RESUMEN
We investigated the direct role of cholesterol lowering on human platelet aggregation by in vitro cholesterol depletion using methyl-beta-cyclodextrin. Collagen and thrombin receptor agonist peptide induced maximal aggregation was significantly decreased in cholesterol depleted platelets. In contrast, anti-CD9 antibody, mAb7, or anti-beta(3) antibody, D3, induced percent maximal aggregation was unaffected by cholesterol depletion. Surface and total alpha(IIb)beta(3) levels were equivalent in both groups. Morphological and ultrastructural analysis of collagen induced aggregates revealed that normal and cholesterol depleted platelets changed shape and aggregated; however, cholesterol depletion impaired microtubule ring formation and aggregate size. Cholesterol depletion also diminished the extent of the open canalicular system and collagen induced platelet ATP release. These data suggest cholesterol depletion impairs platelet aggregation by altering platelet ultrastructure critical in mediating secretion. Temporal differences and differences in tyrosine phosphoprotein levels following collagen stimulation were observed, thereby indicating that platelet signaling was concurrently affected by cholesterol depletion.
Asunto(s)
Plaquetas/ultraestructura , Colesterol/fisiología , Agregación Plaquetaria , beta-Ciclodextrinas , Adenosina Trifosfato/metabolismo , Plaquetas/metabolismo , Plaquetas/fisiología , Tamaño de la Célula , Colágeno/farmacología , Ciclodextrinas/farmacología , Humanos , Microscopía Electrónica de Rastreo , Fosfoproteínas , Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombina/agonistasRESUMEN
In determining the role of Chk in T cell signaling, we have focused on its protein-protein interactions. We detected a tyrosine phosphoprotein that coimmunoprecipitated with Chk from pervanadate stimulated human blastic T cells. Subsequent Western blot analysis identified this tyrosine phosphoprotein as paxillin. Paxillin, a cytoskeletal protein involved in focal adhesions, was first identified as a v-Src substrate in transformed fibroblasts. Interestingly, Chk specifically bound tyrosine phosphorylated paxillin. Consistent with our in vivo data, Chk and paxillin were observed to localize in similar cellular regions prior to and following stimulation. Using GST fusion proteins, we determined that the Chk SH2 domain, not the SH3 domain, bound tyrosine phosphorylated paxillin. Specifically, paxillin bound to the FLVRES motif of the Chk SH2 domain. Using Far Western analysis, we revealed that the Chk SH2 domain directly associates with tyrosine phosphorylated paxillin. Finally, p52(Chk) expression in Csk-deficient mouse embryo fibroblasts decreased total phosphotyrosine levels of paxillin, implying a physiological role for Chk. These studies provide important insight into the role of Chk in tyrosine mediated signaling, as well as T cell physiology.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src) , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteína Tirosina Quinasa CSK , Fraccionamiento Celular , Membrana Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Fibroblastos/metabolismo , Humanos , Ratones , Peso Molecular , Mutación , Paxillin , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Vanadatos/farmacología , Dominios Homologos src/genética , Dominios Homologos src/fisiología , Familia-src QuinasasRESUMEN
Recently, the cDNAs for Lsk, Matk and Hyl, three Csk-related protein tyrosine kinases, have been cloned. We have examined the relationship of Lsk, Matk and Hyl, and found that the gene for each of these proteins is localized to the same region of human chromosome 19. Further, the proteins encoded by Lsk and Matk cDNAs are immunologically similar. These data strongly suggest that Lsk, Hyl and Matk are the same gene product. Previous reports demonstrating expression of Hyl and Matk in hematopoietic lineages led us to investigate the regulation of Lsk expression in response to stem cell factor (SCF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in M07e, a human leukemic cell line. Induction of Lsk/Hyl/Matk protein and mRNA was observed after treatment with SCF but not with GM-CSF. GM-CSF and IL-3, potent mitogens, had no effect on Lsk/Hyl/Matk expression. In contrast, PMA induced Lsk/Hyl/Matk but did not stimulate proliferation. Therefore, induction of Lsk/ Hyl/Matk does not correlate with the capacity to stimulate proliferation. None of the stimuli examined increased Csk protein or mRNA expression. These data demonstrate differential regulation of Csk family members by cytokines and suggest a role for Lsk/ Hyl/Matk in responses mediated by SCF and PMA. Further, our data demonstrate that, as has been seen in blood monocytes, cytokine driven translational control of Lsk/Hyl/ Matk is likely a critical mode of regulation. Lastly, since our studies strongly suggest that the Lsk, Hyl and Matk kinases are related and regulated distinctly from Csk, we and several of the original authors have agreed to rename this kinase the Csk homologous kinase (Chk).
Asunto(s)
Megacariocitos/enzimología , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas pp60(c-src) , Factor de Células Madre/farmacología , Western Blotting , Diferenciación Celular/genética , Mapeo Cromosómico , Cromosomas Humanos Par 19/genética , Clonación Molecular , Inducción Enzimática , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Hibridación Fluorescente in Situ , Interleucina-3/farmacología , Megacariocitos/citología , Pruebas de Precipitina , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/inmunología , ARN Mensajero/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Glutathione S-transferases (GSTs) detoxify diverse electrophilic chemicals, including anticancer drugs. Containing at least 100 microM total GST, the adult rat liver has abundant alpha and mu class GST isoenzymes. We utilized primary cultured rat hepatocytes, maintained in chemically defined medium, to examine direct regulation of GST activities by human growth hormone (hGH). Maintenance of GST activities in this primary cultured hepatocyte system for 8 days allowed subsequent study of GST regulation by hGH. Protein concentration, cell number, DNA content, and viability did not significantly differ (p > 0.05) between the untreated and hGH (2 micrograms/ml)-treated hepatocytes. However, hGH treatment decreased mu GST activity (p < 0.05), whereas alpha GST activity was unaltered. As positive controls for our culture system and to corroborate our findings, we examined phenobarbital induction of GST activities and hGH regulation of certain cytochrome P450-dependent testosterone hydroxylases.