RESUMEN
Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.
Asunto(s)
Proteínas Portadoras/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Lipoilación/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipoilación/efectos de los fármacos , Ratones , Propiolactona/análogos & derivados , Propiolactona/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas RGS/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
The copper (Cu) exporter ATP7B mediates resistance to cisplatin (cDDP) but details of the mechanism are unknown. We explored the role of the CXXC motifs in the metal binding domains (MBDs) of ATP7B by investigating binding of cDDP to the sixth metal binding domain (MBD6) or a variant in which the CXXC motif was converted to SXXS. Platinum measurement showed that cDDP bound to wild type MBD6 but not to the SXXS variant. Wild type ATP7B rendered ovarian 2008 cells resistant to cDDP. In 2008 and in HEK293T cells, wild type ATP7B trafficked from TGN to peripheral locations in response to Cu or cDDP. A variant in which the CXXC motifs in all 6 MBDs were converted to SXXS localized correctly to the TGN but failed to traffic when exposed to either Cu or cDDP. Deletion of either the first 5 MBDs or all 6 MBDs resulted in failure to localize to the TGN. Neither the SXXS variant nor the deletion variant was able to mediate resistance to cDDP. We conclude that cDDP binds to the CXXC motifs of ATP7B and that this interaction is essential to the trafficking of ATP7B and to its ability to mediate resistance to cDDP.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Antineoplásicos/farmacología , Proteínas de Transporte de Catión/metabolismo , Cisplatino/farmacología , Resistencia a Antineoplásicos , Adenosina Trifosfatasas/genética , Secuencias de Aminoácidos , Proteínas de Transporte de Catión/genética , Línea Celular Tumoral , ATPasas Transportadoras de Cobre , Células HEK293 , Humanos , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Previous work has demonstrated that the copper (Cu) transporters Ctr1, Atp7a and Atp7b regulate the cellular pharmacology of cisplatin (CDDP) by mediating its uptake and efflux. It was also shown that, in the process of uptake by Ctr1, CDDP triggers the rapid proteasomal degradation of its own transporter. The current study examined the role of the metallochaperone Atox1 in the regulation of uptake, efflux and subcellular distribution of CDDP by using a pair of fibroblast cell lines established from Atox1(+/+) and Atox1(-/-) mice. Atox1 is a metallochaperone that is known to play a central role in distributing Cu within the cells and was recently shown to act as a Cu-dependent transcription factor. Loss of Atox1 increased Cu accumulation and reduced efflux. In contrast, loss of Atox1 reduced the influx of CDDP and subsequent accumulation in vesicular compartments and in DNA. Loss of Atox1 was found to block the CDDP-induced down regulation of Ctr1. Ctr1 was found to be polyubiquitinated in an Atox1-dependent manner during CDDP exposure. In conclusion, Atox1 is required for the polyubiquitination of Ctr1 and the Ctr1-mediated uptake of CDDP.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Transporte de Catión/metabolismo , Cisplatino/farmacología , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Antineoplásicos/metabolismo , Transporte Biológico , Proteínas de Transporte de Catión/genética , Línea Celular , Cisplatino/metabolismo , Cobre/farmacología , Proteínas Transportadoras de Cobre , Aductos de ADN/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Poliubiquitina/metabolismo , UbiquitinaciónRESUMEN
Abscisic acid (ABA) plays a key role in plant responses to abiotic stress, particularly drought stress. A wide number of ABA-hypersensitive mutants is known, however, only a few of them resist/avoid drought stress. In this work we have generated ABA-hypersensitive drought-avoidant mutants by simultaneous inactivation of two negative regulators of ABA signaling, i.e. the protein phosphatases type 2C (PP2Cs) ABA-INSENSITIVE1 (ABI1) and HYPERSENSITIVE TO ABA1 (HAB1). Two new recessive loss-of-function alleles of ABI1, abi1-2 and abi1-3, were identified in an Arabidopsis (Arabidopsis thaliana) T-DNA collection. These mutants showed enhanced responses to ABA both in seed and vegetative tissues, but only a limited effect on plant drought avoidance. In contrast, generation of double hab1-1 abi1-2 and hab1-1 abi1-3 mutants strongly increased plant responsiveness to ABA. Thus, both hab1-1 abi1-2 and hab1-1 abi1-3 were particularly sensitive to ABA-mediated inhibition of seed germination. Additionally, vegetative responses to ABA were reinforced in the double mutants, which showed a strong hypersensitivity to ABA in growth assays, stomatal closure, and induction of ABA-responsive genes. Transpirational water loss under drought conditions was noticeably reduced in the double mutants as compared to single parental mutants, which resulted in reduced water consumption of whole plants. Taken together, these results reveal cooperative negative regulation of ABA signaling by ABI1 and HAB1 and suggest that fine tuning of ABA signaling can be attained through combined action of PP2Cs. Finally, these results suggest that combined inactivation of specific PP2Cs involved in ABA signaling could provide an approach for improving crop performance under drought stress conditions.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Agua/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Mutación , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/fisiología , Hojas de la Planta/anatomía & histología , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Transpiración de Plantas/fisiología , Transducción de SeñalRESUMEN
To identify new loci in abscisic acid (ABA) signaling, we screened a library of 35ScDNA Arabidopsis (Arabidopsis thaliana)-expressing lines for ABA-insensitive mutants in seed germination assays. One of the identified mutants germinated on 2.5 microm ABA, a concentration that completely inhibits wild-type seed germination. Backcrosses and F2 analyses indicated that the mutant exhibits a dominant phenotype and that the ABA insensitivity was linked to a single T-DNA insertion containing a 35ScDNA fusion. The inserted cDNA corresponds to a full-length cDNA of the AtPP2CA gene, encoding a protein phosphatase type 2C (PP2C). Northern-blot analyses demonstrated that the AtPP2CA transcript is indeed overexpressed in the mutant (named PP2CAox). Two independent homozygous T-DNA insertion lines, pp2ca-1 and pp2ca-2, were recovered from the Arabidopsis Biological Resource Center and shown to lack full-length AtPP2CA expression. A detailed characterization of PP2CAox and the T-DNA disruption mutants demonstrated that, whereas ectopic expression of a 35SAtPP2CA fusion caused ABA insensitivity in seed germination and ABA-induced stomatal closure responses, disruption mutants displayed the opposite phenotype, namely, strong ABA hypersensitivity. Thus our data demonstrate that the PP2CA protein phosphatase is a strong negative regulator of ABA signal transduction. Furthermore, it has been previously shown that the AtPP2CA transcript is down-regulated in the ABA-hypersensitive nuclear mRNA cap-binding protein mutant abh1. We show here that down-regulation of AtPP2CA in abh1 is not due to impaired RNA splicing of AtPP2CA pre-mRNA. Moreover, expression of a 35SAtPP2CA cDNA fusion in abh1 partially suppresses abh1 hypersensitivity, and the data further suggest that additional mechanisms contribute to ABA hypersensitivity of abh1.