RESUMEN
Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.
RESUMEN
The immunomodulatory drug (IMiD) thalidomide and its derivatives lenalidomide and pomalidomide are therapeutic agents used in the treatment of multiple myeloma. Although pomalidomide offers considerable clinical benefits to patients with lenalidomide-resistant multiple myeloma, the molecular mechanisms underlying its superior efficacy remain unclear. Here we show that ARID2, a component of the polybromo-associated BAF (PBAF) chromatin-remodeling complex, is a pomalidomide-induced neosubstrate of CRL4CRBN. BRD7, another subunit of PBAF, is critical for pomalidomide-induced ARID2 degradation. ARID2 is involved in transcriptional regulation of pomalidomide target genes including MYC. Pomalidomide is more effective than lenalidomide in degrading ARID2 and is capable of inhibiting MYC expression and proliferation in lenalidomide-resistant cell lines. Notably, ARID2 expression is associated with a poor prognosis and is higher in chemoresistant minimal residual disease (MRD) populations, and in patients with relapsed/refractory multiple myeloma. These findings suggest that ARID2 is a promising target for overcoming lenalidomide resistance in patients with multiple myeloma.
Asunto(s)
Antineoplásicos/farmacología , Mieloma Múltiple/metabolismo , Talidomida/análogos & derivados , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Antineoplásicos/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Lenalidomida/farmacología , Mieloma Múltiple/tratamiento farmacológico , Mutación , Unión Proteica , Proteolisis/efectos de los fármacos , ARN Mensajero , ARN Interferente Pequeño , Talidomida/metabolismo , Talidomida/farmacología , Factores de Tiempo , Factores de Transcripción/genética , UbiquitinaciónAsunto(s)
Epítopos/genética , Vectores Genéticos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Epítopos/metabolismo , Expresión Génica , Marcación de Gen , Recombinación Homóloga , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoAsunto(s)
Marcadores Genéticos/genética , Mutagénesis Insercional/métodos , Plásmidos/genética , Saccharomyces cerevisiae/genética , Vectores Genéticos , Mutagénesis Insercional/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Selección GenéticaRESUMEN
Skp1/Cul1/F-box (SCF)-type F-box proteins are a component of the Cullin-RING SCF ubiquitin E3 ligase, which is involved in numerous cellular processes. However, the function of non-SCF-type F-box proteins remains largely unknown. The Rab5-like small guanosine 5'-triphosphatase Vps21/Ypt51 is a key regulator of intracellular transportation; however, deletion of its isoforms, Ypt52 and Ypt53, results in only a modest inhibition of intracellular trafficking. The function of these proteins therefore remains largely elusive. Here we analyze the role of a previously uncharacterized non-SCF-type F-box protein, Roy1/Ymr258c, in cell growth and intracellular transport in Saccharomyces cerevisiae. Roy1 binds to Ypt52 under physiological conditions, and Skp1 is indispensable for the association of Roy1 with Ypt52. The vps21Δ yeast cells exhibit severe deficiencies in cell growth and intracellular trafficking, whereas simultaneous deletion of roy1 alleviates the defects caused by deletion of vps21. However, additional disruption of ypt52 in roy1Δvps21Δ cells largely suppresses the cell growth and trafficking observed in roy1Δvps21Δ cells. We demonstrate that Roy1 interacts with guanosine 5'-diphosphate-bound and nucleotide-free Ypt52 and thereby inhibits the formation of guanosine 5'-triphosphate-bound, active Ypt52. These results thus indicate that Roy1 negatively modulates cell viability and intracellular transport by suppressing Ypt52.
