Structure of the MRAS–SHOC2–PP1C phosphatase complex

Abstract

RAS–MAPK signalling is fundamental for cell proliferation and is altered in most human cancers1–3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF–MEK1–14-3-3 complexes4, the intermediate steps that lead to RAF activation remain unclear. The MRAS–SHOC2–PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF–RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in rasopathies—developmental syndromes caused by aberrant MAPK pathway activation6–14—and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)–RAS-driven tumours15–18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS–SHOC2–PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS–GTP molecules recruit RAF–14-3-3 and SHOC2–PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein–protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS–MAPK signalling, therefore providing the structural basis for therapeutic interventions.

Defining bone fide effectors of RAS GTPases

“…Our current understanding of these networks infers a great deal from sequence homology, and more work is required to elucidate which proteins with RBD domains are bone fide H/K/NRAS effectors. It is also emerging that proteins lacking RBDs may serve as RAS effectors, including SHOC2 which complexes using a leucine rich repeat (LRR) domain [ 126–129 ] and hexokinase 1 (HK1) which binds KRAS4A in a manner to be determined. [ 130 ] There are two key questions that future research must address: (1) selectivity of the >50 putative effectors for all RAS superfamily GTPases, and (2) how does GTPase binding elicit effector activation.…”

Structural insights into the role of SHOC2‐MRAS‐PP1C complex in RAF activation

“…1A) [37][38][39][40][41][42][43][44][45][46][47]. To explore the structural and functional role of SHOC2 and MRAS in the recruitment of PP1C to the plasma membrane and the dephosphorylation of the CR2-pS in RAF kinases, four research groups, including us, recently determined the structure of the SMP complex by cryo-EM and X-ray crystallography [22,[48][49][50]. These studies also determined the affinity of SMP assembly by biophysical methods, its dependence on GTP-bound MRAS and that MRAS could be substituted for the canonical H/K/NRAS.…”

Structural Advancement in Shoc2‐MAPK Signaling Pathways in the Treatment of Cancer and Other Diseases

“…[56,57] New Xray structures of the protein are also available in the Protein Data Bank (PDB) (PDB id: 7T7 A, 7SD1, 7TVG and 7TYG). [56,57] A comparison of 3D structure for Shoc2 in its bound form (PDB id7sd0) and unbound form (PDB id: 7 t7a) was conducted to observe any apparent structural changes on bound and unbound form of the Shoc2 protein. This comparison has been shown in Figure 3.…”

“…The dephosphorylation is followed by phosphorylation of MEK and ERK family kinases thereby stimulating the MAPK pathway. The latest cryo EM structures of Shoc2 in complex with MRAS and PP1 C with a resolution of 2.89‐2.95 Å (PDB id: 7SD0 and 7UPI) are available which provides further insights into the complex [56,57] . New X‐ray structures of the protein are also available in the Protein Data Bank (PDB) (PDB id: 7T7 A, 7SD1, 7TVG and 7TYG) [56,57] .…”

“…The latest cryo EM structures of Shoc2 in complex with MRAS and PP1 C with a resolution of 2.89-2.95 Å (PDB id: 7SD0 and 7UPI) are available which provides further insights into the complex. [56,57] New Xray structures of the protein are also available in the Protein Data Bank (PDB) (PDB id: 7T7 A, 7SD1, 7TVG and 7TYG). [56,57] A comparison of 3D structure for Shoc2 in its bound form (PDB id7sd0) and unbound form (PDB id: 7 t7a) was conducted to observe any apparent structural changes on bound and unbound form of the Shoc2 protein.…”