RESUMEN
Accumulating evidence has shown that SHC SH2 domain-binding protein 1 (SHCBP1) functions as an oncogene and participated in the progression of various cancers. Oroxylin A, an active ingredient extracted from Chinese Medicine Scutellaria baicalensis, shows strong anticancer effects on multiple cancers, however, the pharmacological effect of oroxylin A on skin cancer and the regulatory effect of SHCBP1 on this process have never been evaluated. The present study was aimed at elucidating the effect of oroxylin A on carcinogen (DMBA/TPA)-induced skin tumorigenesis, and to further clarify the role of SHCBP1 in oroxylin A induced antitumor effect. Pretreatment with oroxylin A remarkably inhibited DMBA/TPA-induced tumor formation and growth, and significantly reduced tumor incidence and the average number of tumors per mouse. Oroxylin A suppressed DMBA/TPA-induced skin hyperplasia and tumor proliferation. Oroxylin A significantly inhibited the expression of several inflammatory factors in vivo. In vitro experiments found that oroxylin A inhibited TPA-induced cell malignant transformation of skin epidermal JB6 Pâ¯+â¯cells. Besides, oroxylin A significantly suppressed the levels of TPA-induced inflammatory factors in vitro. Mechanistic studies showed that oroxylin A remarkably inhibited TPA-induced increased expression of SHCBP1. Overexpression of SHCBP1 attenuated the oroxylin A-induced anti-inflammatory effect. In addition, TPA increased the expression of nuclear NF-κB p65, and SHCBP1 siRNA notably decreased the nuclear NF-κB p65 expression in JB6 Pâ¯+â¯cells. Collectively, the anti-skin cancer effect of oroxylin A may possibly by inhibiting inflammation via suppression of SHCBP1. Oroxylin A might be a potential candidate compound for the treatment of skin cancer.
Asunto(s)
Antiinflamatorios/uso terapéutico , Antineoplásicos/uso terapéutico , Flavonoides/uso terapéutico , Proteínas Adaptadoras de la Señalización Shc/inmunología , Neoplasias Cutáneas/tratamiento farmacológico , 9,10-Dimetil-1,2-benzantraceno , Animales , Antiinflamatorios/farmacología , Antineoplásicos/farmacología , Carcinógenos , Línea Celular , Citocinas/genética , Citocinas/inmunología , Femenino , Flavonoides/farmacología , Ratones Endogámicos ICR , ARN Interferente Pequeño/genética , Proteínas Adaptadoras de la Señalización Shc/genética , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol , Factor de Transcripción ReIA/inmunologíaRESUMEN
PURPOSE: This study aimed to examine the effects of the Chinese yam extract and adenosine on lipopolysaccharide (LPS)-induced cardiac anomalies and the underlying mechanisms involved. METHODS: Chinese yam extract [1630â¯mg/kg, intragastric (i.g.), 2 times/day] and adenosine (50â¯mg/kg, i.g., 2 times/day) were administered for 3 days, followed by the induction of sepsis by injecting LPS intraperitoneally [10 mg/kg, 2â¯h prior, intraperitoneally (i.p.)]. Also, estrogen receptor (ER)-unspecific antagonist Faslodex (ICI182,780, 0.5â¯mg/kg, i.p.) was administered 30â¯min before the treatments of Chinese yam extract or adenosine to evaluate whether the observed effects elicited by yam and adenosine were mediated via ERs. The heart function and the levels of pro-inflammatory cytokines, reversed mitogen-activated protein kinases (MAPKs), renin-angiotensin system (RAS), apoptosis markers, ER, and SHC/Ras/Raf1 were examined. The antagonistic effect of ICI182,780 (1 µM) and FTS (1 µM) against the Chinese yam extract (0.1 mg/ml) and adenosine (5 µM) in LPS (20 µg/ml, 24â¯h)-induced H9c2 cells was also investigated. RESULTS: The Chinese yam extract and adenosine improved heart function, downregulated pro-inflammatory cytokines, reversed MAPK and RAS, transformed the apoptosis markers, and increased the expression of ER and SHC/Ras/Raf1 following LPS challenge. These effects could be blocked by ICI182,780. FTS could not block the expression of ER on the Chinese yam extract and adenosine interposed on LPS-induced H9c2 cells, demonstrating that ER might be the upstream signaling regulator of SHC/Ras/Raf1. CONCLUSION: The Chinese yam extract and adenosine ameliorated LPS-induced cardiac contractility through the inhibition of RAS and apoptosis possibly via an ER-SHC/Ras/Raf1-dependent mechanism.
