RESUMEN
Extracellular signal-regulated kinase 1/2 (ERK1/2) is activated by various extracellular stimuli including growth factors and cytokines and plays a pivotal role in regulating cell proliferation and differentiation by phosphorylating nuclear transcription factors. Recently, it was reported that activated ERK1/2 also concentrates at adhesion sites and regulates cell spreading and migration. Vinexin is a focal adhesion protein regulating both cell spreading and growth factor signaling. We show here that vinexin was directly phosphorylated by ERK1/2 upon stimulation with growth factors. ERK1/2 phosphorylated the linker region of vinexin between the second and third SH3 domains. Site-directed mutagenesis revealed that ERK2 mainly phosphorylated the serine 189 residue of vinexin beta. Furthermore, vinexin beta interacted with ERK1/2 both in vitro and in vivo. Vinexin interacted with the active but not inactive form of ERK1/2. A putative DEF (docking for ERK FXFP) domain located in the linker region of vinexin was required for the interaction with ERK1/2 and efficient phosphorylation of vinexin beta by ERK2. Finally, we showed that cell adhesion to fibronectin also induced the association of vinexin beta with ERK2 and the phosphorylation of vinexin beta. Furthermore, vinexin and ERK were co-localized to the periphery of cells during cell spreading on fibronectin. Together, these results suggest that vinexin is a novel substrate of ERK2 and may play roles in ERK-dependent cell regulation during cell spreading as well as in growth factor-induced responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Factor de Crecimiento Epidérmico/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Musculares/metabolismo , Animales , Adhesión Celular , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Fibronectinas/química , Eliminación de Gen , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Immunoblotting , Proteínas Luminiscentes/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Musculares/química , Mutagénesis Sitio-Dirigida , Células 3T3 NIH , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Serina/química , Factores de Tiempo , Transfección , Dominios Homologos srcRESUMEN
MDR1 is clinically important because it is involved in multidrug resistance of cancer cells and affects the pharmacokinetics of various drugs. Because MDR1 harnesses adenosine 5'-triphosphate (ATP) hydrolysis for transporting drugs, examining the effect on ATPase activity is imperative for understanding the interactions between drugs and MDR1. However, conventional assay systems for ATPase activity are not sensitive enough for screening drugs using purified MDR1. Here we report a novel method to measure ATPase activity of MDR1 using high-performance liquid chromatography equipped with a titanium dioxide column. The amount of adenosine 5'-diphosphate (ADP) produced by the ATPase reaction was determined within 2 min with a titanium dioxide column (4.6 mm ID x 100 mm). The relationship between ADP amount and chromatogram peak area was linear from 5 pmol to 10 nmol. This method made it possible to reduce the amount of purified MDR1 required for a reaction to 0.5 ng, about 1/20th of the conventional colorimetric inorganic phosphate detection assay. This method is sensitive enough to detect any subtle changes in ATPase activity of MDR1 induced by drugs and can be applied to measure ATPase activity of any protein.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Titanio/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Adenosina Difosfato/análisis , Adenosina Difosfato/química , Animales , Berilio/farmacología , Línea Celular , Cromatografía Líquida de Alta Presión/métodos , Activación Enzimática/efectos de los fármacos , Fluoruros/farmacología , Humanos , Magnesio/farmacología , Nucleótidos/química , Sensibilidad y Especificidad , Verapamilo/farmacologíaRESUMEN
P-glycoprotein/MDR1 was the first member of the ATP-binding cassette (ABC) transporter superfamily to be identified in a eukaryote. In eukaryotes, ABC proteins can be classified into three major groups based on function: transporters, regulators, and channels. MDR1/P-glycoprotein is a prominent member of eukaryotic export-type ABC proteins. MDR1/P-glycoprotein extrudes a very wide array of structurally dissimilar compounds, all lipophilic and ranging in mass from approximately 300 to 2000 Da, including cytotoxic drugs that act on different intracellular targets, steroid hormones, peptide antibiotics, immunosuppressive agents, calcium channel blockers, and others. Nucleotide binding and hydrolysis by MDR1/P-glycoprotein is tightly coupled with its function, substrate transport. ATP binding and hydrolysis were extensively analyzed with the purified MDR1/P-glycoprotein. The vanadate-induced nucleotide trapping method was also applied to study the hydrolysis of ATP by MDR1/P-glycoprotein. When MDR1 hydrolyzes ATP in the presence of excess orthovanadate, an analog of inorganic phosphate, it forms a metastable complex after hydrolysis. Using this method, MDR1/P-glycoprotein can be specifically photoaffinity-labeled in the membrane, if 8-azido-[alpha(32)P]ATP is used as ATP. Visualization of the structure, as well as the biochemical data, is needed to fully understand how MDR1/P-glycoprotein recognizes such a variety of compounds and how it carries its substrates across the membrane using the energy from ATP hydrolysis. To do so, large amounts of pure and stable proteins are required. Heterologous expression systems, which have been used to express P-glycoprotein, are also described.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Animales , Antineoplásicos/metabolismo , Transporte Biológico , Epitelio/metabolismo , Conformación ProteicaRESUMEN
