RESUMO
Proper control of epidermal growth factor receptor (EGFR) signaling is important for maintaining cellular homeostasis. Given that EGFR signaling occurs at the plasma membrane and endosomes following internalization, endosomal trafficking of EGFR spatiotemporally regulates EGFR signaling. In this process, leucine-rich repeat kinase 1 (LRRK1) has multiple roles in kinase activity-dependent transport of EGFR-containing endosomes and kinase-independent sorting of EGFR into the intraluminal vesicles (ILVs) of multivesicular bodies. Active, phosphorylated EGFR inactivates the LRRK1 kinase activity by phosphorylating Y944. In this study, we demonstrate that LRRK1 facilitates EGFR dephosphorylation by PTP1B (also known as PTPN1), an endoplasmic reticulum (ER)-localized protein tyrosine phosphatase, at the ER-endosome contact site, after which EGFR is sorted into the ILVs of endosomes. LRRK1 is required for the PTP1B-EGFR interaction in response to EGF stimulation, resulting in the downregulation of EGFR signaling. Furthermore, PTP1B activates LRRK1 by dephosphorylating pY944 on the contact site, which promotes the transport of EGFR-containing endosomes to the perinuclear region. These findings provide evidence that the ER-endosome contact site functions as a hub for LRRK1-dependent signaling that regulates EGFR trafficking.
Assuntos
Endossomos , Receptores ErbB , Humanos , Células HeLa , Endossomos/metabolismo , Receptores ErbB/metabolismo , Retículo Endoplasmático/metabolismo , Corpos Multivesiculares/metabolismo , Transporte Proteico/fisiologia , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Primary cilia are antenna-like organelles that regulate growth and development via extracellular signals. However, the molecular mechanisms underlying cilia dynamics, particularly those regulating their disassembly, are not well understood. Here, we show that leucine-rich repeat kinase 1 (LRRK1) plays a role in regulating cilia disassembly. The depletion of LRRK1 impairs primary cilia resorption following serum stimulation in cultured cells. Polo-like kinase 1 (PLK1) plays an important role in this process. During ciliary resorption, PLK1 phosphorylates LRRK1 at the primary cilia base, resulting in its activation. We identified nuclear distribution protein nudE-like 1 (NDEL1), which is known to positively regulate cilia disassembly, as a target of LRRK1 phosphorylation. Whereas LRRK1 phosphorylation of NDEL1 on Ser-155 promotes NDEL1 interaction with the intermediate chains of cytoplasmic dynein-2, it is also crucial for triggering ciliary resorption through dynein-2-driven retrograde intraflagellar transport. These findings provide evidence that a novel PLK1-LRRK1-NDEL1 pathway regulates cilia disassembly.
Assuntos
Cílios , Dineínas , Dineínas/metabolismo , Fosforilação , Cílios/metabolismo , Transporte Biológico/fisiologia , Organelas/metabolismoRESUMO
Cell adhesion molecule-related/downregulated by oncogenes (Cdon) is a cell-surface receptor that mediates cell-cell interactions and positively regulates myogenesis. The cytoplasmic region of Cdon interacts with other proteins to form a Cdon/JLP/Bnip-2/CDC42 complex that activates p38 mitogen-activated protein kinase (MAPK) and induces myogenesis. However, Cdon complex may include other proteins during myogenesis. In this study, we found that Cullin 2-interacting protein zinc finger SWIM type containing 8 (ZSWIM8) ubiquitin ligase is induced during C2C12 differentiation and is included in the Cdon complex. We knocked-down Zswim8 in C2C12 cells to determine the effect of ZSWIM8 on differentiation. However, we detected neither ZSWIM8-dependent ubiquitination nor the degradation of Bnip2, Cdon, or JLP. In contrast, ZSWIM8 knockdown accelerated C2C12 differentiation. These results suggest that ZSWIM8 is a Cdon complex-included myogenic protein that prevents C2C12 differentiation without affecting the stability of Bnip2, Cdon, and JLP.