Asunto(s)
Proteínas F-Box/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rab5/antagonistas & inhibidores , Eliminación de Gen , Células HEK293 , Humanos , Unión Proteica , Proteínas Ligasas SKP Cullina F-box/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Eliminación de Secuencia/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Of 20 natural amino acids, leucine is particularly important for promoting cellular protein synthesis. The effect of leucine involves mammalian target of rapamycin (mTOR), a key protein kinase controlling cell growth. Leucine enhances mTOR-mediated phosphorylation of S6K1 and 4E-BP, thereby promoting protein synthesis. However, how the presence of leucine is sensed and transmitted to mTOR is poorly understood. Here, we show evidence that UBR1 and UBR2 might be cellular targets of leucine. UBR1 and UBR2 are E3 ubiquitin ligases that recognize the identity of N-terminal residues and contribute to selective destabilization of target proteins according to the N-end rule. Using leucine-immobilized affinity beads, we identified UBR1 and UBR2 as leucine-binding proteins from leucine-responsive rat hepatoma H4IIE cells. Over-expression of UBR1 or UBR2 resulted in a reduction in mTOR-dependent S6K1 phosphorylation, whereas knockdown of UBR1 or UBR2 increased S6K1 phosphorylation in amino acid-starved human 293T cells. We also found that leucine binds to the substrate-recognition domain of UBR2 and inhibits degradation of N-end rule substrates in vitro. These findings suggest that UBR1 and UBR2 are negative regulators of the leucine-mTOR signaling pathway. Leucine might activate this pathway in part through inhibition of their ubiquitin ligase activity.
Asunto(s)
Ligasas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Ubiquitina-Proteína Ligasas , Animales , Línea Celular , Humanos , Leucina/genética , Leucina/metabolismo , Ligasas/genética , Mamíferos/genética , Mamíferos/metabolismo , Fosforilación , Biosíntesis de Proteínas , Ratas , Transducción de Señal/genética , Sirolimus , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Dia2 is an F-box protein, which is involved in the regulation of DNA replication in the budding yeast Saccharomyces cerevisiae. The function of Dia2, however, remains largely unknown. In this study, we report that Dia2 is associated with the replication fork and regulates replication fork progression. Using modified yeast two-hybrid screening, we have identified components of the replisome (Mrc1, Ctf4 and Mcm2), as Dia2-binding proteins. Mrc1 and Ctf4 were ubiquitinated by SCF(Dia2) both in vivo and in vitro. Domain analysis of Dia2 revealed that the leucine-rich repeat motif was indispensable for the regulation of replisome progression, whereas the tetratricopeptide repeat (TPR) motif was involved in the interaction with replisome components. In addition, the TPR motif was shown to be involved in Dia2 stability; deleting the TPR stabilized Dia2, mimicking the effect of DNA damage. ChIP-on-chip analysis illustrated that Dia2 localizes to the replication fork and regulates fork progression on hydroxyurea treatment. These results demonstrate that Dia2 is involved in the regulation of replisome activity through a direct interaction with replisome components.
Asunto(s)
ADN Superhelicoidal/metabolismo , Proteínas F-Box/química , Proteínas F-Box/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos/fisiología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/fisiología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas F-Box/metabolismo , Leucina/metabolismo , Leucina/fisiología , Estabilidad Proteica , Estructura Terciaria de Proteína/fisiología , Secuencias Repetitivas de Aminoácido/fisiología , Fase S/genética , Fase S/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
SCF-type E3-ubiquitin ligases control numerous cellular processes through the ubiquitin-proteasome pathway. However, the regulation of SCF function remains largely uncharacterized. Here, we report a novel SCF complex-interacting protein, Lag2, in Saccharomyces cerevisiae. Lag2 interacts with the SCF complex under physiological conditions. Lag2 negatively controls the ubiquitylation activities of SCF E3 ligase by interrupting the association of Cdc34 to SCF complex. Overexpression of Lag2 increases unrubylated Cdc53, whereas deletion of lag2, together with the deletions of dcn1 and jab1, results in the accumulation of Rub1-modified Cdc53. In vitro rubylation assays show that Lag2 inhibits the conjugation of Rub1 to Cdc53 in competition with Dcn1, which suggest that Lag2 down-regulates the rubylation of Cdc53 rather than promoting derubylation. Furthermore, Dcn1 hinders the association of Lag2 to Cdc53 in vivo. Finally, the deletion of lag2 combined with the deletion of either dcn1 or rub1 suppresses the growth of yeast cells. These observations thus indicate that Lag2 has a significant function in regulating the SCF complex by controlling its ubiquitin ligase activities and its rubylation cycle.
