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1.
Dis Model Mech ; 9(9): 941-52, 2016 09 01.
Article in English | MEDLINE | ID: mdl-27483357

ABSTRACT

The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALK(F1174L)/MYCN Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.


Subject(s)
Lactams, Macrocyclic/therapeutic use , N-Myc Proto-Oncogene Protein/antagonists & inhibitors , Neuroblastoma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Aminopyridines , Anaplastic Lymphoma Kinase , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Clinical Trials as Topic , Crizotinib , Lactams , Lactams, Macrocyclic/pharmacology , Mice, Inbred BALB C , Mice, Nude , Mutation/genetics , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/pathology , PC12 Cells , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyridines/pharmacology , Pyridines/therapeutic use , Rats , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Xenograft Model Antitumor Assays
2.
J Wound Care ; 10(3): 65-9, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11924353

ABSTRACT

This paper reports on the findings from the first part of a three-phase project that aimed to identify nursing strategies used in the management of malignant wounds. The difficulties relating to the management of these wounds and the significant physical and psychological impact on patients are described. A quantitative postal survey aiming to identify the types of dressing used in the care of malignant wounds was sent to specialist nurses working in oncology and palliative care in New South Wales, Australia. Additional qualitative data showed that the major issues were coping with odour and meeting the financial costs of the dressing products. The long list of products compiled for this research demonstrates the complexities nurses face when selecting dressings for the management of malignant wounds. Furthermore, there are no clear recommendations to guide nursing practice. This study provides a framework for subsequent phases of the project and will hopefully lead to the development of guidelines for best practice in malignant wound management.


Subject(s)
Bandages , Nursing Assessment , Skin Care/methods , Wounds and Injuries/nursing , Wounds and Injuries/pathology , Community Health Nursing/methods , Female , Health Care Surveys , Humans , Male , New South Wales , Palliative Care/methods , Risk Assessment , Surveys and Questionnaires , Wound Healing/physiology
4.
Curr Opin Genet Dev ; 7(2): 281-7, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9115418

ABSTRACT

Aging is a near universal process, yet the molecular mechanisms that underlie cellular senescence have remained elusive. Recent progress in determining the roles of various genetic influences in controlling the rate of cellular aging has made this an exciting time in aging research. Genetic screens designed to isolate long-lived mutants in Saccharomyces cerevisiae and Caenorhabditis elegans have implicated factors involved in transcriptional silencing and the dauer pathway in the control of aging. The gene responsible for Werner's syndrome, a disease with symptoms of premature aging, was isolated and found to be a member of the RecQ subfamily of DNA helicases. The regulation of telomere length and its role in senescence and cellular immortalization has been found to be more complex than expected. In C. elegans, mutations have been isolated in maternal-effect genes that presumably control its biological clocks and can dramatically extend its lifespan. Indeed, aging research within the past year has implicated a variety of mechanisms ranging from the control of gene expression, stress resistance, and DNA metabolism to the overall 'rate of living'.


Subject(s)
Cellular Senescence , Animals , Caenorhabditis elegans , Humans , Mammals , Saccharomyces cerevisiae , Stress, Physiological
5.
Cell ; 84(4): 633-42, 1996 Feb 23.
Article in English | MEDLINE | ID: mdl-8598049

ABSTRACT

We show that sterility is an aging-specific phenotype in S. cerevisiae and, by genetic and physical means, demonstrate that this phenotype results from a loss of silencing in most old cells by the SIR complex at the HM loci. This loss of silencing is specific because transcription of genes, such as ME14 and DCM1, normally induced by sporulation, is not observed, while transcription of HMRa is observed. These findings pinpoint the molecular cause of an aging-specific phenotype in yeast. Further, they provide direct evidence for a breakdown of silencing in old cells, as predicted from earlier findings that SIR4 is a determinant of life span in this organism.


Subject(s)
Saccharomyces cerevisiae/genetics , Transcription, Genetic/physiology , Base Sequence , Cell Separation , Cellular Senescence/genetics , Gene Expression Regulation , Immunomagnetic Separation , Molecular Sequence Data , Phenotype , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/growth & development
6.
EMBO J ; 13(24): 6006-10, 1994 Dec 15.
Article in English | MEDLINE | ID: mdl-7813438

