RESUMO
Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.
Assuntos
Caspase 8/metabolismo , Tamanho Celular , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Apoptose , Células COS , Caspase 8/genética , Chlorocebus aethiops , Ativação Enzimática , Células HeLa , Humanos , Células MCF-7 , Mutação , Canais de Potássio de Domínios Poros em Tandem/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Transdução de Sinais , Especificidade por Substrato , Fatores de Tempo , Transfecção , Xenopus laevisRESUMO
FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Antígenos de Diferenciação/fisiologia , Apoptose , Proteína de Domínio de Morte Associada a Fas/fisiologia , Coração/embriologia , NF-kappa B/metabolismo , Receptores Imunológicos/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus/embriologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Blastômeros/enzimologia , Blastômeros/metabolismo , Proteínas Culina/química , Proteínas Culina/genética , Proteínas Culina/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Morfolinos/genética , NF-kappa B/fisiologia , Fragmentos de Peptídeos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Análise de Sequência de DNA , Deleção de Sequência , Transdução de Sinais , Ativação Transcricional , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
We have previously purified and cloned an apoptosis-inducing protein (AIP) derived from fish infected with the anisakis simplex. Recently, we identified a series of AIP-responsive genes in the HL-60 cell line using a subtractive hybridization method. Here we report the molecular cloning and characterization of one of these genes, which encodes a novel human kelch protein containing 568 amino acid residues, termed hDKIR. The Drosophila Kelch protein localizes to a ring canal structure, which is required for oocyte development. When hDKIR was expressed in cultured-mammalian cells, hDKIR localized to a ring-like structure. Furthermore, when coexpressed with Mayven or Keap1, hDKIR bound to Mayven and recruited Mayven into ring-like structures perfectly. This indicates that kelch homologues can interact with each other in a specific manner and such interaction can affect the subcellular localization of kelch proteins. Finally, domain analysis revealed that both the N-terminal POZ (poxviruses and zinc fingers) and intervening region (IVR) domains of hDKIR are essential for ring-like structure activity, suggesting that the development of the ring-like structure is independent of the ability to bind actin.