RESUMO
Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.
Assuntos
Cálcio/metabolismo , Homeostase , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Espermatogênese , Animais , Calcineurina/metabolismo , Sinalização do Cálcio , Linhagem Celular , Retículo Endoplasmático/metabolismo , Epididimo/metabolismo , Epididimo/patologia , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Espaço Intracelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
The IkappaB kinase (IKK) complex serves as the master regulator for the activation of NF-kappaB by various stimuli. It contains two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKalpha/beta at its activation loop and the K63-linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T-cell receptor (TCR) stimulation. However, the phosphorylation of IKKalpha/beta activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1-independent manner and mediate IKKalpha/beta phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKalpha/beta and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.
Assuntos
Quinase I-kappa B/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/deficiência , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ativação Enzimática , Humanos , Quinase I-kappa B/deficiência , Células Jurkat , Lisina/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Modelos Imunológicos , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Transporte Proteico , Receptores de Antígenos de Linfócitos T/imunologia , Frações Subcelulares/metabolismo , Linfócitos T/enzimologiaRESUMO
Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor-alpha (TNF-alpha)-induced IkappaB kinase (IKK) activation and subsequent activation of transcription factor NF-kappaB. However, the molecular mechanism by which RIP mediates TNF-alpha-induced NF-kappaB activation is not completely defined. In this study, we have found that TAK1 is recruited to the TNF-alpha receptor complex in a RIP-dependent manner following the stimulation of TNF-alpha receptor 1 (TNF-R1). Moreover, a forced recruitment of TAK1 to TNF-R1 in the absence of RIP is sufficient to mediate TNF-alpha-induced NF-kappaB activation, indicating that the major function of RIP is to recruit its downstream kinases to the TNF-R1 complex. Interestingly, we also find that TAK1 and MEKK3 form a functional complex, in which TAK1 regulates autophosphorylation of MEKK3. The TAK1-mediated regulation of MEKK3 phosphorylation is dependent on the kinase activity of TAK1. Although TAK1-MEKK3 interaction is not affected by overexpressed TAB1, TAB1 is required for TAK1 activation and subsequent MEKK3 phosphorylation. Together, we conclude that TAK1 is recruited to the TNF-R1 complex via RIP and likely cooperates with MEKK3 to activate NF-kappaB in TNF-alpha signaling.