ABSTRACT

Protein phosphorylation is commonly used to modulate transcription factor activity. However, all existing genetic evidence for stimulation of transcription factor activity by phosphorylation rests on loss-of-function mutations. To demonstrate conclusively that phosphorylation of a transcription factor potentiates its transactivation potential in vivo, we constructed a c-Jun mutant that is phosphorylated by the cAMP-sensitive protein kinase A (PKA) instead of the UV- and Ras-responsive protein kinase JNK. The transcriptional activity of this mutant is enhanced by PKA, but not by JNK activation. These results provide a positive and conclusive proof that phosphorylation of c-Jun on a critical site (Ser73) located in its activation domain is directly responsible for enhancing its transactivation function.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction , Transcription, Genetic , Transcriptional Activation , Amino Acid Sequence , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , JNK Mitogen-Activated Protein Kinases , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Proto-Oncogene Proteins c-jun/genetics , Recombinant Proteins/metabolism , Substrate Specificity
7.
Mol Cell Biol ; 14(10): 6683-8, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7935387

ABSTRACT

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.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Enzymologic , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Animals , Calcium-Calmodulin-Dependent Protein Kinases/classification , Cells, Cultured , Enzyme Activation , Humans , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 1 , Phosphorylation , Signal Transduction , Substrate Specificity
8.
Nature ; 370(6486): 226-9, 1994 Jul 21.
Article in English | MEDLINE | ID: mdl-8028671

ABSTRACT

A number of signalling pathways stimulate transcription of target genes through nuclear factors whose activities are primarily regulated by phosphorylation. Cyclic AMP regulates the expression of numerous genes, for example, through the protein kinase-A (PKA)-mediated phosphorylation of transcription factor CREB at Ser 133. Although phosphorylation may stimulate transcriptional activators by modulating their nuclear transport or DNA-binding affinity, CREB belongs to a class of proteins whose phosphorylation appears specifically to enhance their trans-activation potential. Recent work describing a phospho-CREB binding protein (CBP) which interacts specifically with the CREB trans-activation domain prompted us to examine whether CBP is necessary for cAMP regulated transcription. We report here that microinjection of an anti-CBP antiserum into fibroblasts can inhibit transcription from a cAMP responsive promoter. Surprisingly, CBP also cooperates with upstream activators such as c-Jun, which are involved in mitogen responsive transcription. We propose that CBP is recruited to the promoter through interaction with certain phosphorylated factors, and that CBP may thus play a critical role in the transmission of inductive signals from cell surface receptor to the transcriptional apparatus.


Subject(s)
Cyclic AMP/metabolism , Gene Expression Regulation , Mitogens/metabolism , Nuclear Proteins/metabolism , Signal Transduction , Trans-Activators , Transcription Factors/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Blotting, Western , CREB-Binding Protein , Cyclic AMP Response Element-Binding Protein/metabolism , Genes, Reporter , HeLa Cells , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/immunology , Phosphorylation , Promoter Regions, Genetic , Proto-Oncogene Proteins c-jun/genetics , Rabbits , Serine/metabolism , Transcription Factors/immunology , Transcription, Genetic
9.
Genes Dev ; 7(11): 2135-48, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8224842

ABSTRACT

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.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Ultraviolet Rays , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line , Cell Line, Transformed , Genes, ras , Glutathione Transferase/biosynthesis , Glutathione Transferase/metabolism , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases , Mice , Molecular Weight , Mutagenesis, Site-Directed , Phosphorylation , Protein Serine-Threonine Kinases/radiation effects , Proto-Oncogene Proteins c-jun/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Serine , Transfection
11.
Cell ; 71(7): 1081-91, 1992 Dec 24.
Article in English | MEDLINE | ID: mdl-1473146

ABSTRACT

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.


Subject(s)
Genes, jun/radiation effects , Genes, ras/radiation effects , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Cell Membrane/metabolism , Enzyme Activation , Genistein , HeLa Cells , Humans , Isoflavones/pharmacology , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Ultraviolet Rays
12.
Trends Biochem Sci ; 17(10): 418-22, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1455510

ABSTRACT

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.


Subject(s)
Blood Proteins/physiology , Proto-Oncogene Proteins c-jun/physiology , Signal Transduction/physiology , Transcription Factors/physiology , Activating Transcription Factors , Animals , Gene Expression Regulation/physiology , Humans , Phosphorylation
13.
Cell ; 70(5): 777-89, 1992 Sep 04.
Article in English | MEDLINE | ID: mdl-1516134

ABSTRACT

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.


Subject(s)
Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-jun/antagonists & inhibitors , Amino Acid Sequence , Base Sequence , Casein Kinase II , Cell Line , Down-Regulation , Enzyme Induction/drug effects , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/pharmacology , Phosphorylation , Protein Serine-Threonine Kinases/biosynthesis , Proto-Oncogene Proteins c-jun/genetics , Trypsin
14.
Mol Cell Biol ; 12(8): 3507-13, 1992 Aug.
Article in English | MEDLINE | ID: mdl-1630458

ABSTRACT

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.