Asunto(s)
Proteínas Cullin/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinación , Proliferación Celular , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Fenotipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/genéticaRESUMEN
Ubiquitin-dependent degradation is implicated in various cellular regulatory mechanisms. The SCF(Cdc4) (Skp1, Cullin/Cdc53, and the F-box protein Cdc4) complex is an ubiquitin ligase complex that acts as a regulator of cell cycle, signal transduction, and transcription. These regulatory mechanisms are not well defined because of the difficulty in identifying the interaction between ubiquitin ligases and their substrates. To identify substrates of the yeast SCF(Cdc4) ubiquitin ligase complex, we refined the yeast two-hybrid system to allow screening Cdc4-substrate interactions under conditions of substrate stabilization, and identified Swi5 as a substrate of the SCF(Cdc4) complex. Swi5 is the transcriptional activator of Sic1, the inhibitor of S phase cyclin-dependent kinases (CDKs). We showed that Swi5 is indeed ubiquitinated and degraded through the SCF(Cdc4) complex. Furthermore, the SCF(Cdc4)-dependent degradation of Swi5 was required to terminate SIC1 transcription at early G(1) phase, which ensured efficient entry into S phase: Hyperaccumulation of Sic1 was noted in cells expressing stabilized Swi5, and expression of stabilized Swi5 delayed S phase entry, which was dominantly suppressed by SIC1 deletion. These findings indicate that the SCF(Cdc4) complex regulates S phase entry not only through degradation of Sic1, but also through degradation of Swi5.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Fase S , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Cullin/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Ciclinas/metabolismo , Proteínas F-Box/metabolismo , Fase G1 , Regulación de la Expresión Génica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos Híbridos , UbiquitinaciónRESUMEN
A change in the protein level of RCAN1 (DSCR1/MCIP/Adapt78/CSP1) has been implicated in oxidative stress-induced cell death in neurons and in the pathogenesis of Alzheimer's disease. The pathogenic processes in neurodegenerative diseases are closely related to oxidative stress and the ubiquitin proteasome system (UPS). Therefore, we investigated whether oxidative stress induces a change in the protein level of RCAN1 through the UPS. H2O2 induced ubiquitination of RCAN1 at the same concentrations as those causing a decrease in RCAN1 in HEK293T cells. beta-TrCP, the F-box protein component of SCF ubiquitin ligase, interacted with RCAN1 in response to H2O2 stimulation. Although FBW4, another F-box protein, interacted with RCAN1, its interaction was independent of H2O2 stimulation. In vitro ubiquitination assay showed that SCFbeta-TrCP but not SCFFBW4 increased ubiquitination of RCAN1, dependent on H2O2 stimulation. In addition, knockdown of beta-TrCP by siRNA abolished the H2O2-induced decrease in RCAN1 in HEK293T cells. We further examined whether RCAN1 undergoes ubiquitination by H2O2 in primary neurons, similarly to that in HEK293T cells. An H2O2-induced decrease in RCAN1 was exhibited also in hippocampal and cortical neurons. Ubiquitination of RCAN1 was induced by 500 muM H2O2, the concentration at which H2O2 induced a decrease in RCAN1 in primary neurons. These results suggest that H2O2 induces SCF beta-TrCP-mediated ubiquitination of RCAN1, leading to a decrease in the protein level of RCAN1.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/metabolismo , Estrés Oxidativo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitinación , Animales , Línea Celular , Proteínas de Unión al ADN , Humanos , Peróxido de Hidrógeno/farmacología , Ratones , Neuronas/metabolismo , ARN Interferente Pequeño/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismoRESUMEN
Apoptosis is often observed in developing tissues. However, it remains unclear how the apoptotic pathway is regulated during development. To clarify this issue, we isolated zebrafish mutants that show extensive apoptosis of retinal cells during their development. pinball eye (piy) is one such mutant, in which retinal stem cells proliferate normally but almost all retinal neurons undergo apoptosis during differentiation. We found that a missense mutation occurred in the small subunit of DNA primase (Prim1) in the piy mutant. DNA primase is essential for DNA replication; however, this mutation does not affect cell proliferation but rather induces neuronal apoptosis. RNA synthesis catalyzed by Prim1 is important for the activation of the DNA damage response, which may activate Ataxia telangiectasia mutated (ATM), Checkpoint kinase 2 (Chk2) and the tumor suppressor p53. We found that the apoptosis induced by the prim1 mutation depends on the ATM-Chk2-p53 apoptotic pathway. These data suggest that the surveillance system of genome integrity strongly influences the cell fate decision between differentiation and apoptosis during retinal neurogenesis in zebrafish.
