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1.
Diabetologia ; 56(1): 31-5, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23073708

ABSTRACT

AIMS/HYPOTHESIS: Exercise-induced hyperinsulinism (EIHI) is a hypoglycaemic disorder characterised by inappropriate insulin secretion following anaerobic exercise or pyruvate load. Activating promoter mutations in the MCT1 gene (also known as SCLA16A1), coding for monocarboxylate transporter 1 (MCT1), were shown to associate with EIHI. Recently, transgenic Mct1 expression in pancreatic beta cells was shown to introduce EIHI symptoms in mice. To date, MCT1 has not been demonstrated in insulin-producing cells from an EIHI patient. METHODS: In vivo insulin secretion was studied during an exercise test before and after the resection of an insulinoma. The presence of MCT1 was analysed using immunohistochemistry followed by laser scanning microscopy, western blot analysis and real-time RT-PCR of MCT1. The presence of MCT1 protein was analysed in four additional insulinoma patients. RESULTS: Clinical testing revealed massive insulin secretion induced by anaerobic exercise preoperatively, but not postoperatively. MCT1 protein was not detected in the patient's normal islets. In contrast, immunoreactivity was clearly observed in the insulinoma tissue. Western blot analysis and real-time RT-PCR showed a four- to fivefold increase in MCT1 in the insulinoma tissue of the EIHI patient compared with human pancreatic islets. MCT1 protein was detected in three of four additional insulinomas. CONCLUSIONS/INTERPRETATION: We show for the first time that an MCT1-expressing insulinoma was associated with EIHI and that MCT1 might be present in most insulinomas. Our data suggest that MCT1 expression in human insulin-producing cells can lead to EIHI and warrant further studies on the role of MCT1 in human insulinoma patients.


Subject(s)
Hyperinsulinism/etiology , Hypoglycemia/etiology , Insulin-Secreting Cells/metabolism , Insulinoma/physiopathology , Monocarboxylic Acid Transporters/metabolism , Motor Activity , Neoplasm Proteins/metabolism , Symporters/metabolism , Adolescent , Exercise Test , Female , Humans , Hyperinsulinism/physiopathology , Hypoglycemia/prevention & control , Insulin-Secreting Cells/pathology , Insulinoma/metabolism , Insulinoma/pathology , Insulinoma/surgery , Male , Middle Aged , Monocarboxylic Acid Transporters/genetics , Sleep Stages , Sleep Wake Disorders/etiology , Sleep Wake Disorders/prevention & control , Symporters/genetics , Treatment Outcome , Unconsciousness/etiology , Unconsciousness/prevention & control
2.
Technol Cancer Res Treat ; 1(4): 297-304, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12625789

ABSTRACT

Colorectal cancer is the second most frequent cancer in Western countries. Exogenous factors play a major role in the aetiology of sporadic colorectal cancer representing about 90% of all cases, hereditary cancers accounting for about 10% of patients. Thus, in the large majority of cases, cell dysfunction in CRC results from multiple rather than single, gene interactions. Numerous cellular events and environmental influences modify gene expression or post-translational protein modifications. Changes like glycosylation of proteins and lipids which are a common feature in colorectal cancer and influence cancer cell behaviour, cannot be directly detected by genetic studies. Better than genomics studies, functional proteomics studies allow the investigation of environmental factors over time, allowing the monitoring of metabolic responses to various stimuli. However, proteomics studies also have several drawbacks: a) current tools only allow narrow-range analyses, b) identification of proteins of interest remains cumbersome, c) protein studies address multiple compounds of high complexity, d) large amount of proteins are necessary to allow analysis, e) protein research require specific tools, e.g. tagged antibodies, that first have to be developed. Some protein tests are already in application for CRC: a classical prognostic test in colorectal cancer is based on the detection and quantification of a single protein (CEA) in body fluids. Recently, a screening assay based on APC protein truncation test has also been proposed. However, studies linking large protein expression patterns with clinical outcome in colorectal cancer are still in their infancy. To be able to predict occurrence of disease, and treatment outcome, more studies on genotype-phenotype correlations are needed both in sporadic and in hereditary colorectal cancer.


