RESUMEN
Here we report that the platelet-derived growth factor beta receptor (betaPDGFR) is not the only tyrosine kinase able to associate with the GTPase-activating protein of Ras (RasGAP). The interaction of non-betaPDGFR kinase(s) with RasGAP was dependent on stimulation with platelet-derived growth factor (PDGF) and seemed to require tyrosine phosphorylation of RasGAP. Because the tyrosine phosphorylation site of RasGAP is in a sequence context that is favoured by the Src homology 2 ('SH2') domain of Src family members, we tested the possibility that Src was the kinase that associated with RasGAP. Indeed, Src interacted with phosphorylated RasGAP fusion proteins; immunodepletion of Src markedly decreased the recovery of the RasGAP-associated kinase activity. Thus PDGF-dependent tyrosine phosphorylation of RasGAP results in the formation of a complex between RasGAP and Src. To begin to address the relevance of these observations, we focused on the consequences of the interaction of Src and RasGAP. We found that a receptor mutant that did not activate Src was unable to efficiently mediate the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Taken together, these observations support the following hypothesis. When RasGAP is recruited to the betaPDGFR, it is phosphorylated and associates with Src. Once bound to RasGAP, Src is no longer able to promote the phosphorylation of PLCgamma. This hypothesis offers a mechanistic explanation for our previously published findings that the recruitment of RasGAP to the betaPDGFR attenuates the tyrosine phosphorylation of PLCgamma. Finally, these findings suggest a novel way in which RasGAP negatively regulates signal relay by the betaPDGFR.
Asunto(s)
Isoenzimas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ácidos Fosfoaminos/análisis , Fosfolipasa C gamma , Fosforilación , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Tirosina , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
Cell shape change and the restructuring of the cytoskeleton are important regulatory responses that influence the growth, differentiation, and commitment to apoptosis of different cell types. MEK kinase 1 (MEKK1) activates the c-Jun NH2-terminal kinase (JNK) pathway in response to exposure of cells to microtubule toxins, including taxol. MEKK1 expression is elevated 3-fold in mitosis and microtubule toxin-treated cells accumulated at G2/M of the cell cycle. Targeted disruption of MEKK1 expression in embryonic stem cells resulted in the loss of JNK activation and increased apoptosis in response to taxol. Targeted disruption of the MEK kinase 2 gene had no effect on activation of the JNK pathway in response to microtubule toxins demonstrating a specific role of MEKK1 in this response. Cytochalasin D-mediated disruption of actin fibers activates JNK and stimulates apoptosis similarly in MEKK1(-/-) and wild type cells. The results show that MEKK1 is required for JNK activation in response to microtubule but not actin fiber toxins in embryonic stem cells. MEKK1 activation can protect cells from apoptosis in response to change in the integrity of the microtubule cytoskeleton.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Citoesqueleto/ultraestructura , Microtúbulos/ultraestructura , Proteínas Quinasas Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/metabolismo , Apoptosis , Células Cultivadas , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos , Microtúbulos/efectos de los fármacos , MitosisRESUMEN
Cloning and characterization of MEKK1 in 1993 revealed that in addition to Raf there were other pathways activated by extracellular stimuli that were responsible for ERK activation. Since then, three additional MEKK family members have been cloned adding even further diversity to the regulation of MAPK pathways. The MEKK family members are regulated by a diverse array of extracellular stimuli ranging from growth factors to DNA damaging stimuli and so are important for the cell to sense exposure to various environmental stimuli. One important aspect of MEKK biology is that they can potentially serve in more than one pathway. Regulation of MEKK family members often involves LMWG proteins, phosphorylation and subcellular localization. With regard to at least MEKK1, serine/threonine kinases such as NIK, GLK and HPK1 appear also to be important for regulation. Of the MEKK family members, the biological role of MEKK1 is best characterized and studies have shown that MEKK1 is important in mediating survival vs. apoptosis, possibly via its ability to regulate transcription factors, the expression of death receptors and their ligands. The biological roles of MEKK2, 3 and 4 are under investigation and undoubtedly homologous deletion of these MEKK family members will be invaluable at determining the biological functions of these MEKKs. At present, the MEKK family members are characterized as localized sensors that control cell responses at the level of gene expression, metabolism and the cytoskeleton
Asunto(s)
MAP Quinasa Quinasa 4 , Quinasa 1 de Quinasa de Quinasa MAP , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Apoptosis , Supervivencia Celular , Humanos , MAP Quinasa Quinasa 2 , MAP Quinasa Quinasa 3 , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Transducción de SeñalRESUMEN
The activation of caspases is a final commitment step for apoptosis. It is now evident that signal transduction pathways involving specific protein kinases modulate the apoptotic response. Both pro-apoptotic and anti-apoptotic pathways integrate environmental cues that control the decision to undergo apoptosis. Pro- and anti-apoptotic signal pathways regulate the activation of the caspases. In this review we describe our current understanding of apoptotic signal transduction.
Asunto(s)
Apoptosis/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Transducción de Señal , Animales , Caspasas/metabolismo , Citocinas/metabolismo , Sustancias de Crecimiento/metabolismo , Humanos , MAP Quinasa Quinasa 3 , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
A quartz crystal microbalance is used in aqueous solutions to monitor the rate of attachment of osteoblasts, bone-forming cells, to the surface of the crystal. Changes in resonant frequency of the crystal are measured for various surface coverages by osteoblasts. Crystal surface coverages are determined by digital image processing of scanning electron micrographs. A linear relationship is established between the surface coverages and the changes in resonant frequency of the crystal. The osteoblasts are observed to behave viscoelastically. Hence, the Sauerbrey equation can not be used to describe the relationship between the change in mass of osteoblasts on the surface and the change in resonant frequency of the crystal. Apparent viscosities at 5.0 MHz are also determined for osteoblasts.