Assuntos
Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Proteína Axina , Humanos , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , UbiquitinaçãoRESUMO
The Fbw7 tumor suppressor gene encodes the substrate recognition subunit of the SCF ubiquitin ligase, which targets for degradation a range of oncogenic proteins in a phosphorylation-dependent manner. Substrate phosphorylation is thought to be the main mechanism that ensures timely destruction of Fbw7 substrates. We show here that PI3K dependent phosphorylation of Fbw7 stimulates its ability to ubiquitinate and degrade its substrates. Mutation of the phosphorylation site destabilizes Fbw7 and attenuates degradation of cyclin E and Myc leading to the enhanced expression of a subset of Myc target genes. We suggest that PI3K-dependent phosphorylation of Fbw7 controls the balance between turnover of Fbw7 and its substrates to fine-tune their activity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Humanos , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
A delicate balance exists between the central dopaminergic and cholinergic neurotransmitter systems with respect to motor function. An imbalance can result in motor dysfunction as observed in Parkinson's disease patients and in patients treated with antipsychotic compounds. Cholinergic receptor antagonists can alleviate extrapyramidal symptoms in Parkinson's disease and motor side effects induced by antipsychotics. The effects of anticholinergics are mediated by muscarinic receptors of which five subtypes (M(1)-M(5)) exist. Muscarinic M(4) receptors are found at high concentrations in motor parts of the striatum, suggesting a role for muscarinic M(4) receptors in the motor side effects of antipsychotics, and in the alleviation of these side effects by anticholinergics. Here we investigated the potential role of the muscarinic M(4) receptor in catalepsy induced by antipsychotics (haloperidol and risperidone) as well as the anti-cataleptic effects of the non-selective anticholinergic drug scopolamine in fully backcrossed muscarinic M(4) receptor knockout mice. The drug-induced catalepsy was strongly attenuated, but not abolished, in M(4) knockout mice as compared to wild-type controls. Scopolamine further attenuated the cataleptic response in M(4) knockout mice, suggesting that non-M(4) muscarinic receptors also participate in the anti-cataleptic effects. In conclusion, these data indicate an important role for M(4) receptors in antipsychotic-induced motor side effects and suggest that M(4) receptors could be a target for future pharmacological treatment of antipsychotic-induced as well as idiopathic parkinsonism.
Assuntos
Antipsicóticos/efeitos adversos , Catalepsia/induzido quimicamente , Catalepsia/metabolismo , Receptor Muscarínico M4/deficiência , Animais , Cruzamento , Catalepsia/patologia , Catalepsia/fisiopatologia , Haloperidol/efeitos adversos , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Receptor Muscarínico M4/antagonistas & inibidores , Risperidona/efeitos adversos , Escopolamina/farmacologiaRESUMO
The SCFFbw7 ubiquitin ligase mediates growth-factor-regulated turnover of the Myc oncoprotein. Here we show that SCFß-TrCP binds to Myc by means of a characteristic phosphodegron and ubiquitylates Myc; this results in enhanced Myc stability. SCFFbw7 and SCFß-TrCP can exert these differential effects through polyubiquitylation of the amino terminus of Myc. Whereas SCFFbw7 with the Cdc34 ubiquitin-conjugating enzyme specifically requires lysine 48 (K48) of ubiquitin, SCFß-TrCP uses the UbcH5 ubiquitin-conjugating enzyme to form heterotypic polyubiquitin chains on Myc. Ubiquitylation of Myc by SCFß-TrCP is required for Myc-dependent acceleration of cell cycle progression after release from an arrest in S phase. Therefore, alternative ubiquitylation events at the N terminus can lead to the ubiquitylation-dependent stabilization of Myc.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Células Cultivadas , Sequência Consenso , Proteína 7 com Repetições F-Box-WD , Fase G2 , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Células NIH 3T3 , Poliubiquitina/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fase S , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Quinase 1 Polo-LikeRESUMO
INTRODUCTION: Muscarinic M(5) receptors are the only muscarinic receptor subtype expressed by dopamine-containing neurons of the ventral tegmental area. These cells play an important role for the reinforcing properties of psychostimulants and M(5) receptors modulate their activity. Previous studies showed that M(5) receptor knockout (M (5) (-/-) ) mice are less sensitive to the reinforcing properties of addictive drugs. MATERIALS AND METHODS: Here, we investigate the role of M(5) receptors in the effects of amphetamine and cocaine on locomotor activity, locomotor sensitization, and dopamine release using M (5) (-/-) mice backcrossed to the C57BL/6NTac strain. STATISTICAL ANALYSES: Sensitization of the locomotor response is considered a model for chronic adaptations to repeated substance exposure, which might be related to drug craving and relapse. The effects of amphetamine on locomotor activity and locomotor sensitization were enhanced in M (5) (-/-) mice, while the effects of cocaine were similar in M (5) (-/-) and wild-type mice. RESULTS: Consistent with the behavioral results, amphetamine-, but not cocaine, -elicited dopamine release in nucleus accumbens was enhanced in M (5) (-/-) mice. DISCUSSION: The different effects of amphetamine and cocaine in M (5) (-/-) mice may be due to the divergent pharmacological profile of the two drugs, where amphetamine, but not cocaine, is able to release intracellular stores of dopamine. In conclusion, we show here for the first time that amphetamine-induced hyperactivity and dopamine release as well as amphetamine sensitization are enhanced in mice lacking the M(5) receptor. These results support the concept that the M(5) receptor modulates effects of addictive drugs.
Assuntos
Anfetamina/farmacologia , Cocaína/farmacologia , Dopamina/metabolismo , Receptor Muscarínico M5/metabolismo , Animais , Inibidores da Captação de Dopamina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Receptor Muscarínico M5/genéticaRESUMO
The cellular levels of the Myc oncoprotein are critical determinants of cell proliferation, cell growth and apoptosis and are tightly regulated by external growth factors. Levels of Myc oncoprotein also decline in response to intracellular stress signals such as DNA damage. We show here that this decline is in part due to proteasomal degradation and that it is mediated by the Fbw7 ubiquitin ligase. We have shown previously that the ubiquitin-specific protease Usp28, binds to the nucleoplasmic isoform of Fbw7, Fbw7alpha, and counteracts its function in mammalian cells. Usp28 dissociates from Fbw7alpha in response to UV irradiation, providing a mechanism how Fbw7-mediated degradation of Myc is enhanced upon DNA damage. Our data extend previous observations that link Myc function to the cellular response to DNA damage.