, Thr-231 and Ser-249, are phosphorylated by casein kinase II (CKII). Substitution of the third site, Ser-243, by Phe interferes with phosphorylation of the inhibitory sites in vivo and by purified CKII in vitro. Microinjection into living cells of synthetic peptides that are specific competitive substrates or inhibitors of CKII results in induction of AP-1 activity and c-Jun expression. Microinjection of CKII suppresses induction of AP-1 by either phorbol ester or an inhibitory peptide. These results suggest that one of the roles of CKII, a major nuclear protein kinase with no known functions, is to attenuate AP-1 activity through phosphorylation of c-Jun.

Amino Acid Sequence↗
PubMed1992

Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB.

We have examined the mechanism by which the transcriptional activity of the cAMP-responsive factor CREB is attenuated following induction with forskolin. Metabolic labeling studies reveal that, after an initial burst of phosphorylation in response to cAMP, CREB is dephosphorylated and transcription of the cAMP-responsive somatostatin gene is correspondingly reduced. The phosphatase inhibitor 1 protein and okadaic acid both prevented the dephosphorylation of CREB at Ser-133 in PC12 cells and also augmented the transcriptional response to cAMP. Of the four Ser/Thr phosphatases described to date, only PP-1 appears to be similarly inhibited by these agents. As PP-1 specifically dephosphorylates CREB at Ser-133 and inhibits cAMP-dependent transcription, we propose that this phosphatase is the major regulator of CREB activity in cAMP-responsive cells.

Cyclic AMP↗
PubMed1992

Differential splicing of the GHF1 primary transcript gives rise to two functionally distinct homeodomain proteins.

The POU domain protein GHF-1 has a critical role in generation, proliferation and phenotypic expression of three pituitary cell types. GHF-1 functions in part by binding to and transactivating the promoters of both the growth hormone (GH) and prolactin (PRL) genes and that of the GHF1 gene itself. We describe a naturally occurring isoform of GHF-1, GHF-2, in which an additional 26 amino acids are inserted into the activation domain of the protein as a result of alternative splicing. GHF-2 retains the DNA binding activity of GHF-1 and can activate the GH promoter but has lost the ability to activate the PRL and GHF1 promoters. These results suggest that GHF-2 may function in differential target gene activation during differentiation of the somatotrophic lineage. Both GHF-1 and GHF-2 transcripts are specifically expressed in the anterior pituitary. Analysis of the genomic GHF1 gene shows that most of the distinct functional domains of GHF-1 (and GHF-2) are encoded by separate exons. Gene segment duplication and exon shuffling may have contributed to the evolution of this cell type-specific transcriptional regulatory gene.

Amino Acid Sequence↗
PubMed1992

Control of transcription factors by signal transduction pathways: the beginning of the end.

Signal transduction pathways regulate gene expression by modulating the activity of nuclear transcription factors. The mechanisms that control the activity of two groups of sequence-specific transcription factors, the AP-1 and CREB/ATF proteins, are described. These factors serve as a paradigm explaining the transfer of regulatory information from the cell surface to the nucleus.

Activating Transcription Factors↗
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↗
PubMed1992

Signal transduction from cell surface to nucleus in development and disease.

Recent studies indicate that extracellular signals affect cell proliferation and differentiation by modulating transcription factor activity via protein phosphorylation cascades. This review discusses the basic outline of the eukaryotic signal transduction systems used to transmit information from the cell surface to the transcriptional machinery in the nucleus. Several examples that illustrate how these pathways control cell proliferation, differentiation and development are discussed.

Animals↗
PubMed1992

Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression.

Myogenin and MyoD belong to a family of muscle-specific helix-loop-helix (HLH) proteins that have the potential to activate muscle-specific genes in nonmyogenic cells. Peptide growth factors can block the ability of myogenin and MyoD to activate their target genes. Here, we show that the growth factor-inducible proto-oncogenes c-fos, c-jun, and junB mimic the effects of exogenous growth factors and suppress trans-activation of the muscle creatine kinase (MCK) enhancer by myogenin and MyoD. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, is an inefficient inhibitor of the trans-activating capacity of myogenin and MyoD. Transcriptional repression by Fos and Jun is specific to myogenic HLH proteins and is not observed with the widely expressed HLH protein E47, which recognizes the same DNA sequence. Repression of the MCK enhancer by Fos and Jun is targeted at the myogenin and MyoD DNA recognition sequence and can be mediated by the amino terminus of c-Jun. Comparison of several myogenin mutants for their responsiveness to Fos and Jun shows that repression is directed at the basic-HLH region. These results indicate that members of the Jun family can be distinguished on the basis of their effects on muscle-specific transcription and suggest there is cross talk between transcription factors that control myogenesis and those involved in cell proliferation.

Animals↗
PubMed1992

Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation.

Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.

Blotting, Northern↗
PubMed1992

Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73.

In resting cells, c-Jun is phosphorylated on five sites. Three of these sites reside next to its DNA binding domain and negatively regulate DNA binding. In response to expression of oncogenic Ha-Ras, phosphorylation of these sites decreases, while phosphorylation of two other sites within c-Jun's activation domain is greatly enhanced. Phosphorylation of these residues, serines 63 and 73, stimulates the transactivation function of c-Jun and is required for oncogenic cooperation with Ha-Ras. We now show that the same changes in c-Jun phosphorylation are elicited by a variety of transforming oncoproteins with distinct biochemical activities. These oncoproteins, v-Sis, v-Src, Ha-Ras, and Raf-1, participate in a signal transduction pathway that leads to increased phosphorylation of serines 63 and 73 on c-Jun. While oncogenic Ha-Ras is a constitutive stimulator of c-Jun activity and phosphorylation, the normal c-Ha-Ras protein is a serum-dependent modulator of c-Jun's activity. c-Jun is therefore a downstream target for a phosphorylation cascade involved in cell proliferation and transformation.

3T3 Cells↗