Subject(s)
Colorectal Neoplasms/diagnosis , Proteome , Colorectal Neoplasms/metabolism , Electrophoresis, Gel, Two-Dimensional , Humans , Immunoblotting , Prognosis , Treatment Outcome , Tumor Suppressor Protein p53/metabolism
3.
Oncogene ; 20(26): 3341-7, 2001 Jun 07.
Article in English | MEDLINE | ID: mdl-11423984

ABSTRACT

The protein product of the tumor suppressor gene p53 is phosphorylated on multiple residues by several protein kinases. Using a battery of 10 antibodies developed against different phosphorylated and acetylated residues of p53, we compared the pattern of p53 phosphorylation and acetylation in tumor-derived cell lines, tumor samples, and non-neoplastic cells. Irrespective of tumor types or the presence of p53 mutation, phosphorylation and acetylation of p53 was substantially higher in samples obtained from tumor tissues than those found in non-transformed samples. Among the 10 sites analysed, phosphorylation of residues 15, 81, 392, and acetylation were among the more frequent modifications. Analysis of two of the more abundant phosphorylation or acetylation sites on p53 is sufficient to detect 72% of tumor-derived p53 proteins. The distinct pattern of p53 phosphorylation and acetylation in human tumors may offer a new means to monitor the status and activity of p53 in the course of tumor development and progression.


Subject(s)
Neoplasm Proteins/chemistry , Neoplasms/metabolism , Protein Processing, Post-Translational , Tumor Suppressor Protein p53/chemistry , Acetylation , Amino Acid Substitution , Blotting, Western , Fibroblasts/metabolism , Genes, p53 , Humans , Mutation, Missense , Neoplasm Proteins/genetics , Neoplasms/pathology , Phosphorylation , Protein Kinases/metabolism , Tumor Cells, Cultured/metabolism
4.
J Biol Chem ; 276(44): 40389-95, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11384992

ABSTRACT

Covalent attachment of SUMO-1 to Mdm2 requires the activation of a heterodimeric Aos1-Uba2 enzyme (ubiquitin-activating enzyme (E1)) followed by the conjugation of Sumo-1 to Mdm2 by Ubc9, a protein with a strong sequence similarity to ubiquitin carrier proteins (E2s). Upon Sumo-1 conjugation, Mdm2 is protected from self-ubiquitination and elicits greater ubiquitin-protein isopeptide ligase (E3) activity toward p53, thereby increasing its oncogenic potential. Because of the biological implication of Mdm2 sumoylation, we mapped Ubc9 binding on Mdm2. Here we demonstrate that Ubc9 can associate with Mdm2 only if amino acids 40-59 within the N terminus of Mdm2 are present. Mdm2 from which amino acids 40-59 have been deleted can no longer be sumoylated. Furthermore, addition of a peptide that corresponds to amino acids 40-59 on Mdm2 to a sumoylation reaction efficiently inhibits Mdm2 sumoylation in vitro and in vivo. In UV-treated cells Mdm2 exhibits reduced association with Ubc9, which coincides with decreased Mdm2 sumoylation. Our findings regarding the association of Ubc9 with Mdm2, and the effect of UV-irradiation on Ubc9 binding, point to an additional level in the regulation of Mdm2 sumoylation under normal growth conditions as well as in response to stress conditions.


Subject(s)
Ligases/metabolism , Nuclear Proteins , Proto-Oncogene Proteins/metabolism , SUMO-1 Protein/metabolism , Ubiquitin-Conjugating Enzymes , 3T3 Cells , Animals , Cell Line, Transformed , Humans , Mice , Protein Binding , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p53/metabolism , Ubiquitin/metabolism , Ultraviolet Rays
5.
Genes Dev ; 15(9): 1067-77, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11331603

ABSTRACT

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258-260 and 393-395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.


Subject(s)
DNA Damage/physiology , Nuclear Proteins , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Amino Acid Substitution , Animals , Antibodies, Monoclonal/pharmacology , Aspartic Acid , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins , Cell Nucleus/metabolism , Cells, Cultured , DNA Damage/radiation effects , DNA-Binding Proteins , Epitope Mapping , Fibroblasts , Humans , Mice , Microinjections , Mutation , Phosphorylation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins c-mdm2 , Serine/metabolism , Tumor Suppressor Proteins
6.
Mol Cell Biol ; 21(8): 2743-54, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11283254