Asunto(s)
Apoptosis , ADN Primasa/genética , Mutación Missense , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Animales Modificados Genéticamente , Quinasa de Punto de Control 2 , Daño del ADN , Embrión no Mamífero , Activación Enzimática/genética , Modelos Biológicos , Neuronas/patología , Retina/citología , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
The highly conserved RCN family of proteins regulates the serine/threonine protein phosphatase calcineurin, which is required for the expression of genes involved in Ca(2+)-dependent processes, such as the control of memory, apoptosis, T cell activation, cell cycle, Ca(2+)-homeostasis, and skeletal and cardiac muscle growth and differentiation. However, RCNs regulate calcineurin through two paradoxical actions: they act as feedback inhibitors of calcineurin, whereas their phosphorylation stimulates calcineurin. Here we show that phosphorylation of yeast RCN, Rcn1, triggers degradation through the SCF(Cdc4) ubiquitin ligase complex. Degradation of phosphorylated Rcn1 is required to mitigate inhibition of calcineurin by Rcn1 and results in activation of calcineurin activity in response to Ca(2+) as well as in reactivation of calcineurin in response to changes in Ca(2+) concentration. The SCF(Cdc4)-dependent degradation required phosphorylation of Rcn1 by Mck1, a member of the GSK3 family of protein kinases, and was promoted by Ca(2+). However, such degradation was counteracted by dephosphorylation of Rcn1, which was promoted by Ca(2+)-stimulated calcineurin. Thus, calcineurin activity is fine-tuned to Ca(2+) signals by mechanisms that have opposite functions. Our results identify the molecular mechanism of Rcn1 phosphorylation-induced stimulation of the phosphatase activity of calcineurin. The results provide insight into the mechanism involved in maintaining proper responses to Ca(2+) signals.
Asunto(s)
Inhibidores de la Calcineurina , Calcineurina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Calcio/farmacología , Proteínas de Ciclo Celular/genética , Proteínas F-Box , Péptidos y Proteínas de Señalización Intracelular , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The members of the transcription factor Foxo family regulate the expression of genes concerned with the stress response, cell cycle and gluconeogenesis. Foxo1 (FKHR) contains 15 consensus phosphorylation sites for the mitogen-activated protein kinase (MAPK) family. Therefore, we hypothesized that MAPKs could directly regulate the transcriptional activity of Foxo1 via phosphorylation. In vitro kinase assay showed that Foxo1 was phosphorylated by extracellular signal-regulated kinase (Erk) and p38 MAPK (p38) but not by c-jun NH2-terminal kinase (JNK). In NIH3T3 cells, epidermal growth factor or anisomycin increased phosphorylation of exogenous Foxo1, which was significantly inhibited by pretreatment with an MEK 1 inhibitor, PD98059, or a p38 inhibitor, SB203580. Two-dimensional phosphopeptide mapping using mutation of phosphorylation sites for MAPK revealed that the nine serine residues in Foxo1 are specifically phosphorylated by Erk and that five of the nine residues are phosphorylated by p38 in vivo. Moreover, we also found that Foxo1 interacts with Ets-1 and functions as a coactivator for Ets-1 on the fetal liver kinase (Flk)-1 promoter in bovine carotid artery endothelial cells. Mutation of the nine phosphorylation sites for Erk in Foxo1 was shown to lead to less binding and synergistic activity for Ets-1 on the Flk-1 promoter when compared with wild-type Foxo1. These results suggest that Foxo1 is specifically phosphorylated by Erk and p38, and that this phosphorylation regulates the function of Foxo1 as a coactivator for Ets-1.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Anisomicina/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Línea Celular , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Flavonoides/farmacología , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Genes Reporteros , Humanos , Imidazoles/farmacología , Luciferasas/metabolismo , Ratones , Células 3T3 NIH , Fosforilación , Inhibidores de la Síntesis de la Proteína/farmacología , Piridinas/farmacologíaRESUMEN
Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4+ cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11+ cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.