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Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Control de la activación de la MAP quinasa por la threonina/tirosina fosfatasa PAC1 inducida por el mitógeno
1Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892.
Nature
|February 17, 1994
Resumen
PAC1 es una fosfatasa que inactiva las proteína-activada por mitógeno (MAP) quinasas. Este hallazgo revela el PAC1
Área de la Ciencia:
- La señalización celular de las células.
- Biología molecular La biología molecular.
- La bioquímica es la bioquímica.
Sus antecedentes:
- La estimulación mitogénica activa las cascadas de señalización intracelular que involucran las proteínas activadas por mitógenos (MAP) kinasas ERK1 y ERK2.
- La activación de la MAP quinasa ocurre a través de la doble fosforilación por la MAP quinasa quinasa (MEK).
- Las quinasas MAP activadas se translocan al núcleo, fosforilando los factores de transcripción.
Objetivo del estudio:
- Para investigar el papel de PAC1, una tirosina fosfatasa nuclear previamente identificada, en la regulación de la MAP quinasa.
- Para determinar si PAC1 funciona como una fisiológicamente relevante MAP quinasa fosfatasa.
Principales métodos:
- Caracterización in vitro del PAC1 recombinante como una fosfatasa de doble especificidad.
- Evaluación de la especificidad del sustrato de PAC1 para la MAP quinasa.
- Estudios in vivo que involucran la expresión constitutiva de PAC1 para evaluar su efecto sobre la actividad de la cinasa MAP.
- Análisis de la expresión del gen reportero regulado por la quinasa MAP después de la expresión de PAC1.
Principales resultados:
- El PAC1 recombinante demostró actividad dual específica de threonina/tirosina fosfatasa con alta especificidad para la MAP quinasa.
- La expresión constitutiva de PAC1 inhibió la actividad de la MAP quinasa estimulada por el factor de crecimiento epidérmico, el acetato de forbol miristilo y el enlace cruzado del receptor de células T.
- La inactivación mediada por PAC1 de la MAP quinasa condujo a la inhibición de la expresión génica del reportero regulado por la MAP quinasa.
Conclusiones:
- PAC1 es una fisiológicamente relevante MAP quinasa fosfatasa.
- PAC1 juega un papel crucial en la regulación de las vías de señalización de la quinasa MAP.
- La inactivación PAC1 de las quinasas MAP tiene un impacto en la regulación de la transcripción aguas abajo.
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