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Published on: November 11, 2018
Las proteínas 14-3-3 interactúan con un motivo híbrido de prenil-fosforilación para inhibir las proteínas G
Philippe Riou1, Svend Kjær, Ritu Garg
1Randall Division of Cell and Molecular Biophysics, New Hunt's House, King's College London, London, UK.
Cell
|April 30, 2013
Resumen
Las proteínas 14-3-3 se unen a Rnd3, una proteína G constitutivamente activa, a través de un sitio C-terminal único. Esta interacción, que involucra farnesilación y fosforilación, inhibe la actividad de Rnd3 al reubicarla en el citosol.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- La señalización celular de las células.
- Bioquímica de las proteínas Bioquímica de las proteínas
Sus antecedentes:
- La señalización de la proteína G generalmente se basa en el cambio de estado de unión GTP/GDP.
- Las Rho GTPasas a menudo están reguladas por RhoGDI.
- Las proteínas Rnd son atípicas, constitutivas de proteínas Rho unidas al GTP con una regulación poco clara.
Objetivo del estudio:
- Para aclarar el mecanismo regulador de las proteínas Rnd, específicamente Rnd3.
- Identificar los socios vinculantes y los sitios de interacción de Rnd3.
- Para entender las consecuencias funcionales de la regulación Rnd3.
Principales métodos:
- Mutagénesis dirigida al sitio y síntesis de péptidos para identificar el sitio de unión 14-3-3.
- Ensayos de fosforilación utilizando una bobina enrollada asociada a Rho que contiene proteína quinasa (ROCK) y proteína quinasa C (PKC).
- Determinación de la estructura cristalina del péptido Rnd3 unido al 14-3-3.
- Estudios de localización celular para evaluar la translocación de Rnd3.
Principales resultados:
- Se identificó un sitio de unión 14-3-3 de alta afinidad en el extremo Rnd3 C, que requiere tanto farnesilación como fosforilación dependiente de ROCK (Ser240).
- Los sitios de fosforilación adicionales (Ser218 por ROCK y/o Ser210 por PKC) también median la unión 14-3-3.
- La estructura cristalina reveló proteínas 14-3-3 que acomodan el grupo farnesilo en una ranura hidrofóbica.
- La unión 14-3-3 inhibe el redondeo celular inducido por Rnd3 al causar su translocación de la membrana plasmática al citosol.
- Se observaron interacciones similares para Rnd1, Rnd2 y Rap1A geranilado.
Conclusiones:
- Las proteínas Rnd son reguladas por las proteínas 14-3-3 a través de un nuevo mecanismo que involucra la modificación del C-terminal y la fosforilación.
- Esta interacción proporciona un modo sin precedentes de inhibición de la proteína G, distinto del interruptor canónico GTP/GDP.
- Los hallazgos revelan una nueva capa de regulación para las GTPasas Rho y la superfamilia Ras.
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