Asunto(s)
Adenosina/farmacología , Dioscorea/química , Medicamentos Herbarios Chinos/farmacología , Cardiopatías/tratamiento farmacológico , Corazón/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Citocinas/metabolismo , Femenino , Fulvestrant/farmacología , Cardiopatías/inducido químicamente , Cardiopatías/fisiopatología , Lipopolisacáridos/toxicidad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ratas Wistar , Receptores de Estrógenos/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteínas ras/metabolismoRESUMEN
In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25µM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.
Asunto(s)
Antioxidantes/farmacología , Blastocisto/efectos de los fármacos , Portadores de Fármacos , Técnicas de Cultivo de Embriones/veterinaria , Fármacos para la Fertilidad Femenina/farmacología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Nanocápsulas , Especies Reactivas de Oxígeno/metabolismo , Tretinoina/farmacología , Animales , Antioxidantes/química , Apoptosis/efectos de los fármacos , Blastocisto/metabolismo , Blastocisto/patología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Bovinos , Química Farmacéutica , Relación Dosis-Respuesta a Droga , Desarrollo Embrionario/efectos de los fármacos , Femenino , Fármacos para la Fertilidad Femenina/química , Regulación del Desarrollo de la Expresión Génica , Nanomedicina , Fosforilación , Embarazo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de Señal/efectos de los fármacos , Tretinoina/química , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Pancreatic cancer is the fourth leading cause of cancer deaths in the US and no significant treatment is currently available. Here, we describe the effect of crocetinic acid, which we purified from commercial saffron compound crocetin using high performance liquid chromatography. Crocetinic acid inhibits proliferation of pancreatic cancer cell lines in a dose- and time-dependent manner. In addition, it induced apoptosis. Moreover, the compound significantly inhibited epidermal growth factor receptor and Akt phosphorylation. Furthermore, crocetinic acid decreased the number and size of the pancospheres in a dose-dependent manner, and suppressed the expression of the marker protein DCLK-1 (Doublecortin Calcium/Calmodulin-Dependent Kinase-1) suggesting that crocetinic acid targets cancer stem cells (CSC). To understand the mechanism of CSC inhibition, the signaling pathways affected by purified crocetinic acid were dissected. Sonic hedgehog (Shh) upon binding to its cognate receptor patched, allows smoothened to accumulate and activate Gli transcription factor. Crocetinic acid inhibited the expression of both Shh and smoothened. Finally, these data were confirmed in vivo where the compound at a dose of 0.5 mg/Kg bw suppressed growth of tumor xenografts. Collectively, these data suggest that purified crocetinic acid inhibits pancreatic CSC, thereby inhibiting pancreatic tumorigenesis.
Asunto(s)
Carotenoides/química , Proteínas Hedgehog/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Cromatografía Líquida de Alta Presión , Crocus/química , Relación Dosis-Respuesta a Droga , Quinasas Similares a Doblecortina , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fosforilación , Extractos Vegetales/química , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de Señal , Receptor Smoothened , Esferoides Celulares/metabolismo , Proteína Transformadora 2 que Contiene Dominios de Homología 2 de Src , Vitamina A/análogos & derivadosRESUMEN
AIMS: The cellular prion protein, PrP(C), whose aberrant isoforms are related to prion diseases of humans and animals, has a still obscure physiological function. Having observed an increased expression of PrP(C) in two in vivo paradigms of heart remodelling, we focused on isolated mouse hearts to ascertain the capacity of PrP(C) to antagonize oxidative damage induced by ischaemic and non-ischaemic protocols. METHODS AND RESULTS: Hearts isolated from mice expressing PrP(C) in variable amounts were subjected to different and complementary oxidative perfusion protocols. Accumulation of reactive oxygen species, oxidation of myofibrillar proteins, and cell death were evaluated. We found that overexpressed PrP(C) reduced oxidative stress and cell death caused by post-ischaemic reperfusion. Conversely, deletion of PrP(C) increased oxidative stress during both ischaemic preconditioning and perfusion (15 min) with H2O2. Supporting its relation with intracellular systems involved in oxidative stress, PrP(C) was found to influence the activity of catalase and, for the first time, the expression of p66(Shc), a protein implicated in oxidative stress-mediated cell death. CONCLUSIONS: Our data demonstrate that PrP(C) contributes to the cardiac mechanisms antagonizing oxidative insults.