Subject(s)
Oncogene Proteins, Viral/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction , 3T3 Cells , Amino Acid Sequence , Animals , Cell Line , Cell Transformation, Neoplastic , Electrophoresis, Polyacrylamide Gel , Methionine/metabolism , Mice , Mutation , Oncogene Protein p21(ras)/metabolism , Oncogene Protein pp60(v-src)/metabolism , Oncogene Proteins v-sis , Phosphates/metabolism , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/isolation & purification , Proto-Oncogene Proteins c-raf , Retroviridae Proteins, Oncogenic/metabolism , Serine , Transcriptional Activation
15.
Nature ; 354(6353): 494-6, 1991 Dec 12.
Article in English | MEDLINE | ID: mdl-1749429

ABSTRACT

Recent advances indicate a link between tumour promoters, transformation, and AP-1 activity. Protein kinase C activation increases AP-1 DNA-binding activity independently of new protein synthesis. AP-1 is also stimulated by transforming oncoproteins and growth factors. These proteins are thought to participate in a signalling cascade affecting the nuclear AP-1 complex composed of the Jun and Fos proteins. Because c-Jun is the most potent transactivator in the AP-1 complex and is elevated in Ha-ras-transformed cells, in which c-Fos is downregulated, we focused on it as a potential target. c-Jun could convert input from an oncogenic signalling cascade into changes in gene expression. Indeed, transformation of rat embryo fibroblasts by c-Jun requires an intact transcriptional activation domain and cooperation with oncogenic Ha-ras. Expression of oncogenic Ha-ras augments transactivation by c-Jun and stimulates its phosphorylation. Here we describe the mapping of the Ha-ras-responsive phosphorylation sites to serines 63 and 73 of c-Jun. Site-directed mutagenesis indicates that phosphorylation of these serines is essential for stimulation of c-Jun activity and for cooperation with Ha-ras in ocogenic transformation.


Subject(s)
Cell Transformation, Neoplastic , Gene Expression Regulation, Neoplastic/physiology , Oncogene Protein p21(ras)/physiology , Proto-Oncogene Proteins c-jun/physiology , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Proto-Oncogene Proteins c-jun/metabolism , Rats , Serine/metabolism , Transcription, Genetic , Transcriptional Activation
16.
Nature ; 351(6322): 122-7, 1991 May 09.
Article in English | MEDLINE | ID: mdl-1903181

ABSTRACT

Ha-Ras augments c-Jun-mediated transactivation by potentiating the activity of the c-Jun activation domain. Ha-Ras also causes a corresponding increase in phosphorylation of specific sites in that part of the c-Jun protein. A Ha-Ras-induced protein kinase cascade resulting in hyperphosphorylation of the c-Jun activation domain could explain how these oncoproteins cooperate to transform rat embryo fibroblasts.


Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogenes/genetics , Transcription Factors/metabolism , Animals , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , DNA-Binding Proteins/genetics , Phosphorylation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fos , Proto-Oncogene Proteins c-jun , Proto-Oncogene Proteins p21(ras)/genetics , Transcription Factors/genetics , Transcriptional Activation , Transfection
17.
Cell ; 64(3): 573-84, 1991 Feb 08.
Article in English | MEDLINE | ID: mdl-1846781

ABSTRACT

In resting human epithelial and fibroblastic cells, c-Jun is phosphorylated on serine and threonine at five sites, three of which are phosphorylated in vitro by glycogen synthase kinase 3 (GSK-3). These three sites are nested within a single tryptic peptide located just upstream of the basic region of the c-Jun DNA-binding domain (residues 227-252). Activation of protein kinase C results in rapid, site-specific dephosphorylation of c-Jun at one or more of these three sites and is coincident with increased AP-1-binding activity. Phosphorylation of recombinant human c-Jun proteins in vitro by GSK-3 decreases their DNA-binding activity. Mutation of serine 243 to phenylalanine blocks phosphorylation of all three sites in vivo and increases the inherent trans-activation ability of c-Jun at least 10-fold. We propose that c-Jun is present in resting cells in a latent, phosphorylated form that can be activated by site-specific dephosphorylation in response to protein kinase C activation.