Assuntos
Diferenciação Celular/fisiologia , Desenvolvimento Muscular/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Células K562 , Sistema de Sinalização das MAP Quinases/fisiologia , Ligação Proteica/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The breast cancer susceptibility protein BRCA1 and its partner BRCA1-associated RING domain protein 1 (BARD1) form an E3-ubiquitin (Ub) ligase complex that acts as a tumor suppressor in mitotic cells. However, the roles of BRCA1-BARD1 in postmitotic cells, such as neurons, remain poorly defined. Here, we report that BRC-1 and BRD-1, the Caenorhabditis elegans orthologs of BRCA1 and BARD1, are required for adult-specific axon regeneration, which is positively regulated by the EGL-30 Gqα-diacylglycerol (DAG) signaling pathway. This pathway is downregulated by DAG kinase (DGK), which converts DAG to phosphatidic acid (PA). We demonstrate that inactivation of DGK-3 suppresses the brc-1 brd-1 defect in axon regeneration, suggesting that BRC-1-BRD-1 inhibits DGK-3 function. Indeed, we show that BRC-1-BRD-1 poly-ubiquitylates DGK-3 in a manner dependent on its E3 ligase activity, causing DGK-3 degradation. Furthermore, we find that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. These results suggest that the BRC-1-BRD-1 complex regulates axon regeneration in concert with the Gqα-DAG signaling network. Thus, this study describes a new role for breast cancer proteins in fully differentiated neurons and the molecular mechanism underlying the regulation of axon regeneration in response to nerve injury.SIGNIFICANCE STATEMENT BRCA1-BRCA1-associated RING domain protein 1 (BARD1) is an E3-ubiquitin (Ub) ligase complex acting as a tumor suppressor in mitotic cells. The roles of BRCA1-BARD1 in postmitotic cells, such as neurons, remain poorly defined. We show here that Caenorhabditis elegans BRC-1/BRCA1 and BRD-1/BARD1 are required for adult-specific axon regeneration, a process that requires high diacylglycerol (DAG) levels in injured neurons. The DAG kinase (DGK)-3 inhibits axon regeneration by reducing DAG levels. We find that BRC-1-BRD-1 poly-ubiquitylates and degrades DGK-3, thereby keeping DAG levels elevated and promoting axon regeneration. Furthermore, we demonstrate that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. Thus, this study describes a new role for BRCA1-BARD1 in fully-differentiated neurons.
Assuntos
Axônios/metabolismo , Diacilglicerol Quinase/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regeneração Nervosa/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Células COS , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Chlorocebus aethiops , Diacilglicerol Quinase/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
In Caenorhabditis elegans, the JNK MAP kinase (MAPK) pathway is important for axon regeneration. The JNK pathway is activated by a signaling cascade consisting of the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury in a process involving the transcription factors ETS-4 and CEBP-1. Here, we find that svh-14/mxl-1, a gene encoding a Max-like transcription factor, is required for activation of svh-2 expression in response to axonal injury. We show that MXL-1 binds to and inhibits the function of TDPT-1, a C. elegans homolog of mammalian tyrosyl-DNA phosphodiesterase 2 [TDP2; also called Ets1-associated protein II (EAPII)]. Deletion of tdpt-1 suppresses the mxl-1 defect, but not the ets-4 defect, in axon regeneration. TDPT-1 induces SUMOylation of ETS-4, which inhibits ETS-4 transcriptional activity, and MXL-1 counteracts this effect. Thus, TDPT-1 interacts with two different transcription factors in axon regeneration.
