Identification and Characterization of a B‐Raf Kinase Alpha Helix Critical for the Activity of MEK Kinase in MAPK Signaling

Abstract

In the mitogen‐activated protein kinase (MAPK) pathway, an oncogenic V600E mutation in B‐Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B‐Raf accounts for more than half of all melanomas and ~3% of all cancers and many drugs target the ATP‐binding site of the enzyme for its inhibition. Since B‐Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we used information from the available B‐Raf‐MEK crystal structure to generate an active form of B‐RafV600E that can be expressed using a bacterial expression system. In doing so, we identified an alpha helix on B‐Raf, found at the B‐Raf‐MEK interface, that is critical for their interaction and the oncogenic activity of B‐RafV600E. We introduced mutations along this alpha helix to pinpoint regions that are important for the B‐Raf‐MEK interaction and tested their effects on binding and phosphorylation. We performed binding experiments between B‐Raf mutants and MEK using pull downs and biolayer interferometry. We also assessed phosphorylation levels of MEK, as well as its downstream target ERK, in vitro and in cells. These studies showed that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. This result suggests that this B‐Raf alpha helix binding site on MEK could be a site to target for drug development to treat B‐RafV600E‐induced melanomas. Our cell‐based data with a point mutation in B‐Raf further suggests that combination therapies with ATP‐competitive inhibitors would be useful to further reduce B‐Raf activity and prevent the development of resistance.