RESUMEN
The PAR-1-MARK pathway controls cell polarity through the phosphorylation of microtubule-associated proteins. Rho-Rac guanine nucleotide exchange factor 2 (ARHGEF2), which activates Ras homolog family member A (RHOA), is anchored to the microtubule network and sequestered in an inhibited state through binding to dynein light chain Tctex-1 type 1 (DYNLT1). We showed in mammalian cells that liver kinase B1 (LKB1) activated the microtubule affinity-regulating kinase 3 (MARK3), which in turn phosphorylated ARHGEF2 at Ser151 This modification disrupted the interaction between ARHGEF2 and DYNLT1 by generating a 14-3-3 binding site in ARHGEF2, thus causing ARHGEF2 to dissociate from microtubules. Phosphorylation of ARHGEF2 by MARK3 stimulated RHOA activation and the formation of stress fibers and focal adhesions, and was required for organized cellular architecture in three-dimensional culture. Protein phosphatase 2A (PP2A) dephosphorylated Ser151 in ARHGEF2 to restore the inhibited state. Thus, we have identified a regulatory switch controlled by MARK3 that couples microtubules to the actin cytoskeleton to establish epithelial cell polarity through ARHGEF2.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Polaridad Celular/fisiología , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Células COS , Chlorocebus aethiops , Dineínas/genética , Dineínas/metabolismo , Adhesiones Focales/metabolismo , Células HEK293 , Humanos , Fosforilación , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Serina/metabolismo , Fibras de Estrés/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The B-Raf kinase is a Ras pathway effector activated by mutation in numerous human cancers and certain developmental disorders. Here we report that normal and oncogenic B-Raf proteins are subject to a regulatory cycle of extracellular signal-regulated kinase (ERK)-dependent feedback phosphorylation, followed by PP2A- and Pin1-dependent dephosphorylation/recycling. We identify four S/TP sites of B-Raf phosphorylated by activated ERK and find that feedback phosphorylation of B-Raf inhibits binding to activated Ras and disrupts heterodimerization with C-Raf, which is dependent on the B-Raf pS729/14-3-3 binding site. Moreover, we find that events influencing Raf heterodimerization can alter the transforming potential of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity but have no significant effect on proteins with high kinase activity, such as V600E B-Raf. Mutation of the feedback sites or overexpression of the Pin1 prolyl-isomerase, which facilitates B-Raf dephosphorylation/recycling, resulted in increased transformation, whereas mutation of the S729/14-3-3 binding site or expression of dominant negative Pin1 reduced transformation. Mutation of each feedback site caused increased transformation and correlated with enhanced heterodimerization and activation of C-Raf. Finally, we find that B-Raf and C-Raf proteins containing mutations identified in certain developmental disorders constitutively heterodimerize and that their signaling activity can also be modulated by feedback phosphorylation.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Retroalimentación Fisiológica , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Butadienos/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Mutación , Células 3T3 NIH , Peptidilprolil Isomerasa de Interacción con NIMA , Nitrilos/farmacología , Isomerasa de Peptidilprolil/genética , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Multimerización de Proteína , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/genética , Transducción de SeñalRESUMEN
Protein scaffolds have emerged as important regulators of MAPK cascades, facilitating kinase activation and providing crucial spatio/temporal control to their signaling outputs. Using a proteomics approach to compare the binding partners of the two mammalian KSR scaffolds, we find that both KSR1 and KSR2 interact with the kinase components of the ERK cascade and have a common function in promoting RTK-mediated ERK signaling. Strikingly, we find that the protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2 uniquely contributes to Ca2+-mediated ERK signaling. Calcineurin dephosphorylates KSR2 on specific sites in response to Ca2+ signals, thus regulating KSR2 localization and activity. Moreover, we find that depletion of endogenous KSR2 impairs Ca2+-mediated ERK activation and ERK-dependent signaling responses in INS1 pancreatic beta-cells and NG108 neuroblastoma cells. These findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new mechanism whereby Ca2+ impacts Ras to ERK pathway signaling.