RESUMEN
The LKB1 gene encodes a serine/threonine kinase mutated in Peutz-Jeghers cancer syndrome. Despite several proposed models for LKB1 function in development and in tumour suppression, the detailed molecular action of LKB1 remains undefined. Here, we report the identification and characterization of an LKB1-specific adaptor protein and substrate, STRAD (STe20 Related ADaptor). STRAD consists of a STE20- like kinase domain, but lacks several residues that are indispensable for intrinsic catalytic activity. Endogenous LKB1 and STRAD form a complex in which STRAD activates LKB1, resulting in phosphorylation of both partners. STRAD determines the subcellular localization of wild-type, but not mutant LKB1, translocating it from nucleus to cytoplasm. One LKB1 mutation previously identified in a Peutz-Jeghers family that does not compromise its kinase activity is shown here to interfere with LKB1 binding to STRAD, and hence with STRAD-dependent regulation. Removal of endogenous STRAD by siRNA abrogates the LKB1-induced G(1) arrest. Our results imply that STRAD plays a key role in regulating the tumour suppressor activities of LKB1.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencia de Aminoácidos , Animales , Células COS , Ciclo Celular/fisiología , Línea Celular , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Sustancias Macromoleculares , Datos de Secuencia Molecular , Síndrome de Peutz-Jeghers/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
The role of the protein kinase Akt in cell migration is incompletely understood. Here we show that sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T(236) residue. Transactivation of EDG-1 by Akt is not required for G(i)-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequestered Akt and acted as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration.
Asunto(s)
Quimiotaxis/fisiología , Endotelio Vascular/citología , Proteínas Inmediatas-Precoces/metabolismo , Lisofosfolípidos , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G , Transducción de Señal/fisiología , Actinas/metabolismo , Animales , Línea Celular , Endotelio Vascular/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Modelos Biológicos , Neovascularización Fisiológica/fisiología , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Receptores de Superficie Celular , Receptores Lisofosfolípidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacología , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Peutz-Jeghers syndrome is an inherited cancer syndrome that results in a greatly increased risk of developing tumors in those affected. The causative gene is a protein kinase termed LKB1, predicted to function as a tumor suppressor. The mechanism by which LKB1 is regulated in cells is not known. Here, we demonstrate that stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induced phosphorylation of endogenously expressed LKB1 at Ser(431). We present pharmacological and genetic evidence that p90(RSK) mediated this phosphorylation in response to agonists that activate ERK1/2 and that cAMP-dependent protein kinase mediated this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and we demonstrate that full-length LKB1 expressed in 293 cells was prenylated by addition of a farnesyl group to Cys(433). Our data suggest that phosphorylation of LKB1 at Ser(431) does not affect farnesylation and that farnesylation does not affect phosphorylation at Ser(431). Phosphorylation of LKB1 at Ser(431) did not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser(431) was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated was still able to suppress the growth of cells.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cisteína/química , Mutación , Síndrome de Peutz-Jeghers/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa , Proteínas Quinasas S6 Ribosómicas/metabolismo , Sulfonamidas , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Animales , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular , Línea Celular , Clonación Molecular , Colforsina/farmacología , AMP Cíclico/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Isoquinolinas/farmacología , Ácido Mevalónico/farmacología , Ratones , Modelos Biológicos , Fosforilación , Pruebas de Precipitina , Prenilación de Proteína , Ratas , Serina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , TransfecciónRESUMEN
Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and