RESUMEN
Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1-HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Daño del ADN/fisiología , Activación Enzimática/fisiología , Isomerasa de Peptidilprolil/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular , Vectores Genéticos , Humanos , Microscopía Fluorescente , Peptidilprolil Isomerasa de Interacción con NIMA , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
The tumour suppressor HIPK2 is an important regulator of cell death induced by DNA damage, but how its activity is regulated remains largely unclear. Here we demonstrate that HIPK2 is an unstable protein that colocalizes and interacts with the E3 ubiquitin ligase Siah-1 in unstressed cells. Siah-1 knockdown increases HIPK2 stability and steady-state levels, whereas Siah-1 expression facilitates HIPK2 polyubiquitination, degradation and thereby inactivation. During recovery from sublethal DNA damage, HIPK2, which is stabilized on DNA damage, is degraded through a Siah-1-dependent, p53-controlled pathway. Downregulation of Siah-1 inhibits HIPK2 degradation and recovery from damage, driving the cells into apoptosis. We have also demonstrated that DNA damage triggers disruption of the HIPK2-Siah-1 complex, resulting in HIPK2 stabilization and activation. Disruption of the HIPK2-Siah-1 complex is mediated by the ATM/ATR pathway and involves ATM/ATR-dependent phosphorylation of Siah-1 at Ser 19. Our results provide a molecular framework for HIPK2 regulation in unstressed and damaged cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Daño del ADN , Proteínas de Unión al ADN/genética , Estabilidad de Enzimas , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Ratones , Proteínas Nucleares/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina/metabolismo , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Inflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected coactivator function for the nuclear receptor interacting protein 140 (RIP140) for nuclear factor kappaB (NFkappaB), a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that RIP140 suppresses the expression of metabolic gene networks, but we have found that genetic as well as acute deficiency of RIP140 leads to the inhibition of the proinflammatory program in macrophages. The ability of RIP140 to function as a coactivator for cytokine gene promoter activity relies on direct protein-protein interactions with the NFkappaB subunit RelA and histone acetylase cAMP-responsive element binding protein (CREB)-binding protein (CBP). RIP140-dependent control of proinflammatory gene expression via RelA/CBP may, therefore, represent a molecular rational for the cellular integration of metabolic and inflammatory pathways.
