Abstract 969: B-Raf activation loop phosphorylation is crucial for efficient MAPK signalling in vivo

Abstract

Abstract Raf proteins are evolutionary conserved protein serine/threonine kinases and represent the MAPKKKs of the ERK-pathway. This pathway plays a crucial role in embryogenesis and is deregulated in multiple diseases. B-Raf represents the most potent MEK kinase of the Raf family and mutations in B-Raf or its upstream activators (e.g. Ras) are well-described in malignancies. So far, targeting the kinase function directly is flawed by paradoxical ERK activation in presence of oncogenic Ras. Therefore, a better understanding of its activation and finding an alternative targeting strategy is crucial. Phosphorylation of the T599VKS602-motif within the B-Raf activation loop is essential for full in vitro kinase activity stimulated by oncogenic Ras and is mimicked by oncogenic mutations such as V600E. To validate the importance of activation loop phosphorylation in vivo, we established a novel conditional BrafAVKA knock-in mouse that, upon Cre-mediated recombination, expresses the B-RafAVKA protein in which T599 and S602 are replaced by alanine residues. Strikingly and in strong contrast to Braf knock-out mice, homozygous BrafAVKA mice are viable and show no early developmental defects suggesting that kinase impairment but not loss of the protein can be tolerated during embryogenesis. Additionally, these findings suggest that the B-RafAVKA mutant protein does not provoke a paradoxical ERK activation like the kinase dead B-RafD594A. However, neurological abnormalities such as ataxia or aberrant locomotion occur in older animals. This phenotype correlates with decreased MEK/ERK phosphorylation in total brain lysates. Also, signalling capacity of the ERK MAPK module, immediate early gene induction and proliferation are impaired in B-RafAVKA expressing MEFs. Our findings support the concept that T599VKS602-motif phosphorylation is necessary for full B-Raf kinase activity in vivo. Therefore, blocking activation loop phosphorylation could be used to suppress aberrant ERK-pathway activity, e.g. by oncogenic Ras or RTKs, and circumventing paradoxical ERK activation in vivo. Citation Format: Martin Koehler, Michael Roering, Sandra Braun, Tilman Brummer. B-Raf activation loop phosphorylation is crucial for efficient MAPK signalling in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 969. doi:10.1158/1538-7445.AM2015-969