ABSTRACT

The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Animals , Base Sequence , Binding Sites , Cell Division , Cell Line , DNA Primers/genetics , Drug Stability , Dual-Specificity Phosphatases , Genes, p53 , Humans , Intracellular Signaling Peptides and Proteins , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 4 , Mass Spectrometry , Mice , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase Phosphatases , Mitogen-Activated Protein Kinases/genetics , Mutation , Neoplasms/genetics , Neoplasms/metabolism , Oligonucleotides, Antisense/pharmacology , Phosphorylation , Protein Tyrosine Phosphatases/metabolism , Stress, Physiological/genetics , Stress, Physiological/metabolism , Threonine/chemistry , Transcription, Genetic , Tumor Suppressor Protein p53/genetics , Ultraviolet Rays
7.
J Biol Chem ; 276(17): 13852-7, 2001 Apr 27.
Article in English | MEDLINE | ID: mdl-11278372

ABSTRACT

Regulation of the stability of p53 is key to its tumor-suppressing activities. mdm2 directly binds to the amino-terminal region of p53 and targets it for degradation through the ubiquitin-proteasome pathway. The coactivator protein TAF(II)31 binds to p53 at the amino-terminal region that is also required for interaction with mdm2. In this report, we demonstrate that expression of TAF(II)31 inhibits mdm2-mediated ubiquitination of p53 and increases p53 levels. TAF(II)31-mediated p53 stabilization results in activation of p53-mediated transcriptional activity and leads to p53-dependent growth arrest in fibroblasts. UV-induced stabilization of p53 coincides with an increase in p53-associated TAF(II)31 and a corresponding decrease in mdm2-p53 interaction. Non-p53 binding mutant of TAF(II)31 fails to stabilize p53. Our results suggest that direct interaction of TAF(II)31 and p53 not only mediates p53 transcriptional activation but also protects p53 from mdm2-mediated degradation, thereby resulting in activation of p53 functions.


Subject(s)
Gene Expression Regulation , Nuclear Proteins , TATA-Binding Protein Associated Factors , Trans-Activators/metabolism , Transcription Factor TFIID , Tumor Suppressor Protein p53/metabolism , 3T3 Cells , Animals , Blotting, Northern , Blotting, Western , Cell Cycle , Cell Division , Fibroblasts/metabolism , Flow Cytometry , Genes, Reporter , Genes, p53/genetics , Genetic Vectors , Mice , Mice, Inbred BALB C , Mutation , Plasmids/metabolism , Precipitin Tests , Protein Binding , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Suppression, Genetic , Transcription, Genetic , Transcriptional Activation , Transfection , Ubiquitins/metabolism , Ultraviolet Rays
8.
Cell ; 101(7): 753-62, 2000 Jun 23.
Article in English | MEDLINE | ID: mdl-10892746

ABSTRACT

Mdm2 is an E3 ubiquitin ligase for the p53 tumor suppressor protein. We demonstrate that Mdm2 is conjugated with SUMO-1 (sumoylated) at Lys-446, which is located within the RING finger domain and plays a critical role in Mdm2 self-ubiquitination. Whereas mutant Mdm2(K446R) is stabilized, it elicits increased degradation of p53 and concomitant inhibition of p53-mediated apoptosis. In vitro sumoylation of Mdm2 abrogates its self-ubiquitination and increases its ubiquitin ligase activity toward p53. Radiation caused a dose- and time-dependent decrease in the degree of Mdm2 SUMO-1 modification, which is inversely correlated with the levels of p53. Our results suggest that the maintenance of the intrinsic activity of a RING finger E3 ubiquitin ligase is sumoylation dependent and that reduced Mdm2 sumoylation in response to DNA damage contributes to p53 stability.


Subject(s)
Nuclear Proteins , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Ubiquitins/metabolism , Cell Line, Transformed , DNA Damage , Humans , Protein Binding , Proto-Oncogene Proteins c-mdm2 , SUMO-1 Protein
9.
J Biol Chem ; 275(22): 16590-6, 2000 Jun 02.
Article in English | MEDLINE | ID: mdl-10748185