Subject(s)
DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Protein Kinase C/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinases , DNA Mutational Analysis , Enzyme Activation , Glycogen Synthase Kinases , HeLa Cells , Humans , In Vitro Techniques , Molecular Sequence Data , Oligonucleotides/chemistry , Phosphoproteins/metabolism , Phosphorylation , Phosphoserine/metabolism , Protein Binding , Protein Kinases/metabolism , Proto-Oncogene Proteins c-jun , Tetradecanoylphorbol Acetate/pharmacology
18.
Cell ; 62(6): 1205-15, 1990 Sep 21.
Article in English | MEDLINE | ID: mdl-2169352

ABSTRACT

Glucocorticoids are potent inhibitors of collagenase induction by phorbol esters and inflammatory mediators. The target for this negative effect is the AP-1 site within the collagenase promoter, which also mediates its induction. Negative regulation is due to repression of AP-1 activity by the glucocorticoid receptor (GCR). While the GCR is a potent inhibitor of AP-1 activity (Jun/Fos), both c-Jun and c-Fos are potent repressors of GCR activity. In vitro experiments using purified GCR and c-Jun proteins suggest that mutual repression is due to direct interaction between the two. Direct interaction between GCR and either c-Jun or c-Fos is demonstrated by cross-linking and coimmunoprecipitation. These findings reveal a cross talk between two major signal transduction systems used to control gene transcription in response to extracellular stimuli, and a novel mechanism for transcriptional repression.


Subject(s)
DNA-Binding Proteins/genetics , Dexamethasone/pharmacology , Microbial Collagenase/genetics , Proto-Oncogenes , Receptors, Glucocorticoid/physiology , Transcription Factors/genetics , Transcription, Genetic , Animals , Cell Line , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Enzymologic , HeLa Cells/drug effects , HeLa Cells/enzymology , Humans , Microbial Collagenase/biosynthesis , Plasmids , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fos , Proto-Oncogene Proteins c-jun , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Transfection
19.
J Cell Biochem ; 42(4): 193-206, 1990 Apr.
Article in English | MEDLINE | ID: mdl-2111328

ABSTRACT

Transcription factor AP-1 mediates induction of a set of genes in response to the phorbol ester tumor promoter TPA. Recently, AP-1 preparations from HeLa cells were shown to contain a product of the c-JUN protooncogene (Jun/AP-1) which forms a tight complex with the Fos protein. In this paper, we examine the role of the Fos protein in the DNA-binding activity of the AP-1 complex. We show that the DNA-binding activity of bacterially expressed trpE-Jun fusion proteins is increased many-fold upon their interaction with Fos (or a Fos-related antigen) expressed from a baculovirus vector. The site of Fos interaction is within the DNA-binding domain of Jun/AP-1, and anti-Fos antibodies interfere with the binding of affinity purified AP-1 to DNA. These results suggest that, by associating with Jun/AP-1, Fos is responsible for the formation of a multimeric protein complex that has greater affinity for the target sequence than does Jun/AP-1 alone.


Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogenes , Transcription Factors/metabolism , Genetic Vectors , HeLa Cells , Humans , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins c-fos , Proto-Oncogene Proteins c-jun , Transfection
20.
Genes Dev ; 3(12B): 2091-100, 1989 Dec.
Article in English | MEDLINE | ID: mdl-2516828

ABSTRACT

The cFos proto-oncoprotein associates with cJun to form a heterodimer with increased DNA binding and transcriptional activities. It has been suggested that dimerization of these proteins is mediated by the interdigitation of an orderly repeat of leucine residues forming a leucine zipper. In agreement with this model, we find that binding to the AP-1 site requires dimerization of these proteins. Although cFos, itself, does not seem to dimerize and bind to the AP-1 site, Jun: Fos heterodimers have higher stability than Jun homodimers, which accounts for their increased DNA binding activity. Mutational analysis indicates that at least three of the repeated leucines of cJun are important for homodimer formation. However, these residues can be mutated without affecting formation of Jun: Fos heterodimers. In addition, several other residues present between the leucines are also important for both homo- and heterodimerization. These findings provide support for the recent proposal that these proteins dimerize via formation of a coiled coil and suggest that residues other than leucines provide specificity for this interaction. Assuming that dimerization is required for proper alignment of the DNA recognition sites, we generated a cJun mutant containing a small insertion between the dimerization and the DNA recognition domains. This mutant fails to bind DNA, but it acts as a trans-dominant inhibitor of cJun and cFos because it still dimerizes with the wild-type proteins.


Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Proto-Oncogene Proteins/metabolism , Retroviridae Proteins, Oncogenic/metabolism , Transcription Factors/metabolism , Alleles , Amino Acid Sequence , Amino Acids/metabolism , DNA Mutational Analysis , DNA-Binding Proteins/genetics , Leucine/metabolism , Macromolecular Substances , Molecular Sequence Data , Neoplastic Stem Cells , Oncogene Protein p65(gag-jun) , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-fos , Retroviridae Proteins, Oncogenic/genetics , Transcription Factors/genetics
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