Assuntos
Axônios/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Regeneração Nervosa , Diester Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Modelos Biológicos , Neurônios Motores/metabolismo , Fosforilação , Ligação Proteica , Transcrição GênicaRESUMO
Ligand-induced activation of epidermal growth factor receptor (EGFR) initiates trafficking events that re-localize the receptor from the cell surface to intracellular endocytic compartments. EGFR-containing endosomes are transported to lysosomes for degradation by the dynein-dynactin motor protein complex. However, this cargo-dependent endosomal trafficking mechanism remains largely uncharacterized. Here, we show that GTP-bound Rab7 is phosphorylated on S72 by leucine-rich repeat kinase 1 (LRRK1) at the endosomal membrane. This phosphorylation promotes the interaction of Rab7 (herein referring to Rab7a) with its effector RILP, resulting in recruitment of the dynein-dynactin complex to Rab7-positive vesicles. This, in turn, facilitates the dynein-driven transport of EGFR-containing endosomes toward the perinuclear region. These findings reveal a mechanism regulating the cargo-specific trafficking of endosomes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Receptores ErbB/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosforilação , Transporte Proteico/fisiologia , proteínas de unión al GTP Rab7RESUMO
The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the mechanisms regulating axon regeneration are not well understood. Here, we identify the brc-2 gene encoding a homolog of the mammalian BRCA2 tumor suppressor as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that the RHO-1/Rho GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC-4/MLC) phosphorylation signaling pathway regulates axon regeneration. BRC-2 functions between RHO-1 and LET-502, suggesting that BRC-2 is required for the activation of LET-502 by RHO-1-GTP. We also find that one component that interacts with BRC-2, the ALP (α-actinin-associated LIM protein)/Enigma protein ALP-1, is required for regeneration and acts between LET-502 and MLC-4 phosphorylation. Furthermore, we demonstrate that ALP-1 associates with LET-502 and MLC-4. Thus, ALP-1 serves as a platform to activate MLC-4 phosphorylation mediated by the RHO-1-LET-502 signaling pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Proteínas com Domínio LIM/genética , Cadeias Leves de Miosina/genética , Regeneração Nervosa/genética , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Axotomia/métodos , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas com Domínio LIM/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Cadeias Leves de Miosina/metabolismo , Crescimento Neuronal/genética , Plasticidade Neuronal/genética , Fosforilação , Ligação Proteica , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
The molecular mechanisms underlying the ability of axons to regenerate after injury remain poorly understood. Here we show that in Caenorhabditis elegans, axotomy induces ectopic expression of serotonin (5-HT) in axotomized non-serotonergic neurons via HIF-1, a hypoxia-inducible transcription factor, and that 5-HT subsequently promotes axon regeneration by autocrine signalling through the SER-7 5-HT receptor. Furthermore, we identify the rhgf-1 and rga-5 genes, encoding homologues of RhoGEF and RhoGAP, respectively, as regulators of axon regeneration. We demonstrate that one pathway initiated by SER-7 acts upstream of the C. elegans RhoA homolog RHO-1 in neuron regeneration, which functions via G12α and RHGF-1. In this pathway, RHO-1 inhibits diacylglycerol kinase, resulting in an increase in diacylglycerol. SER-7 also promotes axon regeneration by activating the cyclic AMP (cAMP) signalling pathway. Thus, HIF-1-mediated activation of 5-HT signalling promotes axon regeneration by activating both the RhoA and cAMP pathways.
Assuntos
Axônios/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regeneração Nervosa , Doenças Neurodegenerativas/metabolismo , Serotonina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Axotomia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. In Caenorhabditis elegans, the JNK and p38 MAPK pathways are important for axon regeneration. Axonal injury induces expression of the svh-2 gene encoding a receptor tyrosine kinase, stimulation of which by the SVH-1 growth factor leads to activation of the JNK pathway. Here, we identify ETS-4 and CEBP-1, related to mammalian Ets and C/EBP, respectively, as transcriptional activators of svh-2 expression following axon injury. ETS-4 and CEBP-1 function downstream of the cAMP and Ca2+-p38 MAPK pathways, respectively. We show that PKA-dependent phosphorylation of ETS-4 promotes its complex formation with CEBP-1. Furthermore, activation of both cAMP and Ca2+ signaling is required for activation of svh-2 expression. Thus, the cAMP/Ca2+ signaling pathways cooperatively activate the JNK pathway, which then promotes axon regeneration.