ABSTRACT

The stress-activated protein kinase JNK plays an important role in the stability and activities of key regulatory proteins, including c-Jun, ATF2, and p53. To better understand mechanisms underlying the regulation of JNK activities, we studied the effect of expression of the amino-terminal JNK fragment (N-JNK; amino acids 1-206) on the stability and activities of JNK substrates under nonstressed growth conditions, as well as after exposure to hydrogen peroxide. Mouse fibroblasts that express N-JNK under tetracycline-off (tet-off) inducible promoter exhibited elevated expression of c-Jun, ATF2, and p53 upon tetracycline removal. This increased coincided with elevated transcriptional activities of p53, but not of c-Jun or ATF2, as reflected in luciferase activities of p21(Waf1/Cip1)-Luc, AP1-Luc, and Jun2-Luc, respectively. Expression of N-JNK in cells that were treated with H(2)O(2) impaired transcriptional output as reflected in a delayed and lower level of c-Jun-, limited ATF2-, and reduced p53-transcriptional activities. N-JNK elicited an increase in H(2)O(2)-induced cell death, which is p53-dependent, because it was not seen in p53 null cells yet could be observed upon coexpression of p53 and N-JNK. The ability to alter the activity of ATF2, c-Jun, and p53 and the degree of stress-induced cell death by a JNK-derived fragment identifies new means to elucidate the nature of JNK regulation and to alter the cellular response to stress.


Subject(s)
Cell Death/drug effects , Cyclic AMP Response Element-Binding Protein/metabolism , Hydrogen Peroxide/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Transcription Factors/metabolism , Tumor Suppressor Protein p53/metabolism , 3T3 Cells , Activating Transcription Factor 2 , Animals , JNK Mitogen-Activated Protein Kinases , Mice , Mitogen-Activated Protein Kinases/chemistry , Transcriptional Activation
10.
Cancer Res ; 60(4): 896-900, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10706102

ABSTRACT

p53 phosphorylation and association with proteins is implicated in its stability and activity. We have compared the association of DNA-bound and overall pools of p53 with murine double minute 2 (Mdm2), c-Jun NH2-terminal kinase (JNK), p300/CBP, and p14ARF during cell cycle progression. Whereas DNA-bound p53 associates with JNK at G0-G1 and with Mdm2 and p300 during S and G2-M phases, the general pool of p53 was found in complex with JNK and Mdm2 almost throughout the cell cycle. Phosphorylation of p53 at serines 9, 15, and 20 is at the highest levels at G1 and at serines 37 and 392 during G2-M phase. Whereas a high dose of UV irradiation was required for phosphorylation of serines 15 and 392 between 8 and 24 h after treatment, a low dose caused immediate phosphorylation on serines 9, 20, and 372. These dynamic changes in the phosphorylation of p53 are expected to play a pivotal role in p53 association, stability, and function.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Nuclear Proteins/metabolism , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Tumor Suppressor Protein p53/metabolism , Cell Cycle , Humans , JNK Mitogen-Activated Protein Kinases , Phosphorylation , Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p14ARF , Ultraviolet Rays
11.
J Mol Biol ; 295(4): 1009-21, 2000 Jan 28.
Article in English | MEDLINE | ID: mdl-10656807

ABSTRACT

Identification of Mdm2 and JNK as proteins that target degradation of wt p53 prompted us to examine their effect on mutant p53, which exhibits a prolonged half-life. Of five mutant p53 forms studied for association with the targeting molecules, two no longer bound to Mdm2 and JNK. Three mutant forms, which exhibit high expression levels, showed lower affinity for association with Mdm2 and JNK in concordance with greater affinity to p14(ARF), which is among the stabilizing p53 molecules. Monitoring mutant p53 stability in vitro confirmed that, while certain forms of mutant p53 are no longer affected by either JNK or Mdm2, others are targeted for degradation by JNK/Mdm2, albeit at lower efficiency when compared with wt p53. Expression of wt p53 in tumor cells revealed a short half-life, suggesting that the targeting molecules are functional. Forced expression of mutant p53 in p53 null cells confirmed pattern of association with JNK/Mdm2 and prolonged half-life, as found in the tumor cells. Over-expression of Mdm2 in either tumor (which do express endogenous functional Mdm2) or in p53 null cells decreased the stability of mutant p53 suggesting that, despite its expression, Mdm2/JNK are insufficient (amount/affinity) for targeting mutant p53 degradation. Based on both in vitro and in vivo analyses, we conclude that the prolonged half-life of mutant p53 depends on the nature of the mutation, which either alters association with targeting molecules, ratio between p53 and targeting/stabilizing molecules or targeting efficiency.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Nuclear Proteins , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Cell Membrane/metabolism , Fibroblasts , Genes, p53 , Half-Life , Humans , JNK Mitogen-Activated Protein Kinases , Kinetics , Protein Conformation , Proto-Oncogene Proteins c-mdm2 , Recombinant Proteins/metabolism , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p14ARF , Tumor Suppressor Protein p53/genetics , Zinc Fingers
12.
Eur J Clin Invest ; 30(1): 11-7, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10619996

