Hauseman, Zack, Fodor, Michelle, Viscomi, Jessica, Bleu, Melusine, Galli, Giorgio, Tordella, Luca, Thoma, Claudio and King, Daniel (2022) Cooperative assembly and structure of the RAS-SHOC2-PP1c holophosphatase provides mechanistic insights into disease-relevant mutations. Nature, 609. pp. 416-423. ISSN 1476-4687

Abstract

The RAS-RAF-MAPK signaling pathway is fundamental for cell proliferation and altered in a
majority of human cancers. However, our mechanistic understanding of how RAS proteins
activate RAF kinases is still incomplete. Recent elegant studies revealed structural details on an
inhibitory RAF complex with 14-3-3, although steps leading to RAF
activation remain unclear. As a potential activator of RAF, MRAS-SHOC2-PP1c holophosphatase
activity has been postulated to de-phosphorylate RAF on a specific Serine residue resulting in 14-
3-3 release enabling RAF activation. Despite its importance, to date structural evidence for such a
holophosphatase complex and its organization is missing. Here we reveal a 1.95 Å high-resolution
X-ray crystal structure of the ternary MRAS-SHOC2-PP1c complex showing SHOC2 enclosing in
its concave side both PP1c and MRAS. Biophysical characterization indicates a cooperative
assembly requiring active GTP-loaded MRAS, an observation we could expand to other RAS
isoforms, measuring different affinities. Our findings support a RAS-driven and multimeric model
for RAF activation in which two neighboring RAS-GTP molecules can simultaneously recruit RAF-
14-3-3 and SHOC2-PP1c to produce downstream pathway activation. Finally, MRAS, SHOC2 and
PP1c are mutated in Rasopathies, a spectrum of developmental syndromes caused by aberrant
MAPK pathway activation and SHOC2 itself has recently emerged as potential target in RTK-RAS
driven tumors. We find that Rasopathy mutations reside at protein-protein interfaces within the
holophosphatase and result in affinity changes. Collectively our findings shed light on a
fundamental mechanism of RAS biology and on mechanisms for clinically observed enhanced
RAS-MAPK signaling, thus providing the structural basis for therapeutic interventions.

Item Type:	Article
Date Deposited:	27 Sep 2022 00:45
Last Modified:	27 Sep 2022 00:45
URI:	https://oak.novartis.com/id/eprint/46762