Asunto(s)
Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , Proteínas PrPC/metabolismo , Animales , Catalasa/metabolismo , Muerte Celular , Modelos Animales de Enfermedad , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Musculares/metabolismo , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/patología , Proteínas PrPC/deficiencia , Proteínas PrPC/genética , Conejos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Factores de TiempoRESUMEN
Ultraviolet A (UVA) radiation contributes to skin photoaging. Baicalin, a plant-derived flavonoid, effectively absorbs UV rays and has been shown to have anti-oxidant and anti-inflammatory properties that may delay the photoaging process. In the current study, cultured human skin fibroblasts were incubated with 50 µg/ml baicalin 24 hours prior to 10 J/cm(2) UVA irradiation. In order to examine the efficacy of baicalin treatment in delaying UVA-induced photoaging, we investigated aging-related markers, cell cycle changes, anti-oxidant activity, telomere length, and DNA damage markers. UVA radiation caused an increased proportion of ß-Gal positive cells and reduced telomere length in human skin fibroblasts. In addition, UVA radiation inhibited TGF-ß1 secretion, induced G1 phase arrest, reduced SOD and GSH-Px levels, increased MDA levels, enhanced the expression of MMP-1, TIMP-1, p66, p53, and p16 mRNA, reduced c-myc mRNA expression, elevated p53 and p16 protein expression, and reduced c-myc protein expression. Baicalin treatment effectively protected human fibroblasts from these UVA radiation-induced aging responses, suggesting that the underlying mechanism involves the inhibition of oxidative damage and regulation of the expression of senescence-related genes, including those encoding for p53, p66(Shc) and p16.
Asunto(s)
Senescencia Celular/genética , Senescencia Celular/efectos de la radiación , Fibroblastos/efectos de la radiación , Flavonoides/farmacología , Envejecimiento de la Piel/genética , Envejecimiento de la Piel/efectos de la radiación , Piel/citología , Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Antiinflamatorios no Esteroideos , Antioxidantes , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Daño del ADN/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/efectos de la radiación , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Homeostasis del Telómero/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVES: Ischemia/reperfusion is a leading cause of liver damage after surgical intervention, trauma, and transplantation. It has been reported that the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 attenuates myocardial, cerebral, and renal ischemia/reperfusion damage. This study aimed to investigate the involvement of sirtuin 1-mediated p66shc inhibition in liver ischemia/reperfusion and explore the effect of carnosic acid and ischemic preconditioning on liver ischemia/reperfusion-induced damage. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats and HepG2 cells. INTERVENTIONS: The rats were subjected to 45 minutes of ischemia to 70% of the liver, followed by 3-hour reperfusion. The HepG2 cells were subjected to hypoxia/reoxygenation-induced injury. MEASUREMENTS AND MAIN RESULTS: In the rats with liver ischemia/reperfusion injury, carnosic acid pretreatment and ischemic preconditioning dramatically reduced the serum aminotransferase activity and proinflammatory chemokine levels and improved the liver histological evaluations. Carnosic acid and ischemic preconditioning also increased manganese superoxide dismutase and Bcl-xL, but down-regulated cleaved caspase-3. Interestingly, the protective effect of carnosic acid and ischemic preconditioning was positively associated with sirtuin 1 activation. By contrast, p66shc, a kinase that promotes oxidative injury and apoptosis, was inhibited by carnosic acid and ischemic preconditioning. Sirtuin 1 small interfering RNA knockdown experiments confirmed that carnosic acid increased sirtuin 1-mediated repression of p66shc in HepG2 cells and that the protective effect of carnosic acid against hypoxia/reoxygenation injury was inhibited by the sirtuin 1 inhibitor nicotinamide. These results suggest that carnosic acid protects hepatocytes from hypoxia/reoxygenation damage through sirtuin 1-mediated p66shc suppression. To support this notion, we further demonstrated that the sirtuin 1 activator resveratrol achieved a protective effect similar to that of carnosic acid against hypoxia/reoxygenation injury, whereas sirtuin 1 small interfering RNA and nicotinamide had the opposite effect. CONCLUSIONS: Carnosic acid and ischemic preconditioning protect against ischemia/reperfusion-induced liver injury. Mechanistically, the protective effect involves the sirtuin 1-mediated inhibition of p66shc, suggesting that this pathway is a novel potential therapeutic target for protecting the liver from ischemia/reperfusion injury.