ABSTRACT

BACKGROUND: Formation of neointima after balloon angioplasty is regulated via inducible transcription factors (ITF), such as c-Jun and c-Fos, depending on mitogen activated protein (MAP) kinases, which have been shown to be activated after balloon injury. The precise localization of activated MAP-kinases and concomitant expression of transcription factors in the vessel wall remains to be elucidated. We have now studied the localization and time-dependent expression of MAP-kinases together with corresponding ITFs in the rat carotid angioplasty model. DESIGN: Animals were sacrificed at 0. 5, 6 and 24 h and 3, 5, 7, 14 and 28 days after injury. Cryocut sections were stained using antibodies directed against c-Jun, phosphorylated c-Jun, c-Fos, c-Jun amino-terminal kinase (JNK), extracellular signal related kinase (ERK), von Willebrand factor, ki67 antigen, and alpha-actin. RESULTS: c-Jun expression was strongly induced in smooth muscle cells (SMC) 30 min after injury and remained upregulated for 24 h, thereafter dropping to basal levels at day 3. Re-expression was observed at day 7 and 14 but not day 28. Expression patterns of JNK and phosphorylated c-Jun were highly congruent to that of c-Jun. In contrast, c-Fos expression was restricted to 30 min and, less pronounced than c-Jun and JNK, was visible after 7 days. Also, its expression was congruent with the presence of ERK. CONCLUSIONS: These findings demonstrate a clear association between MAP-kinases and their transcription factor substrates in vivo with a predominant association of JNK and c-Jun with sustained SMC proliferation.


Subject(s)
JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase Kinases/physiology , Mitogen-Activated Protein Kinases/physiology , Proto-Oncogene Proteins c-fos/physiology , Proto-Oncogene Proteins c-jun/physiology , Tunica Intima/pathology , Animals , Catheterization , Cell Division , MAP Kinase Kinase 4 , Male , Mitogen-Activated Protein Kinase 1/analysis , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinase Kinases/analysis , Mitogen-Activated Protein Kinases/analysis , Proto-Oncogene Proteins c-jun/analysis , Rats , Rats, Sprague-Dawley
13.
Oncogene ; 17(19): 2543-7, 1998 Nov 12.
Article in English | MEDLINE | ID: mdl-9824166

ABSTRACT

Key to p53 ability to mediate its multiple cellular functions lies in its stability. In the present study we have elucidated the mechanism by which Mdm2 regulates p53 degradation. Using in vitro and in vivo ubiquitination assays we demonstrate that Mdm2 association with p53 targets p53 ubiquitination. Exposure of cells to UV-irradiation inhibits this targeting. Mdm2 which is deficient in p53 binding failed to target p53 ubiquitination, suggesting that the association is essential for Mdm2 targeting ability. While mdm2-p53 complex is found in non-stressed cells, the amount of p53-bound mdm2 is decreased after UV-irradiation, further pointing to the relationship between mdm2 binding and p53 level. Similar to Swiss 3T3 cells, the dissociation of mdm2-p53 complex was also found in UV-treated Scid cells, lacking functional DNA-PK, suggesting that DNA-PK is not sufficient for dissociating mdm2 from p53. Together our studies point to the role of Mdm2, as one of p53-associated proteins, in targeting p53 ubiquitination.


Subject(s)
DNA-Binding Proteins , Nuclear Proteins , Proto-Oncogene Proteins/physiology , Tumor Suppressor Protein p53/metabolism , 3T3 Cells , Animals , DNA Damage , DNA-Activated Protein Kinase , Fibroblasts , Macromolecular Substances , Mice , Mice, Inbred BALB C , Mice, SCID , Phosphorylation , Protein Conformation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Recombinant Fusion Proteins/metabolism , Tumor Suppressor Protein p53/radiation effects , Ubiquitins/metabolism , Ultraviolet Rays
14.
Brain Res Mol Brain Res ; 62(2): 158-66, 1998 Nov 20.
Article in English | MEDLINE | ID: mdl-9813301