ABCA1 (ATP-binding cassette transporter A1) mediates the release of cellular cholesterol and phospholipid to form high density lipoprotein. Functions of ABCA1 are highly regulated at the transcriptional and post-transcriptional levels, and the synthesized ABCA1 protein turns over rapidly with a half-life of 1-2 h. To examine whether the functions of ABCA1 are modulated by associated proteins, a yeast two-hybrid library was screened with the C-terminal 120 amino acids of ABCA1. Two PDZ (PSD95-Discs large-ZO1) proteins, alpha1-syntrophin and Lin7, were found to interact with ABCA1. Immunoprecipitation revealed that alpha1-syntrophin interacted with ABCA1 strongly and that the interaction was via the C-terminal three amino acids SYV of ABCA1. Co-expression of alpha1-syntrophin in human embryonic kidney 293 cells retarded degradation of ABCA1 and made the half-life of ABCA1 five times longer than in the cells not expressing alpha1-syntrophin. This effect is not common among PDZ-containing proteins interacting with ABCA1, because Lin7, which was also found to interact with the C terminus region of ABCA1, did not have a significant effect on the half-life of ABCA1. Co-expression of alpha1-syntrophin significantly increased the apoA-I-mediated release of cholesterol. ABCA1 was co-immunoprecipitated with alpha1-syntrophin from mouse brain. These results suggest that alpha1-syntrophin is involved in intracellular signaling, which determines the stability of ABCA1 and modulates cellular cholesterol release.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de la Membrana/fisiología , Proteínas Musculares/fisiología , Transportador 1 de Casete de Unión a ATP , Proteínas Adaptadoras Transductoras de Señales , Animales , Encéfalo/metabolismo , Proteínas de Unión al Calcio , Línea Celular , Colesterol/metabolismo , Humanos , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/metabolismo , Fosfolípidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Procesamiento Postranscripcional del ARN , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Transfección , Técnicas del Sistema de Dos Híbridos , Proteínas de Transporte VesicularRESUMEN
ABCA7 is expressed predominantly in myelo-lymphatic tissues or reticuloendothelial cells. Physiological role and function of this protein are not fully understood. We isolated the full-length cDNA (type I) and a splicing variant cDNA (type II) of human ABCA7, and developed monoclonal antibodies against extracellular domain (ECD)1 of ABCA7. RT-PCR experiments suggested that human ABCA7 gene produced the type II mRNA in a tissue-specific manner. Immunostaining revealed that the type I ABCA7, expressed in HEK293 cells, was localized to the plasma membrane and ECD1 was exposed to the extracellular space as was the case for ABCA1. HEK293 cells expressing type I ABCA7 showed apoA-I-dependent cholesterol and phospholipid release. In contrast, type II ABCA7 appeared to be localized mainly in endoplasmic reticulum and did not show apoA-I-dependent cholesterol and phospholipid release. Alternative splicing could be involved in the post-transcriptional regulation of the expression and function of human ABCA7.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Regulación de la Expresión Génica/fisiología , Riñón/metabolismo , Fosfolípidos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Apolipoproteína A-I/farmacología , Línea Celular , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Riñón/efectos de los fármacos , Riñón/embriología , Metabolismo de los Lípidos , ARN Mensajero/metabolismo , Distribución Tisular , Transcripción Genética/fisiologíaRESUMEN