Asunto(s)
Abietanos/farmacología , Isquemia/enzimología , Hígado/efectos de los fármacos , Niacinamida/farmacología , Extractos Vegetales/farmacología , Daño por Reperfusión/enzimología , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Sirtuina 1/fisiología , Análisis de Varianza , Animales , Caspasas/sangre , Inhibidores Enzimáticos/farmacología , Células Hep G2 , Humanos , Etiquetado Corte-Fin in Situ , Isquemia/patología , Precondicionamiento Isquémico , Hígado/enzimología , Hígado/patología , Masculino , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Resveratrol , Proteínas Adaptadoras de la Señalización Shc/antagonistas & inhibidores , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Estilbenos/farmacología , Superóxido Dismutasa/sangre , Transaminasas/sangreRESUMEN
Diallyl trisulfide (DATS) is an organosulfur compound isolated from garlic, and has been shown to have anticancer activity both in vitro and in vivo. The aim of this study was to compare cytotoxic effects of DATS on prostate cancer cells PC-3 and noncancerous human prostate epithelial cells PNT1A. PC-3 prostate cancer and noncancerous human prostate epithelial cells PNT1A were used in the study. We observed that PNT1A cells had higher resistance to DATS-induced cell death than PC-3 cells. Investigating signaling pathways involved in the cell death we observed that p66Shc phosphorylation at serine 36 and extracellular signal-regulated kinase 1/2 activation induced by DATS, were significantly attenuated in PNT1A cells as compared to PC-3 cells. Moreover, DATS-induced Akt inactivation was also significantly reduced in PNT1A cells. In addition to that, DATS-induced reactive oxygen species generation was nearly completely abolished in PNT1A cells. Interestingly, DATS induced only slight decrease in the level of ferritin H, whereas ferritin L was elevated. These data suggest that cytotoxicity of DATS toward PNT1A cells is strongly reduced as opposed to PC-3 cancer cells, which corresponds to the lower activation of prodeath signaling pathway mediated by the adaptor protein p66Shc in the noncancerous PNT1A cells.
Asunto(s)
Compuestos Alílicos/farmacología , Antineoplásicos/farmacología , Células Epiteliales/efectos de los fármacos , Extractos Vegetales/farmacología , Neoplasias de la Próstata/metabolismo , Transducción de Señal , Sulfuros/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Ferritinas/metabolismo , Ajo/química , Humanos , Masculino , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/genética , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
We hypothesized that hypoxia induced testicular damage is mediated by an activated NADPH oxidase (NOX), therefore, APO (apocynin) an inhibitor of NOX and raisanberine (RS), a calcium influx inhibitor were tested if they could attenuate hypoxic toxicity to the testis. Male Sprague-Dawley rats were exposed to hypoxia (10±0.5% O2) for 17d and intervened with APO and RS in the last 6d. Histological changes and expression of pro-inflammation factors were evaluated in vivo. Biomarkers in isolated Leydig cells incubated with H2O2 were also assayed in vitro. Hypoxic rats displayed lower serum testosterone and higher LH and FSH. Upregulation of p22/p47(phox), NOX2, MMP9, PERK and p66Shc was associated with downregulation of StAR, 3ß-HSD and Cx43 in the hypoxia testis, revealed by Western blot and immunohistochemical assay, respectively. APO and RS at least partially normalize hypoxia caused male hypogonadism by suppressing ER stress, and p66Shc in testes.
Asunto(s)
Acetofenonas/uso terapéutico , Berberina/análogos & derivados , Bloqueadores de los Canales de Calcio/uso terapéutico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Eunuquismo/tratamiento farmacológico , NADPH Oxidasas/antagonistas & inhibidores , Testículo/efectos de los fármacos , 3-Hidroxiesteroide Deshidrogenasas/biosíntesis , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Acetofenonas/farmacología , Animales , Berberina/farmacología , Berberina/uso terapéutico , Biomarcadores/sangre , Biomarcadores/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Eunuquismo/sangre , Eunuquismo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/patología , Masculino , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteínas Adaptadoras de la Señalización Shc/agonistas , Proteínas Adaptadoras de la Señalización Shc/antagonistas & inhibidores , Proteínas Adaptadoras de la Señalización Shc/genética , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Testículo/metabolismo , Testículo/patología , Testosterona/sangreRESUMEN
The in vitro production of mammalian embryos suffers from low efficiency, with 50-70% of all fertilized oocytes failing to develop to the blastocyst stage. This high rate of developmental failure is due, in part, to the effects of oxidative stress generated by reactive oxygen species (ROS). The p66Shc adaptor protein controls oxidative stress response by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidants. This study explored the relationship between p66Shc levels, redox state, and developmental potential in early bovine embryos. Embryo developmental potential was established based on observing their time of first cleavage. P66Shc, catalase, and mitochondrial-specific, manganese-superoxide dismutate (MnSOD) levels were compared between embryos with high and low developmental potentials. Additionally, p66Shc, catalase, and MnSOD content were assayed following a variety of oxidative stress-inducing and-alleviating conditions. Increased developmental potential correlated with significantly lower p66Shc content, significantly higher levels of catalase and MnSOD, and significantly lower intracellular ROS levels (MitoSOX staining) and reduced DNA damage (γ-H2A.X(phospho S139) immunostaining). p66Shc content was increased by either high (20%) O(2) culture or H(2)O(2) treatment, and significantly decreased by supplementing culture media with the antioxidant polyethylene glycol-conjugated catalase. While the abundance of p66Shc varied according to pro/anti-oxidant culture conditions, antioxidant content varied only according to developmental potential. This discrepancy has important implications regarding ongoing efforts towards maximizing in vitro embryo production.