ABSTRACT

The activating transcription factor 2 (ATF-2) protein, a neuronal constitutively expressed CRE-binding transcription factor, is essential for the intact development of the mammalian brain. ATF-2 is activated by c-Jun N-terminal kinases and modulates both the induction of the c-jun gene and the function of the c-Jun protein, a mediator of neuronal death and survival. Here we show by immunocytochemistry and Western blotting that ATF-2 is rapidly suppressed in neurons within 1-4 h following neuronal stress such as transient focal ischemia by occlusion of the medial cerebral artery, mechanical injury of the neuroparenchym, stimulation of adult dorsal root ganglion neurons in vitro by doxorubicin as well as within 24 h following nerve fiber transection. ATF-2 reappears and regains basal levels between 12 h and 72 h following ischemia, between 50 and 100 days following axotomy, but remains absent around the site of mechanical injury during the process of degeneration. Following ischemia and tissue injury, ATF-2-IR also disappeared in areas remote from the affected brain compartments indicating the regulation of its expression by diffusible molecules. These findings demonstrate that the rapid and persistent down-regulation of ATF-2 is a constituent of the long-term neuronal stress response and that the reappearance of ATF-2 after weeks is a marker for the normalization of neuronal gene transcription following brain injury.


Subject(s)
Brain Injuries/genetics , Cyclic AMP Response Element-Binding Protein/biosynthesis , Gene Expression Regulation/drug effects , Mitogen-Activated Protein Kinases , Nerve Tissue Proteins/biosynthesis , Neurons/metabolism , Transcription Factors/biosynthesis , Activating Transcription Factor 2 , Animals , Axotomy , Brain Injuries/metabolism , Brain Ischemia/genetics , Brain Ischemia/pathology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/genetics , Doxorubicin/toxicity , Facial Nerve Injuries , Ganglia, Spinal/cytology , Ganglia, Spinal/drug effects , Genes, jun , JNK Mitogen-Activated Protein Kinases , Male , Nerve Crush , Nerve Tissue Proteins/genetics , Prosencephalon/injuries , Rats , Rats, Sprague-Dawley , Transcription Factors/genetics , Transcription, Genetic/drug effects
15.
Genes Dev ; 12(17): 2658-63, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9732264

ABSTRACT

In this study we elucidated the role of nonactive JNK in regulating p53 stability. The amount of p53-JNK complex was inversely correlated with p53 level. A peptide corresponding to the JNK binding site on p53 efficiently blocked ubiquitination of p53. Similarly, p53 lacking the JNK binding site exhibits a longer half-life than p53(wt). Outcompeting JNK association with p53 increased the level of p53, whereas overexpression of a phosphorylation mutant form of JNK inhibited p53 accumulation. JNK-p53 and Mdm2-p53 complexes were preferentially found in G0/G1 and S/G2M phases of the cell cycle, respectively. Altogether, these data indicate that JNK is an Mdm2-independent regulator of p53 stability in nonstressed cells.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases , Nuclear Proteins , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Ubiquitins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Cell Line , JNK Mitogen-Activated Protein Kinases , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phosphorylation , Proto-Oncogene Proteins c-mdm2 , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reticulocytes/metabolism , Sequence Deletion , Spodoptera , Transfection , Tumor Suppressor Protein p53/chemistry
16.
Neuroscience ; 84(1): 163-76, 1998 May.
Article in English | MEDLINE | ID: mdl-9522371

