RESUMEN
PTPmu, an Ig superfamily receptor protein-tyrosine phosphatase, promotes cell-cell adhesion and interacts with the cadherin-catenin complex. The signaling pathway downstream of PTPmu is unknown; therefore, we used a yeast two-hybrid screen to identify additional PTPmu interacting proteins. The membrane-proximal catalytic domain of PTPmu was used as bait. Sequencing of two positive clones identified the scaffolding protein RACK1 (receptor for activated protein C kinase) as a PTPmu interacting protein. We demonstrate that RACK1 interacts with PTPmu when co-expressed in a recombinant baculovirus expression system. RACK1 is known to bind to the src protein-tyrosine kinase. This study demonstrates that PTPmu association with RACK1 is disrupted by the presence of constituitively active src. RACK1 is thought to be a scaffolding protein that recruits proteins to the plasma membrane via an unknown mechanism. We have shown that the association of endogenous PTPmu and RACK1 in a lung cell line is increased at high cell density. We also demonstrate that the recruitment of RACK1 to both the plasma membrane and cell-cell contact sites is dependent upon the presence of the PTP mu protein in these cells. Therefore, PTPmu may be one of the proteins that recruits RACK1 to points of cell-cell contact, which may be important for PTPmu-dependent signaling in response to cell-cell adhesion.
Asunto(s)
Adhesión Celular , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Línea Celular , Proteínas de Unión al GTP , Humanos , Inmunohistoquímica , Visón , Unión Proteica , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores , Receptores de Cinasa C Activada , Receptores de Superficie Celular , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPmu associates with the cadherin-catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S. M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977- 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPmu interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPmu and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPmu and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPmu from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPmu in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513-1517) have asserted that the association we observed between PTPmu and the cadherin-catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPmu, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPmu obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPmu antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPmu and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.
Asunto(s)
Cadherinas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Anticuerpos Monoclonales , Cadherinas/aislamiento & purificación , Línea Celular , Línea Celular Transformada , Cerebelo , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Ratones , Proteínas Tirosina Fosfatasas/aislamiento & purificación , Ratas , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecciónRESUMEN
Gene targeting was used to create a null allele at the epidermal growth factor receptor locus (Egfr). The phenotype was dependent on genetic background. EGFR deficiency on a CF-1 background resulted in peri-implantation death due to degeneration of the inner cell mass. On a 129/Sv background, homozygous mutants died at mid-gestation due to placental defects; on a CD-1 background, the mutants lived for up to 3 weeks and showed abnormalities in skin, kidney, brain, liver, and gastrointestinal tract. The multiple abnormalities associated with EGFR deficiency indicate that the receptor is involved in a wide range of cellular activities.
Asunto(s)
Anomalías Múltiples/genética , Desarrollo Embrionario y Fetal , Receptores ErbB/genética , Receptores ErbB/fisiología , Marcación de Gen , Animales , Secuencia de Bases , Encéfalo/anomalías , Encéfalo/citología , División Celular , Sistema Digestivo/citología , Anomalías del Sistema Digestivo , Receptores ErbB/deficiencia , Femenino , Cabello/anomalías , Homocigoto , Riñón/citología , Pulmón/citología , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , Fenotipo , Piel/citología , Anomalías CutáneasRESUMEN
Previous experiments have indicated that zidovudine is cytotoxic to early murine embryos both in vivo and in vitro. Newer nucleoside analogs (ddI, ddC, and d4T) with antiretroviral activity were tested to determine whether they had similar toxicity. Exposure of two-cell embryos to each of these three drugs inhibited blastocyst formation only at concentrations > or = to 100 microM. Sublethal preblastocyst exposure to d4T resulted in failure to develop beyond the blastocyst stage at 10 microM; no effect was seen with ddC or ddI at concentrations up to 100 microM. In each instance, however, cytotoxicity of all three drugs was significantly less than with zidovudine at equivalent concentration. These experiments suggest that newer antiretroviral nucleosides may be safer to use in early pregnancy than zidovudine.
Asunto(s)
Antivirales/toxicidad , Embrión de Mamíferos/efectos de los fármacos , Nucleósidos/toxicidad , Animales , Técnicas de Cultivo , Didanosina/toxicidad , VIH/efectos de los fármacos , Ratones , Nucleósidos/uso terapéutico , Estavudina/toxicidad , Zalcitabina/toxicidad , Zidovudina/toxicidadRESUMEN
It previously has been demonstrated that zidovudine (AZT) is lethal to early murine embryos. The effect of the drug on pre- and postimplantation embryos was examined to delineate the timing of this toxicity and to investigate its possible mechanisms. Embryos exposed in the whole mouse during preblastocyst development were unable to proceed beyond the blastocyst stage. Similarly, when two-cell embryos harvested from unexposed females were exposed to low-concentration (1 microM) AZT in vitro over 24 h, development beyond the blastocyst stage was inhibited. In contrast, drug exposure during in vitro blastocyst and postblastocyst development resulted in little or no morphologic toxicity. Further investigation revealed that preblastocyst AZT exposure resulted in the development of blastocysts with significantly lower cell numbers than control embryos. While embryonic exposure to AZT at the blastocyst and postblastocyst stages also resulted in retarded cell division, the effects were milder than those recorded after preblastocyst exposure. These data demonstrate that the critical period of AZT toxicity toward murine embryos is between ovulation and implantation and indicate that AZT directly suppresses cell division in the preimplantation embryo.