Negative regulation of RAF kinase activity by ATP is overcome by 14-3-3-induced dimerization

Area of Science:

• Molecular Biology
• Structural Biology
• Cancer Research

Background:

• The RAS-RAF-MEK-ERK signaling pathway is crucial in cell growth and frequently dysregulated in cancers.
• Adenosine triphosphate (ATP) plays a key role in regulating RAF kinase activity by preventing dimer formation.

Purpose of the Study:

• To elucidate the structural mechanisms by which ATP and 14-3-3 protein regulate BRAF kinase activity.
• To understand how oncogenic BRAF mutations disrupt these regulatory mechanisms, leading to pathway activation.

Main Methods:

• X-ray crystallography was used to determine the structures of human BRAF kinase-domain (BRAF^KD) complexed with MEK and an ATP analog (AMP-PCP) at 2.9-Å resolution.
• A second crystal structure of the BRAF^KD-14-3-3 complex was determined at 2.5-Å resolution.

Main Results:

• The BRAF^KD-ATP analog structure revealed that ATP binding induces an inactive, monomeric BRAF^KD conformation.
• The BRAF^KD-14-3-3 structure demonstrated that dimeric 14-3-3 protein promotes BRAF^KD dimerization, thereby increasing kinase activity and overcoming ATP's inhibitory effect.
• Oncogenic BRAF mutations appear to alter ATP interactions, lowering the threshold for RAF dimerization and pathway activation.

Conclusions:

• This study provides a structural framework for understanding BRAF regulation by ATP and 14-3-3 protein.
• The findings offer insights into the mechanisms of oncogenic BRAF mutations and suggest new strategies for developing targeted cancer therapies.