ABSTRACT

The expression of c-Jun, JunB, JunD, c-Fos and ATF-2 transcription factors was studied in L4/L5 dorsal root ganglion neurons of adult rats, in order to determine the extent to know to which extend the expression of transcription factors in vitro parallels the pathophysiological expression in vivo. First, dorsal root ganglia were dissociated and cultured for up to 15 days in vitro (culture). Second, the dorsal root and the peripheral nerve fibres were transected close at the dorsal root ganglia, and the completely axotomized dorsal root ganglia were kept in artificial cerebrospinal fluid for up to 24 h. This procedure (explantation) preserves the intraganglionic morphology intact. Culture evoked a persistent expression of c-Jun and JunD in the majority of small neurons independent on neurite extension, In contrast, the number of large neurons with c-Jun decreased and with JunD increased with incubation time. JunB and c-Fos, which were also visible in the majority of neurons, strongly decreased with culture time in both small and large neurons. ATF-2 was visible in the vast majority of neurons and did not change during the observation period. Incubation with brain-derived neurotrophic factor for 15 days reduced JunB expression and raised c-Fos expression, but did not affect c-Jun or JunD labelings. Explantation of dorsal root ganglia evoked a dramatic and rapid induction of c-Jun in neurons located in the periphery of the ganglia, an area that showed prominent apoptosis as visualized by transferase dUTP nick end-labelling, followed by a delayed increase in neurons of the central parts of dorsal root ganglia. Expression of JunB showed a dramatic increase within 2 h in the whole ganglion, but disappeared within the following hours. JunD dropped from its basal levels within 4 h and was almost absent after 8 h. c-Fos did not appear until 6 h, when transferase dUTP nick end-labelling also became detectable, and remained visible in a rather small number of neurons. As with culture, incubation of explanted dorsal root ganglia with brain-derived neurotrophic factor prevented the initial rise in JunB, accelerated and enhanced c-Fos expression, but did not alter c-Jun and JunD expression. Immunoreactivity of ATF-2 declined or disappeared in those dorsal root ganglia compartments that showed a rise in c-Jun and transferase dUTP nick end-labelling. These findings demonstrate that inducible transcription factors such as Jun and Fos proteins are differentially expressed in adult neurons in vitro when compared to pathophysiological conditions in vivo such as nerve fibre transection (axotomy or rhizotomy). Moreover, the comparison between the explantation and culture experiments suggests that it is the complete axotomy of neurons that provokes those expression patterns found in neuronal cultures of adult neurons. The rapid and persisting expression of c-Jun during neurite extension and apoptosis points at the activation of a pivotal program that might be determined by the presence or absence of ATF-2 and that is involved in regeneration or degeneration.


Subject(s)
Axotomy , Cyclic AMP Response Element-Binding Protein/metabolism , Ganglia, Spinal/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Transcription Factors/metabolism , Activating Transcription Factor 2 , Animals , Apoptosis/physiology , Brain-Derived Neurotrophic Factor/metabolism , Brain-Derived Neurotrophic Factor/pharmacology , Cells, Cultured , Ganglia, Spinal/cytology , Ganglia, Spinal/drug effects , Genetic Techniques , Male , Neurons/metabolism , Rats , Rats, Sprague-Dawley
17.
Neuroscience ; 81(1): 199-212, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9300412

ABSTRACT

The expression of the constitutive transcription factors activating transcription factor-2 (ATF-2), serum response factor (SRF) and cAMP/Ca response element binding factor (CREB), and the phosphorylation of SRF and CREB were studied in the untreated adult rat nervous system and following seizure activities and neurodegenerative stimuli. In the untreated rat, intense nuclear SRF immunoreactivity was present in the vast majority of neurons in the forebrain, cortex, striatum, amygdala and hippocampus, and in some scattered neurons in the medulla and spinal cord. In contrast, SRF immunoreactivity was absent in the midline areas of the forebrain, e.g., the globus pallidum and septum, and in the hypothalamus, thalamus, mesencephalon and motoneurons. Nuclear ATF-2 was expressed at high levels in apparently all neurons, but not glial cells, throughout the neuraxis except for those neuronal populations which exhibit a high basal level of c-Jun, i.e. dentate gyrus and the motoneurons of cranial and somatosensory neurons. CREB immunoreactivity was present at a rather uniform intensity in all neuronal and glial cells throughout the neuraxis. Two hours, but not 5 h or 24 h, following systemic application of kainic acid, an increase in SRF was detectable by western blot analysis in hippocampal and cortical homogenates whereas the expression of ATF-2 and CREB did not change. Phosphorylation of CREB at serine 133 and of SRF at serine 103 were studied with specific antisera. In untreated rats, intense phosphoCREB and phosphoSRF immunoreactivities labelled many glial cells and/or neurons with the highest levels in the dentate gyrus, the entorhinal cortex and the retrosplenial cortex. Following kainate-induced seizures, phosphoSRF-IR but not phosphoCREB-IR transiently increased between 0.5 h and 2 h. Following transection of peripheral or central nerve fibres such as optic nerve, medial forebrain bundle, vagal and facial nerve fibres, ATF-2 rapidly decreased in the axotomized neurons during that period when c-Jun was rapidly expressed. SRF remained unchanged and CREB disappeared in some axotomized subpopulations. Similar to axotomy, c-Jun increased and ATF-2 decreased in cultured adult dorsal root ganglion neurons following ultraviolet irradiation. The distribution of SRF and ATF-2 suggests that their putative target genes c-fos, junB, krox-24 and c-jun can be independently regulated from SRF and ATF-2. The suppression of ATF-2 and the expression of c-Jun following axotomy and ultraviolet irradiation might be part of a novel neuronal stress response in the brain that strongly resembles the stress response characterized in non-neuronal cells.