Vinexin is a recently identified cytoskeletal protein and plays a key role in the regulation of cytoskeletal organization and signal transduction. Vinexin localizes at sites of cell-extracellular matrix adhesion in NIH3T3 fibroblasts and at sites of cell-cell contact in epithelial LLC-PK1 cells. Expression of vinexin promotes the formation of actin stress fiber, but the role of vinexin at sites of cell-cell contact is unclear. Here we identified lp-dlg/KIAA0583 as a novel binding partner for vinexin by using yeast two-hybrid screening. lp-dlg/KIAA0583 has a NH2-terminal coiled-coil-like domain, in addition to four PDZ domains, an Src homology (SH) 3 domain, and a guanylate kinase domain, which are conserved structures in membrane-associated guanylate kinase family proteins. The third SH3 domain of vinexin bound to the region between the second and third PDZ domain of lp-dlg, which contains a proline-rich sequence. lp-dlg colocalized with vinexin at sites of cell-cell contact in LLC-PK1 cells. Furthermore, lp-dlg colocalized with beta-catenin, a major adherens junction protein, in LLC-PK1 cells. Co-immunoprecipitation experiments revealed that both endogenous and epitope-tagged deletion mutants of lp-dlg/KIAA0583 associated with beta-catenin. We also showed that these three proteins could form a ternary complex. Together these findings suggest that lp-dlg/KIAA0583 is a novel scaffolding protein that can link the vinexin-vinculin complex and beta-catenin at sites of cell-cell contact.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Nucleósido-Fosfato Quinasa/química , Proteínas/genética , Transactivadores/metabolismo , Proteínas Supresoras de Tumor , Células 3T3 , Secuencia de Aminoácidos , Animales , Northern Blotting , Células COS , Comunicación Celular , Línea Celular , Membrana Celular/metabolismo , Clonación Molecular , Proteínas del Citoesqueleto/química , ADN Complementario/metabolismo , Homólogo 1 de la Proteína Discs Large , Células Epiteliales/metabolismo , Eliminación de Gen , Genes Supresores de Tumor , Glutatión Transferasa/metabolismo , Guanilato-Quinasas , Humanos , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas Musculares/química , Nucleósido-Fosfato Quinasa/genética , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Distribución Tisular , Transactivadores/química , Técnicas del Sistema de Dos Híbridos , beta Catenina , Dominios Homologos srcRESUMEN
The C-half of cisplatin resistance-associated overexpressed protein (CROP), an SR-related protein, comprises domains rich in arginine and glutamate residues (RE domain), and is rich in arginine and serine residues (RS domain). We analyzed the role of the individual domains of CROP in cellular localization, subnuclear localization, and protein-protein interaction. CROP fused with green fluorescent protein, GFP-CROP, localized exclusively to the nucleus and showed a speckled intranuclear distribution. The yeast two-hybrid system revealed that CROP interacted with SF2/ASF, an SR protein involved in RNA splicing, as well as CROP itself. The RE and RS domains were necessary for both the intranuclear speckled distribution and the protein-protein interaction. CROP was phosphorylated by mSRPK1, mSRPK2, and Clk1 in vitro, and when cells were treated with cisplatin the subnuclear distribution of GFP-CROP was changed. These results suggest that cisplatin affects RNA splicing by changing the subnuclear distribution of SR proteins including CROP.
Asunto(s)
Antineoplásicos/metabolismo , Arginina/metabolismo , Cisplatino/metabolismo , Proteínas Nucleares/metabolismo , Serina/metabolismo , Animales , Células COS , Humanos , Mutación , Proteínas Nucleares/genética , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Empalme del ARN , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión/metabolismo , Factores de Empalme Serina-Arginina , Técnicas del Sistema de Dos HíbridosRESUMEN
ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha-(32)P]ATP was stimulated by adding ortho-vanadate in the presence of Mn(2+) as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Mutación , Fracciones Subcelulares/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Glicosilación , Humanos , Hidrólisis , Unión Proteica , Transporte de ProteínasRESUMEN
Dubin-Johnson syndrome (DJS) is a hereditary disease characterized by hyperbilirubinemia. We investigated the consequences of 2 missense mutations, R768W and Q1382R, of nucleotide-binding domains (NBDs) of the multidrug resistance protein 2 (MRP2; ABCC2) that were previously identified in patients with DJS. Pulse chase analysis revealed that the precursor form of the wild-type and Q1382R MRP2 were converted to the mature form, which is resistant to endoglycosidase H (Endo H) in about 60 minutes. However, the precursor form of the R768W MRP2, which is sensitive to endoglycosidase H, was degraded within 120 minutes and did not mature to the fully glycosylated form. Proteasome inhibitors inhibited the degradation of the precursor form of the R768W MRP2. Unlike the R768W MRP2, the Q1382R MRP2 was mainly localized on the apical membrane in the wild-type form. However, efflux of glutathione monochlorobimane (GS-MCLB) and ATP-dependent leukotriene C(4) (LTC(4)) uptake into plasma membrane vesicles from cells expressing the Q1382R MRP2 were markedly reduced, suggesting that the Q1382R MRP2 on the apical membrane was nonfunctional. Vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP in the wild-type MRP2 was stimulated by estradiol glucuronide (E(2)17betaG) in a concentration-dependent manner but that in the Q1382R MRP2 was not. In conclusion, the R768W mutation causes deficient maturation and impaired sorting, and the Q1382R mutation does not affect maturation or sorting but impairs the substrate-induced ATP hydrolysis.