Asunto(s)
Bovinos/embriología , Espacio Intracelular/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Animales , Catalasa/análisis , Catalasa/genética , Catalasa/metabolismo , Daño del ADN , Técnicas de Cultivo de Embriones , Embrión de Mamíferos , Peróxido de Hidrógeno/metabolismo , Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Adaptadoras de la Señalización Shc/análisis , Proteínas Adaptadoras de la Señalización Shc/genética , Superóxido Dismutasa/análisis , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxidos/análisis , Superóxidos/metabolismoRESUMEN
BACKGROUND: Intestinal ischemia/reperfusion (I/R) causes severe histological injury, reactive oxygen species activation, and cell apoptosis in the lung. In this study, we investigated, using a murine intestinal I/R model, the effect of a polyphenolic compound, protocatechuic acid (PCA), in modulation of ShcA and in protection of the lung from I/R-induced injury. METHODS: Fifty ICR mice were randomly divided into five groups, including a control group, intestinal I/R group, control + PCA group, I/R + PCA low-dose group, and I/R + PCA high-dose group. The I/R and I/R + PCA groups were subjected to mesenteric arterial ischemia for 45 minutes and reperfusion for 90 minutes. The control and control + PCA groups underwent a surgical procedure that included isolation of the superior mesenteric artery without occlusion. In all PCA-pretreated groups, the mice received intraperitoneal PCA administration for three consecutive days. Serum specimens were collected for measuring tumor necrosis factor-α and interleukin 6, while lung tissues were harvested for histopathologic assessment including glutathione (GSH) and GSH peroxidase assay. Lung expression of p66shc, phosphorylated p66shc, manganese superoxide dismutase, caspace-3, and Bcl-xL were determined by Western blotting for protein level and semiquantitative reverse transcription-polymerase chain reaction analysis for mRNA level. RESULTS: PCA pretreatment markedly reduced I/R-induced lung injury as indicated by histological alterations; the decreases in tumor necrosis factor-α, interleukin 6, and caspase-3 expression levels; and the increases in GSH, GSH peroxidase, manganese superoxide dismutase, and Bcl-xL levels in the lung. Moreover, PCA treatment down-regulated p66shc expression and phosphorylation. CONCLUSION: PCA has a significant protective effect in lung injury induced by intestinal I/R. The protective effect of PCA may be attributed to the suppression of p66shc and the modulation of downstream antioxidative/antiapoptotic factors.
Asunto(s)
Anticarcinógenos/uso terapéutico , Hidroxibenzoatos/uso terapéutico , Lesión Pulmonar/metabolismo , Lesión Pulmonar/prevención & control , Daño por Reperfusión/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Animales , Modelos Animales de Enfermedad , Lesión Pulmonar/etiología , Masculino , Ratones , Ratones Endogámicos ICR , ARN Mensajero/metabolismo , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Proteínas Adaptadoras de la Señalización Shc/genética , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Superóxido Dismutasa/metabolismo , Proteína bcl-X/metabolismoRESUMEN
OBJECTIVES: We investigated the effect of argirein on acute inflammation edema and examined that aquaporin 4 (AQP4), p66Shc and activating transcription factor (ATF-6) might be involved in carrageenan-induced rat paw inflammation and be reversed by argirein, rhein and indometacin, but not L-arginine. METHODS: Inflammation was produced by carrageenan injected into rat paw and treated orally with argirein (100 mg/kg), rhein (100 mg/kg), L-arginine (100 mg/kg) or indometacin (5 mg/kg). Inflammatory oedema and biomarkers were examined. KEY FINDINGS: Swelling was reduced by argirein, rhein and indometacin; argirein was more effective than rhein at 1 h following medication. Activation of AQP4, p66Shc, ATF-6, NADPH oxidase subunits p22phox, gp91phox and matrix metalloproteinase 2 (P < 0.01) was significant and was suppressed by arginine, rhein and indometacin but not by l-arginine. CONCLUSIONS: Activated AQP4, endoplasmic reticulum stress and p66Shc were actively implicated in the inflammation and these were suppressed by argirein, and its activity is favorable due to synergism in combination with L-arginine.
