RESUMEN
The genes encoding a family of proteins termed proline-rich γ-carboxyglutamic acid (PRRG) proteins were identified and characterized more than a decade ago, but their functions remain unknown. These novel membrane proteins have an extracellular γ-carboxyglutamic acid (Gla) protein domain and cytosolic WW binding motifs. We screened WW domain arrays for cytosolic binding partners for PRRG4 and identified novel protein-protein interactions for the protein. We also uncovered a new WW binding motif in PRRG4 that is essential for these newly found protein-protein interactions. Several of the PRRG-interacting proteins we identified are essential for a variety of physiologic processes. Our findings indicate possible novel and previously unidentified functions for PRRG proteins.
Asunto(s)
Proteínas de la Membrana/metabolismo , Secuencias de Aminoácidos , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The MAPK ERK2 can enter and exit the nucleus by an energy-independent process that is facilitated by direct interactions with nuclear pore proteins. Several studies also suggest that the localization of ERK2 can be influenced by carrier proteins. Using import reconstitution assays, we examined a group of ERK2 mutants defective in known protein interactions to determine structural properties of ERK2 that contribute to its nuclear entry. ERK2 mutants defective in binding to substrates near the active site or to basic/hydrophobic docking (D) motifs were imported normally. Several ERK2 mutants defective in interactions with FXF motifs displayed slowed rates of nuclear import. The import-impaired mutants also showed reduced binding to a recombinant C-terminal fragment of nucleoporin 153 that is rich in FXF motifs. Despite the deficit revealed in some mutants via reconstitution assays, all but one of the ERK2 mutants accumulated in nuclei of stimulated cells in a manner comparable with the wild type protein; the mutant most defective in import remained in the cytoplasm. These results further support the idea that direct interactions with nucleoporins are involved in ERK2 nuclear entry and that multiple events contribute to the ligand-dependent relocalization of these protein kinases.
Asunto(s)
Sustitución de Aminoácidos , Núcleo Celular/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Mutación Missense , Proteínas de Complejo Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Secuencias de Aminoácidos/genética , Animales , Sitios de Unión/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteínas de Complejo Poro Nuclear/genética , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Spatial regulation of ERK1/2 MAP kinases is an essential yet largely unveiled mechanism for ensuring the fidelity and specificity of their signals. Mxi2 is a p38alpha isoform with the ability to bind ERK1/2. Herein we show that Mxi2 has profound effects on ERK1/2 nucleocytoplasmic distribution, promoting their accumulation in the nucleus. Downregulation of endogenous Mxi2 by RNAi causes a marked reduction of ERK1/2 in the nucleus, accompanied by a pronounced decline in cellular proliferation. We demonstrate that Mxi2 functions in nuclear shuttling of ERK1/2 by enhancing the nuclear accumulation of both phosphorylated and unphosphorylated forms in the absence of stimulation. This process requires the direct interaction of both proteins and a high-affinity binding of Mxi2 to ERK-binding sites in nucleoporins, In this respect, Mxi2 acts antagonistically to PEA15, displacing it from ERK1/2 complexes. These results point to Mxi2 as a key spatial regulator for ERK1/2 and disclose an unprecedented stimulus-independent mechanism for ERK nuclear import.
Asunto(s)
Núcleo Celular/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Biológicos , Transporte Activo de Núcleo Celular/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular , Núcleo Celular/fisiología , Chlorocebus aethiops , Perros , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Fosfoproteínas/metabolismo , Interferencia de ARNRESUMEN
The mitogen-activated protein (MAP) kinases ERK1 and ERK2 often accumulate in the nuclei of stimulated cells to mediate changes in transcription. The mechanisms underlying stimulus-dependent redistribution of these kinases remain unclear. We have used a permeabilized cell reconstitution assay in HeLa cells and human foreskin fibroblasts to explore the processes by which ERK2 enters and exits the nucleus. We previously reported that entry of unphosphorylated ERK2 into the nucleus occurs by facilitated diffusion not requiring cytosolic transport factors. We find that export, like import, can occur by an energy- and carrier-independent mechanism. An energy-dependent mechanism of ERK2 export can also be distinguished, mediated at least in part through the exportin CRM1. We have also examined import and export of thiophosphorylated, active ERK2. Import of active ERK2 is significantly enhanced by the addition of exogenous transport factors and an energy regeneration system. These studies support a model in which multiple constitutive and regulated processes control the subcellular distribution of ERK2.
Asunto(s)
Núcleo Celular/enzimología , Secuencia de Bases , Cartilla de ADN , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , FosforilaciónRESUMEN
The transcriptional ETS2 repressor factor (ERF) is phosphorylated by Erks both in vivo and in vitro. This phosphorylation determines the subcellular localization and biological function of ERF. Here, we show that active and inactive Erk2 proteins bind ERF with high affinity through a hydrophobic pocket formed by the alphaF and alphaG helices and the activation loop of Erk2. We have identified two FXF motifs on ERF that mediate the specific interaction with Erks. One of these motifs is utilized only by active Erks, whereas the other mediates the association with inactive Erks but also contributes to interaction with active Erks. Mutation of the phenylalanines of these motifs to alanines resulted in decreased association and phosphorylation of ERF by Erks both in cells and in vitro. ERF proteins carrying these mutations exhibited increased nuclear accumulation and increased inhibition of cellular proliferation. Expression of ERF regions harboring these motifs could inhibit Erk activity in cells. Our data suggest that, in the proper context, FXF motifs can mediate a strong and specific interaction not only with active but also inactive Erks and that these interactions determine protein function in vivo.
Asunto(s)
Proteína Proto-Oncogénica c-ets-2/fisiología , Secuencias de Aminoácidos , Animales , Células COS , Proliferación Celular , Chlorocebus aethiops , Ratones , Células 3T3 NIH , Fenilalanina/química , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteína Proto-Oncogénica c-ets-2/metabolismo , RatasRESUMEN
Mast cells play a critical role in host defense against a number of pathogens. Increased mast cell infiltration has been described in allergic asthma, in rheumatoid arthritis, and during helminthes infection. Despite the importance of mast cells in allergic disease and defense against infection, little is known about the mechanisms by which mast cells migrate to various tissues under steady state conditions or during infection or inflammation. Here, we show that activation of c-Kit by its ligand, stem cell factor (SCF), cooperates with alpha4 integrin in inducing directed migration of mast cells on fibronectin. A reduction in migration and activation of a small G protein, Rac, was observed in mast cells derived from class IA phosphoinositide-3 kinase (PI-3kinase)-deficient mice in response to SCF stimulation and in mast cells expressing the dominant-negative Rac (RacN17), as well as in mast cells deficient in the hematopoietic-specific small G protein, Rac2. In addition, a PI-3kinase inhibitor inhibited alpha4- as well as SCF-induced migration in a dose-dependent fashion. In contrast, a mitogen-activated protein kinase (MAPK) inhibitor had little effect. Consistent with the pharmacologic results, abrogating the binding of the p85alpha subunit of class IA PI-3kinase to c-Kit also resulted in inhibition of SCF-induced migration on fibronectin. These genetic and biochemical data demonstrate that both c-Kit and alpha4 integrin signaling are linked to class IA PI-3kinase and Rac pathways and regulate integrin-directed (haptotactic) migration in mast cells.