Subject(s)
Brain Chemistry/physiology , Cyclic AMP Response Element-Binding Protein/biosynthesis , DNA-Binding Proteins/biosynthesis , Epilepsy, Generalized/physiopathology , Nuclear Proteins/biosynthesis , Transcription Factors/biosynthesis , Activating Transcription Factor 2 , Age Factors , Animals , Antibody Specificity , Axotomy , Brain Chemistry/radiation effects , Cyclic AMP Response Element-Binding Protein/immunology , Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Epilepsy, Generalized/metabolism , Excitatory Amino Acid Agonists/pharmacology , Kainic Acid/pharmacology , Leucine Zippers/immunology , Male , Nerve Degeneration/drug therapy , Nerve Degeneration/pathology , Nerve Degeneration/radiotherapy , Nerve Fibers/pathology , Nerve Fibers/radiation effects , Nuclear Proteins/immunology , Nuclear Proteins/metabolism , Phosphoproteins/analysis , Phosphoproteins/immunology , Phosphorylation , Proto-Oncogene Proteins c-jun/biosynthesis , Proto-Oncogene Proteins c-jun/metabolism , Rats , Rats, Sprague-Dawley , Serum Response Factor , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Transcription Factors/immunology , Transcription Factors/metabolism , Ultraviolet Rays
18.
Brain Res Mol Brain Res ; 48(1): 7-16, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9379852

ABSTRACT

In adult male rats, the expression of the neuropeptide galanin and its co-localization with the c-Jun transcription factor and the NADPH-diaphorase, the marker enzyme for the nitric oxide synthase (NOS), was investigated by immunohistochemistry in axotomized neurons following unilateral stereotaxic transection of the (a) mamillo-thalamic tract, (b) medial forebrain bundle, (c) fimbria fornix bundle and (d) sciatic nerve. This surgical procedure resulted in axotomy of neurons of (a) mamillary ncl. (MnM), (b) substantia nigra compacta (SNC) and paraventricular ncl. of thalamic (PF) neurons, (c) medial septum (MS) and vertical diagonal band of Broca (VDB), and (d) sciatic motoneurons and dorsal root ganglia (DRG). In all of these axotomized neuronal subpopulations, expression of c-Jun appeared between 24 and 36 h post-axotomy and persisted on substantial levels for 15 days in the SNC and for 30-50 days in the MnM, PF, MS, VBD, sciatic DRG and motoneurons. Expression of galanin was seen in axotomized MnM, MS and DRG, but not in SNC, PF and sciatic motoneurons. Galanin-immunoreactivity (IR) appeared between 3 and 5 days after nerve fiber transection and persisted up to 50 days in the MnM, MS and DRGs. The cytoplasmic galanin-IR was almost completely restricted to those neurons showing a nuclear c-Jun expression. Moreover, galanin expression showed a long-lasting co-localization with those neurons that exhibited an increased NADPH-diaphorase reactivity in the MnM and DRG or a residual NADPH-diaphorase reactivity in MS post-axotomy. Very similar to galanin, NADPH-diaphorase was not affected by axotomy in the SNC, PF or sciatic motoneurons. Our findings suggest a common mechanism for galanin and NOS (NADPH-diaphorase activity) expression. Since the galanin promotor contains an AP-1 binding site, c-Jun might trigger the lasting induction of galanin in NOS-positive central neurons that survive the axotomy-evoked injury.


Subject(s)
Axons/physiology , Brain/physiology , Galanin/biosynthesis , NADPH Dehydrogenase/biosynthesis , Nerve Fibers/physiology , Neurons/metabolism , Proto-Oncogene Proteins c-jun/biosynthesis , Sciatic Nerve/physiology , Thalamic Nuclei/physiology , Animals , Galanin/analysis , Ganglia, Spinal/physiology , Male , Motor Neurons/cytology , Motor Neurons/metabolism , NADPH Dehydrogenase/analysis , Neurons/cytology , Neurons, Afferent/physiology , Nitric Oxide Synthase/analysis , Paraventricular Hypothalamic Nucleus/physiology , Prosencephalon/physiology , Proto-Oncogene Proteins c-jun/analysis , Rats , Rats, Sprague-Dawley , Substantia Nigra/physiology , Time Factors
19.
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