Assuntos
Axônios/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Caenorhabditis elegans/genética , Regeneração/genética , Fatores de Transcrição/genética , Animais , Axônios/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/biossíntese , Sinalização do Cálcio/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Sistema de Sinalização das MAP Quinases/genética , Neurônios/metabolismo , Fosforilação , Transdução de Sinais/genética , Fatores de Transcrição/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
MAP kinases are integral to the mechanisms by which cells respond to a wide variety of environmental stresses. In Caenorhabditis elegans, the KGB-1 JNK signaling pathway regulates the response to heavy metal stress. In this study, we identified FOS-1, a bZIP transcription factor, as a target of KGB-1-mediated phosphorylation. We further identified two transcriptional targets of the KGB-1 pathway, kreg-1 and kreg-2/lys-3, which are required for the defense against heavy metal stress. FOS-1 plays a critical role in the transcriptional repression of the kreg-1 gene by recruiting histone deacetylase (HDAC) to its promoter. KGB-1 phosphorylation prevents FOS-1 dimerization and promoter binding, resulting in promoter derepression. Thus, HDAC behaves as a co-repressor modulating FOS-1-mediated transcriptional regulation. This study describes the direct link from JNK signaling, Fos phosphorylation, and regulation of kreg gene transcription, which modulates the stress response in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Histona Desacetilases , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-fos , Estresse Fisiológico/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/fisiologia , Ativação TranscricionalRESUMO
Mutation of leucine-rich repeat kinase 2 (LRRK2) is the leading genetic cause of Parkinson's Disease (PD), manifested as age-dependent dopaminergic neurodegeneration, but the underlying molecular mechanisms remain unclear. Multiple roles of LRRK2 may contribute to dopaminergic neurodegeneration. Endoplasmic reticulum (ER) stress has also been linked to PD pathogenesis, but its interactive mechanism with PD genetic factors is largely unknown. Here, we used C. elegans, human neuroblastoma cells and murine cortical neurons to determine the role of LRRK2 in maintaining dopaminergic neuron viability. We found that LRRK2 acts to protect neuroblastoma cells and C. elegans dopaminergic neurons from the toxicity of 6-hydroxydopamine and/or human α-synuclein, possibly through the p38 pathway, by supporting upregulation of GRP78, a key cell survival molecule during ER stress. A pathogenic LRRK2 mutant (G2019S), however, caused chronic p38 activation that led to death of murine neurons and age-related dopaminergic-specific neurodegeneration in nematodes. These observations establish a critical functional link between LRRK2 and ER stress.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Neurônios Dopaminérgicos/metabolismo , Estresse do Retículo Endoplasmático , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular Tumoral , Células Cultivadas , Neurônios Dopaminérgicos/patologia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Microscopia Confocal , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Degeneração Neural/induzido quimicamente , Oxidopamina , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Kinesin-1 is a microtubule plus-end-directed motor that transports various cargos along the axon. Previous studies have elucidated the physical and genetic interactions between kinesin-1 and cytoplasmic dynein, a microtubule minus-end-directed motor, in neuronal cells. However, the physiological importance of kinesin-1 in the dynein-dependent retrograde transport of cargo molecules remains obscure. Here, we show that Caenorhabditis elegans kinesin-1 forms a complex with dynein via its interaction with UNC-16, which binds to the dynein light intermediate (DLI) chain. Both kinesin-1 and UNC-16 are required for localization of DLI-1 at the plus ends of nerve process microtubules. In addition, retrograde transport of APL-1 depends on kinesin-1, UNC-16, and dynein. These results suggest that kinesin-1 mediates the anterograde transport of dynein using UNC-16 as a scaffold and that dynein in turn mediates the retrograde transport of cargo molecules in vivo. Thus, UNC-16 functions as an adaptor for kinesin-1-mediated transport of dynein.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Dineínas do Citoplasma/metabolismo , Cinesinas/metabolismo , Animais , Animais Geneticamente Modificados , Transporte Biológico , Células COS , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Chlorocebus aethiops , Dineínas do Citoplasma/genética , Dineínas/metabolismo , Regulação da Expressão Gênica/fisiologia , Imunoprecipitação/métodos , Cinesinas/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mecanorreceptores/citologia , Mecanorreceptores/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microinjeções/métodos , Terminações Pré-Sinápticas/metabolismo , Ligação Proteica , Transporte Proteico/genética , Transfecção/métodosRESUMO