Asunto(s)
Antraquinonas/uso terapéutico , Acuaporina 4/metabolismo , Arginina/uso terapéutico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Indometacina/uso terapéutico , Inflamación/tratamiento farmacológico , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Factor de Transcripción Activador 6/metabolismo , Animales , Antraquinonas/farmacología , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Arginina/farmacología , Biomarcadores/metabolismo , Carragenina , Combinación de Medicamentos , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Edema/tratamiento farmacológico , Femenino , Indometacina/farmacología , Inflamación/inducido químicamente , Inflamación/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , NADPH Oxidasas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
We investigated the anti-inflammatory activities of argirein and rhein on inflammatory edema in rat paw which was caused by complete adjuvant, compared with ibuprofen. We hypothesized that the adjuvant-induced inflammation is attributed to upregulation of activating transcript factor 6 (ATF6; a chaperone for endoplasmic reticulum (ER) stress), p66Shc (an adaptive protein modulating oxidative stress), and NADPH oxidase subunits p22phox and gp91phox in the inflamed tissues. Biomarkers were measured in the rat paw in association with monitoring swellings. The primary inflammatory edema of the injected paw occurred rapidly and sustained over a couple of days, and the secondary inflammation developed 2 weeks later. The inflammatory edema was accompanied by upregulation of cytokines including ATF6, p66Shc, p22phox, gp91phox, and MMP-2 and an increase in ratio of p-Akt/Akt in the afflicted paw. These were suppressed by either argirein and rhein or ibuprofen. These findings indicate that ER stress, upregulated p66Shc, and phosphorylated Akt are actively implicated in the inflammatory zone caused by adjuvant injection. These biomarkers were causal factors responsible for inflammation of the afflicted paw and were suppressed by a supermolecule argirein and rhein, and the anti-inflammatory activities of the two compounds were comparable to that of ibuprofen.
Asunto(s)
Antiinflamatorios/farmacología , Estrés del Retículo Endoplásmico , Inflamación/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Factor de Transcripción Activador 6/metabolismo , Adyuvantes Inmunológicos , Animales , Antraquinonas/farmacología , Antiinflamatorios/uso terapéutico , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/tratamiento farmacológico , Edema/inducido químicamente , Edema/tratamiento farmacológico , Edema/patología , Femenino , Ibuprofeno/farmacología , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, Höhn and Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target GAPs (GTPase-activating proteins) for inactivation and those that result in activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link between PE and GAP inactivation is missing and GEF stimulation is hard to reconcile with the observation that dominant-negative S17N-Ras does not compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation by PE. We have addressed this controversy and carried out an in-depth biochemical study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in growth-arrested COS-7 cells. These data disclose the existence of two separate GEF systems that operate independently from each other to accomplish PE-dependent formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We document that COS-7 cells do not express RasGRP and present evidence that the PE-responsive GEF system may involve PKC-dependent phosphorylation of Sos. More fundamentally, these observations shed new light on enigmatic issues such as the inefficacy of S17N-Ras in blocking PE action or the role of the EGFR in heterologous agonist activation of the Ras/ERK pathway.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Ésteres del Forbol/farmacología , Proteínas ras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Chlorocebus aethiops , Medio de Cultivo Libre de Suero , Activación Enzimática/efectos de los fármacos , Receptores ErbB/metabolismo , Guanosina Trifosfato/metabolismo , Sistema de Señalización de MAP Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Unión Proteica , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Activación Transcripcional , Proteínas ras/genéticaRESUMEN
INTRODUCTION: Shc adapter proteins are secondary messenger proteins involved in various cellular pathways, including those mediating receptor tyrosine kinase signaling and apoptosis in response to stress. We have previously reported that high levels of tyrosine-phosphorylated Shc (PY-Shc) and low levels of its inhibitory p66 Shc isoform are strongly prognostic for identifying both early node-negative and more advanced, node-positive, primary breast cancers with high risk for recurrence. Because aberrant activation of tyrosine kinases upstream of Shc signaling proteins has been implicated in resistance to tamoxifen--the most widely prescribed drug for treatment of estrogen receptor-positive breast cancer--we hypothesized that Shc isoforms may identify patients at increased risk of relapsing despite tamoxifen treatment. METHODS: Immunohistochemical analyses of PY-Shc and p66 Shc were performed on archival primary breast