SHOC2–MRAS–PP1 complex positively regulates RAF activity and contributes to Noonan syndrome pathogenesis

Abstract

Dephosphorylation of the inhibitory “S259” site on RAF kinases (S259 on CRAF, S365 on BRAF) plays a key role in RAF activation. The MRAS GTPase, a close relative of RAS oncoproteins, interacts with SHOC2 and protein phosphatase 1 (PP1) to form a heterotrimeric holoenzyme that dephosphorylates this S259 RAF site. MRAS and SHOC2 function as PP1 regulatory subunits providing the complex with striking specificity against RAF. MRAS also functions as a targeting subunit as membrane localization is required for efficient RAF dephosphorylation and ERK pathway regulation in cells. SHOC2’s predicted structure shows remarkable similarities to the A subunit of PP2A, suggesting a case of convergent structural evolution with the PP2A heterotrimer. We have identified multiple regions in SHOC2 involved in complex formation as well as residues in MRAS switch I and the interswitch region that help account for MRAS’s unique effector specificity for SHOC2–PP1. MRAS, SHOC2, and PPP1CB are mutated in Noonan syndrome, and we show that syndromic mutations invariably promote complex formation with each other, but not necessarily with other interactors. Thus, Noonan syndrome in individuals with SHOC2, MRAS, or PPPC1B mutations is likely driven at the biochemical level by enhanced ternary complex formation and highlights the crucial role of this phosphatase holoenzyme in RAF S259 dephosphorylation, ERK pathway dynamics, and normal human development.

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“…All 199 but two of these 16 de novo SNVs cause missense changes, all but two of these genes cause 200 disease by an altered-function mechanism, and these DNMs were depleted for CpGs relative to 201 all recurrent mutations. Two of the genes with highly recurrent de novo SNVs, SHOC2 and 202 PPP1CB, encode interacting proteins that are known to play a role in regulating the RAS-MAPK 203 pathway, and pathogenic variants in these genes are associated with a Noonan-like 204 syndrome 33 . Moreover, two of these recurrent DNMs are in the same gene SMAD4, which 205 encodes a key component of the TGF-beta signalling pathway, potentially expanding the 206 pathophysiology of germline selection beyond the RAS-MAPK pathway.…”

Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome

“…More recently, the use of wholeexome sequencing (WES) has allowed the discovery of RASopathy-associated genes encoding signal transducers or modulators that do not belong to the canonical RAS-MAPK pathway, but when functionally perturbed, are predicted to impact RAS signaling by still poorly characterized circuits. 20,[23][24][25][26][27][28][29] A remarkable finding of the molecular genetics of NS and other RASopathies is the occurrence of conserved themes in the mechanism of disease. This applies in particular to mutations affecting genes encoding the various members of the RAS superfamily of GTPases that have been implicated in these disorders, including KRAS, HRAS, NRAS, RRAS, MRAS, and CDC42.…”

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“…RASopathy-associated SHOC2, MRAS, and PPP1CB mutants cause enhanced MEK phosphorylation by promoting an augmented or more stable binding of the three proteins forming the MRAS/SHOC2/ PPP1CB complex (Young et al, 2018). Of note, MRAS/SHOC2/PP1C complex-dependent and complex-independent mechanisms of RAF activation exist, which are thought to differentially modulate RAS-ERK signaling output in varying cellular contexts.…”

“…PP1C is required for RAF1 activation and the PP1C/SHOC2/MRAS complex specifically promotes RAF1 dephosphorylation at a conserved inhibitory serine residue (Ser259), which in turn enables protein dimerization and activation. RASopathy‐associated SHOC2, MRAS, and PPP1CB mutants cause enhanced MEK phosphorylation by promoting an augmented or more stable binding of the three proteins forming the MRAS/SHOC2/PPP1CB complex (Young et al, ). Of note, MRAS/SHOC2/PP1C complex‐dependent and complex‐independent mechanisms of RAF activation exist, which are thought to differentially modulate RAS–ERK signaling output in varying cellular contexts.…”