It has not been determined yet whether the ERK-MAPK pathway regulates longevity of metazoans. Here, we show that the Caenorhabditis elegans ERK cascade promotes longevity through the two longevity-promoting transcription factors, SKN-1 and DAF-16. We find that RNAi of three genes, which constitute the ERK cascade (lin-45/RAF1, mek-2/MEK1/2, and mpk-1/ERK1/2), results in reduction of life span. Moreover, RNAi of lip-1, the gene encoding a MAPK phosphatase that inactivates MPK-1, increases life span. Epistasis analyses show that the ERK (MPK-1) cascade-mediated life span extension requires SKN-1, whose function is mediated, at least partly, through DAF-2/DAF-16 insulin-like signaling. MPK-1 phosphorylates SKN-1 on the key sites that are required for SKN-1 nuclear accumulation. Our results also show that one mechanism by which SKN-1 regulates insulin-like signaling is through the regulation of expression of insulin-like peptides. Our findings thus identify a novel ERK-MAPK-mediated signaling pathway that promotes longevity.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Longevidade/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Receptor de Insulina/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Epistasia Genética/fisiologia , Fatores de Transcrição Forkhead , Insulina/genética , Insulina/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação/fisiologia , Receptor de Insulina/genética , Fatores de Transcrição/genética , Quinases raf/genética , Quinases raf/metabolismoRESUMO
Fos and Jun are components of activator protein-1 (AP-1) and play crucial roles in the regulation of many cellular, developmental, and physiological processes. Caenorhabditis elegans fos-1 has been shown to act in uterine and vulval development. Here, we provide evidence that C. elegans fos-1 and jun-1 control ovulation, a tightly regulated rhythmic program in animals. Knockdown of fos-1 or jun-1 blocks dilation of the distal spermathecal valve, a critical step for the entry of mature oocytes into the spermatheca for fertilization. Furthermore, fos-1 and jun-1 regulate the spermathecal-specific expression of plc-1, a gene that encodes a phospholipase C (PLC) isozyme that is rate-limiting for inositol triphosphate production and ovulation, and overexpression of PLC-1 rescues the ovulation defect in fos-1(RNAi) worms. Unlike fos-1, regulation of ovulation by jun-1 requires genetic interactions with eri-1 and lin-15B, which are involved in the RNA interference pathway and chromatin remodeling, respectively. At least two isoforms of jun-1 are coexpressed with fos-1b in the spermatheca, and different AP-1 dimers formed between these isoforms have distinct effects on the activation of a reporter gene. These findings uncover a novel role for FOS-1 and JUN-1 in the reproductive system and establish C. elegans as a model for studying AP-1 dimerization.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , Regulação da Expressão Gênica , Ovulação/fisiologia , Fosfoinositídeo Fosfolipase C/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Montagem e Desmontagem da Cromatina , Inositol 1,4,5-Trifosfato/metabolismo , Mutação , Fosfoinositídeo Fosfolipase C/genética , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and to a wide variety of environmental stresses. MAPK cascades can be inactivated at the MAPK activation step by members of the MAPK phosphatase (MKP) family. However, the components that act in MKP-regulated pathways have not been well characterized in the context of whole organisms. Here we characterize the Caenorhabditis elegans vhp-1 gene, encoding an MKP that acts preferentially on the c-Jun N-terminal kinase (JNK) and p38 MAPKs. We found that animals defective in vhp-1 are arrested during larval development. This vhp-1 defect is suppressed by loss-of-function mutations in the kgb-1, mek-1, and mlk-1 genes encoding a JNK-like MAPK, an MKK7-type MAPKK, and an MLK-type MAPKKK, respectively. The genetic and biochemical data presented here demonstrate a critical role for VHP-1 in the KGB-1 pathway. Loss-of-function mutations in each component in the KGB-1 pathway result in hypersensitivity to heavy metals. These results suggest that VHP-1 plays a pivotal role in the integration and fine-tuning of the stress response regulated by the KGB-1 MAPK pathway.