Asunto(s)
Adenosina Trifosfato/metabolismo , Ictericia Idiopática Crónica/genética , Ictericia Idiopática Crónica/metabolismo , Proteínas Mitocondriales , Transporte de Proteínas/genética , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae , Animales , Transporte Biológico/genética , Membrana Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Vesículas Citoplasmáticas/metabolismo , Expresión Génica , Glicosilación , Humanos , Células LLC-PK1 , Hígado/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Mutación Missense , Etiquetas de Fotoafinidad , Complejo de la Endopetidasa Proteasomal , Estructura Terciaria de Proteína , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Porcinos , Vanadatos/farmacologíaRESUMEN
The 70-kDa peroxisomal membrane protein (PMP70) and adrenoleukodystrophy protein (ALDP), half-size ATP-binding cassette transporters, are involved in metabolic transport of long and very long chain fatty acids into peroxisomes. We examined the interaction of peroxisomal ATP-binding cassette transporters with ATP using rat liver peroxisomes. PMP70 was photoaffinity-labeled at similar efficiencies with 8-azido-[alpha-32P]ATP and 8-azido-[gamma-32P]ATP when peroxisomes were incubated with these nucleotides at 37 degrees C in the absence Mg2+ and exposed to UV light without removing unbound nucleotides. The photoaffinity-labeled PMP70 and ALDP were co-immunoprecipitated together with other peroxisomal proteins, which also showed tight ATP binding properties. Addition of Mg2+ reduced the photoaffinity labeling of PMP70 with 8-azido-[gamma-32P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[alpha-32P]ATP by only 20%. However, two-thirds of nucleotide (probably ADP) was dissociated during removal of unbound nucleotides. These results suggest that ATP binds to PMP70 tightly in the absence of Mg2+, the bound ATP is hydrolyzed to ADP in the presence of Mg2+, and the produced ADP is dissociated from PMP70, which allows ATP hydrolysis turnover. Properties of photoaffinity labeling of ALDP were essentially similar to those of PMP70. Vanadate-induced nucleotide trapping in PMP70 and ALDP was not observed. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s). ATP binding/hydrolysis by and phosphorylation of PMP70 and ALDP are involved in the regulation of fatty acid transport into peroxisomes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de la Membrana/química , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/química , Animales , Hidrólisis , Hígado/metabolismo , Magnesio/metabolismo , Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Tirosina/metabolismo , Rayos Ultravioleta , Vanadatos/farmacologíaRESUMEN
Adaptor proteins, composed of two or more protein-protein interacting modules without enzymatic activity, regulate various cellular functions. Vinexin, CAP/ponsin, and ArgBP2 constitute a novel adaptor protein family. They have a novel conserved region homologous to the active peptide sorbin, as well as three SH3 (src homology 3) domains. A number of proteins binding to this adaptor family have been identified. There is accumulating evidence that this protein family regulates cell adhesion, cytoskeletal organization, and growth factor signaling. This review will summarize the structure and the function of proteins in this family.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Citoesqueleto/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas Musculares/metabolismo , Proteínas/metabolismo , Transducción de Señal/fisiología , Animales , Adhesión Celular/fisiología , Citoesqueleto/metabolismo , Transportador de Glucosa de Tipo 4 , Sustancias de Crecimiento/metabolismo , Humanos , Insulina/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares/química , Transporte de Proteínas , Proteínas de Unión al ARN , Dominios Homologos srcRESUMEN
ERK is activated by soluble growth factors in adherent cells. However, activation of ERK is barely detectable and not sufficient for cell proliferation in non-adherent cells. Here, we show that exogenous expression of vinexin beta, a novel focal adhesion protein, allows anchorage-independent ERK2 activation stimulated by epidermal growth factor. In contrast, expression of vinexin beta had no effect on ERK2 activation in adherent cells, suggesting that vinexin beta regulates the anchorage dependence of ERK2 activation. Analyses using deletion mutants demonstrated that a linker region between the second and third SH3 domains of vinexin beta, but not the SH3 domains, is required for this function of vinexin beta. To evaluate the pathway regulating the anchorage dependence of ERK2 activation, we used a dominant-negative mutant of p21-activated kinase (PAK) and a specific inhibitor (H89) of cAMP-dependent protein kinase (PKA) because PAK and PKA are known to regulate the anchorage dependence of ERK2 activation. The dominant-negative mutant of PAK suppressed the anchorage-independent ERK2 activation induced by expression of vinexin beta. The dominant-negative mutant of vinexin beta inhibited the anchorage-independent ERK2 activation induced by the PKA inhibitor. Together, these observations indicate that vinexin beta plays a key role in regulating the anchorage dependence of ERK2 activation through PKA-PAK signaling.