cancer tumors from a population-based cohort (60 patients, 9 relapses) and, for validation, an independent external cohort (31 patients, 13 relapses) in which all patients received tamoxifen as a sole systemic adjuvant prior to relapse. RESULTS: By univariate and multivariate analyses, the Shc proteins were very strong and independent predictors of treatment failure in both the population-based cohort (interquartile hazard ratio = 8.3, 95% confidence interval [CI] 1.8 to 38, P = 0.007) and the validating cohort (interquartile relative risk = 12.1, 95% CI 1.7 to 86, P = 0.013). CONCLUSION: These results suggest that the levels of PY-Shc and p66 Shc proteins in primary tumors identify patients at high risk for relapsing despite treatment with tamoxifen and therefore with further validation may be useful in guiding clinicians to select alternative adjuvant treatment strategies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/metabolismo , Recurrencia Local de Neoplasia , Tamoxifeno/uso terapéutico , Estudios de Casos y Controles , Femenino , Humanos , Fosfoproteínas/biosíntesis , Fosforilación , Isoformas de Proteínas , Receptores de Estrógenos , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Tirosina/metabolismoRESUMEN
DDR2 is a receptor tyrosine kinase whose activating ligands are various collagens. DDR2-mediated cellular signaling has been shown to require Src activity. However, the precise mechanism underlying the Src dependence of DDR2 signaling is unknown. Here, using baculoviral co-expression of the DDR2 cytosolic domain and Src, we show that Src targets three tyrosine residues (Tyr-736, Tyr-740, and Tyr-741) in the activation loop of DDR2 for phosphorylation. This phosphorylation by Src stimulates DDR2 cis-autophosphorylation of additional tyrosine residues. In vitro Shc binding assays demonstrate that phosphotyrosines resulting from DDR2 autophosphorylation are involved in Shc binding to the DDR2 cytosolic domain. Mutating tyrosine 740 of DDR2 to phenylalanine stimulates autophosphorylation of DDR2 to an extent similar to that resulting from Src phosphorylation of DDR2. In addition, the DDR2 Y740F mutant protein displays collagen-independent, constitutively activated signaling. These findings suggest that tyrosine 740 inhibits DDR2 autophosphorylation. Collectively, our findings are consistent with the following mechanism for Src-dependent DDR2 activation and signaling: 1) ligand binding promotes phosphorylation of Tyr-740 in the DDR2 activation loop by Src; 2) Tyr-740 phosphorylation stimulates intramolecular autophosphorylation of DDR2; 3) DDR2 autophosphorylation generates cytosolic domain phosphotyrosines that promote the formation of DDR2 cytosolic domain-Shc signaling complexes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Mitogénicos/química , Receptores Mitogénicos/metabolismo , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Receptores con Dominio Discoidina , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Células 3T3 NIH , Fosforilación , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Mitogénicos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Spodoptera , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Transfección , Tirosina/químicaRESUMEN
p66(shc-/-) mice exhibit prolonged lifespan and increased resistance to oxidative and hypoxic stress. To investigate p66(shc) involvement in human longevity, p66(shc) mRNA and protein were evaluated in fibroblasts from young people, elderly and centenarians, exposed to oxidative or hypoxic stress. Unexpectedly, centenarians showed the highest basal levels of p66(shc). Oxidative stress induced p66(shc) in all samples. At variance, hypoxic stress caused p66(shc) reduction only in cells from centenarians. These changes occurred in absence of any modification of p66(shc) promoter methylation pattern. Intriguingly, in cells from centenarians, p66(shc) induction was affected by p53 codon 72 polymorphism. Thus, cells from centenarians present a peculiar regulation of p66(shc), suggesting that its role in mammalian longevity is more complex than previously thought.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Fibroblastos/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Western Blotting , Células Cultivadas , Codón , ADN/química , ADN/metabolismo , Metilación de ADN , ADN Complementario/metabolismo , Deferoxamina/farmacología , Desoxirribosa/metabolismo , Humanos , Hipoxia , Longevidad , Persona de Mediana Edad , Estrés Oxidativo , Regiones Promotoras Genéticas , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Sulfitos/química , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K(m) and k(cat), revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k(cat) approximately 5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of approximately 5-fold in the specificity constants (k(cat)/K(m)) for EGFR peptides, whereas approximately 15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins approximately 4-6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Receptores ErbB/metabolismo , Fosfoproteínas/metabolismo , Animales , Sitios de Unión , Western Blotting , Células COS , Dominio Catalítico , Línea Celular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Factor de Crecimiento Epidérmico/metabolismo , Epítopos/química , Glutatión Transferasa/metabolismo , Cinética , Ligandos , Ratones , Péptidos/química , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Especificidad por Sustrato , Factores de Tiempo , Tirosina/químicaRESUMEN
