Raf-1 activates MAP kinase-kinase

Area of Science:

• Cellular Biology
• Molecular Oncology
• Signal Transduction

Background:

• The c-raf-1 protooncogene encodes a serine/threonine protein kinase activated by extracellular stimuli.
• The physiological substrates of c-Raf-1, a key signaling molecule, remain largely unknown.
• Mitogen-activated protein (MAP) kinases Erk1 and Erk2 are activated via phosphorylation by MAP kinase-kinase (MAPK-K).

Purpose of the Study:

• To identify the physiological substrates of the c-Raf-1 protein kinase.
• To investigate the role of c-Raf-1 in the activation of MAPK-K and Erk1/2.
• To elucidate the upstream activators of MAPK-K in cellular signaling.

Main Methods:

• Analysis of constitutively active MAPK-K and Erk1/2 in v-raf-transformed cells.
• Enzymatic assays using partially purified MAPK-K and phosphatase 2A.
• In vitro kinase assays with purified c-Raf-1 to assess MAPK-K reactivation.
• Cation-exchange chromatography to characterize MAPK-K and its interaction with c-Raf-1.

Main Results:

• MAPK-K and Erk1/2 were found to be constitutively active in v-raf-transformed cells.
• Phosphatase 2A deactivated MAPK-K, but purified c-Raf-1 could reactivate it in vitro.
• c-Raf-1 mediated the selective phosphorylation of a 50,000 M(r) polypeptide co-eluting with MAPK-K.
• These findings demonstrate c-Raf-1 as an immediate upstream activator of MAPK-K.

Conclusions:

• c-Raf-1 is identified as the direct upstream activator of MAPK-K.
• MAPK-K is the first identified physiological substrate of the c-raf-1 protooncogene product.
• This study clarifies a critical step in the MAP kinase signaling cascade regulated by c-Raf-1.