A loss of functional androgen receptor and an enhanced expression of growth factor receptors and associated ligands are causal genetic events in prostate cancer (PCA) progression. These genetic alterations lead to an epigenetic mechanism where a feedback autocrine loop between membrane receptor and ligand (e.g. EGFR-TGFalpha) results in a constitutive activation of MAPK-Elk1-AP1-mediated mitogenic signaling in human PCA at an advanced and androgen-independent stage. We rationalized that inhibiting these epigenetic events could be useful in controlling advanced PCA growth. Recently, we found that grape seed extract (GSE), a dietary supplement rich in flavonoid procyanidins, inhibits advanced and androgen-independent human PCA DU145 cell growth in culture and nude mice. Here, we performed detailed mechanistic studies to define the effect of GSE on EGFR-Shc-MAPK-Elk1-AP1-mediated mitogenic signaling in DU145 cells. Pretreatment of serum-starved cells with GSE resulted in 70% to almost complete inhibition of EGF-induced EGFR activation and 50% to complete inhibition of Shc activation, which corroborated with a comparable decrease in EGF-induced Shc binding to EGFR. Conversely, EGF-induced ERK1/2 phosphorylation was inhibited only by lower doses of GSE; in fact, higher doses showed an increase. Additional studies showed that GSE alone causes a dose- and time-dependent increase in ERK1/2 phosphorylation in starved DU145 cells that is inhibited by an MEK1 inhibitor PD98059. Independent of this increase in ERK1/2 phosphorylation, GSE showed a strong inhibition of ERK1/2 kinase activity to Elk1 in both cellular and cell-free systems. GSE treatment of cells also inhibited both EGF-induced and constitutively active Elk1 phosphorylation and AP1 activation. GSE treatment also showed DNA synthesis inhibition in starved and EGF-stimulated cells as well as loss of cell viability and apoptotic death that was further increased by adding MEK1 inhibitor. Since GSE strongly induced apoptosis independent of its affect on an increase in phospho-ERK1/2, we hypothesized that apoptotic effect of GSE could be by other mechanism(s) including its effect on stress-associated MAPK, the JNK. Indeed, GSE-treated cells showed a strong and sustained increase in phospho-JNK1/JNK2 levels, JNK activity and phospho-cJun levels. An inhibition of GSE-induced JNK activation by a novel JNK inhibitor SP600125 resulted in a significant reversal of GSE-induced apoptotic death suggesting the involvement of JNK activation by GSE in its apoptosis response. Together, these results suggest that anticancer effects of GSE in PCA be mediated via impairment of EGFR-ERK1/2-Elk1-AP1-mediated mitogenic signaling and activation of JNK causing growth inhibition and apoptosis, respectively.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Proteínas de Unión al ADN , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Extractos Vegetales/farmacología , Neoplasias de la Próstata/patología , Transducción de Señal/efectos de los fármacos , Factores de Transcripción , Vitis/química , Adenocarcinoma/metabolismo , Antracenos/farmacología , División Celular/efectos de los fármacos , Sistema Libre de Células , Medio de Cultivo Libre de Suero/farmacología , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/efectos de los fármacos , Receptores ErbB/metabolismo , Flavonoides/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 1 , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Semillas/química , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Factor de Transcripción AP-1/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Proteína Elk-1 con Dominio etsRESUMEN
v-Src transforms fibroblasts in vitro and causes tumor formation in the animal by tyrosine phosphorylation of critical cellular substrates. Exactly how v-Src interacts with these substrates remains unknown. One of its substrates, the adaptor protein Shc, is thought to play a crucial role during cellular transformation by v-Src by linking v-Src to Ras. We used Shc proteins with mutations in either the phosphotyrosine binding (PTB) or Src homology 2 domain to determine that phosphorylation of Shc in v-Src-expressing cells depends on the presence of a functional PTB domain. We purified a 100-kDa Shc PTB-binding protein from Src-transformed cells that was identified as the beta chain of the low density lipoprotein receptor-related protein LRP1. LRP1 acts as an import receptor for a variety of proteins and is involved in clearance of the beta-amyloid precursor protein. This study shows that LRP1 is tyrosine-phosphorylated in v-Src-transformed cells and that tyrosine-phosphorylated LRP1 binds in vivo and in vitro to Shc. The association between Shc and LRP1 may provide a mechanism for recruitment of Shc to the plasma membrane where it is phosphorylated by v-Src. It is at the membrane that Shc is thought to be involved in Ras activation. These observations further suggest that LRP1 could function as a signaling receptor and may provide new avenues to investigate its possible role during embryonal development and the onset of Alzheimer's disease.