RAS G-domains allosterically contribute to the recognition of lipid headgroups and acyl chains

Abstract

Mutant RAS are major contributors to cancer and signal primarily from nanoclusters on the plasma membrane (PM). Their C-terminal membrane anchors are main features of membrane association. However, the same RAS isoform bound to different guanine nucleotides spatially segregate. Different RAS nanoclusters all enrich a phospholipid, phosphatidylserine (PS). These findings suggest more complex membrane interactions. Our electron microscopy-spatial analysis shows that wild-types, G12V mutants, and membrane anchors of isoforms HRAS, KRAS4A, and KRAS4B prefer distinct PS species. Mechanistically, reorientation of KRAS4B G-domain exposes distinct residues, such as Arg 135 in orientation state 1 (OS1) and Arg 73/Arg 102 in OS2, to the PM and differentially facilitates the recognition of PS acyl chains. Allele-specific oncogenic mutations of KRAS4B also shift G-domain reorientation equilibrium. Indeed, KRAS4BG12V, KRAS4BG12D, KRAS4BG12C, KRAS4BG13D, and KRAS4BQ61H associate with PM lipids with headgroup and acyl chain specificities. Distribution of these KRAS4B oncogenic mutants favors different nanoscale membrane topography. Thus, RAS G-domains allosterically facilitate membrane lateral distribution.

Lysophosphatidylcholine acyltransferase 1 suppresses nanoclustering and function of KRAS

“…Fig.2B shows that gold labeling of GFP-KRAS G12V per 1 μm 2 of PM area in cells expressing LPCAT1 was also significantly lower than that in the V2-expressing cells, suggesting that LPCAT1 expression significantly mislocalizes GFP-KRAS G12V from the PM. Another RAS isoform, HRAS, distributes to spatially separate nanoclusters enriched with different lipids than KRAS 9,10,15,16,19,20 . LPCAT1 expression partially decreased the nanoclustering of GFP-HRAS G12V (Fig.2C), while having no effect on the PM localization of GFP-HRAS G12V (Fig.2D).…”

“…EGFR dimerization/oligomerization occur in the L o domains or lipid rafts enriched with cholesterol and saturated lipids, which in turn promotes autophosphorylation and signaling 33,42 . On the other hand, KRAS activities occur away from cholesterol in cells 9,10,12,15,17–20 . We recently showed that the PM nanoclusters of KRAS oncogenic mutants, such as KRAS G12C , KRAS G12D , KRAS G12V , KRAS G13D and KRAS Q61H , contain PS species with the unsaturated sn-2 chains 20 .…”

“…On the other hand, KRAS activities occur away from cholesterol in cells 9,10,12,15,17–20 . We recently showed that the PM nanoclusters of KRAS oncogenic mutants, such as KRAS G12C , KRAS G12D , KRAS G12V , KRAS G13D and KRAS Q61H , contain PS species with the unsaturated sn-2 chains 20 . Surface plasmon resonance (SPR) further revealed that the purified KRAS more efficiently binds to model bilayers comprising the unsaturated PS species, but not the saturated PS species 21 .…”

“…Trapping KRAS to lipid rafts abolishes effector binding and the KRAS-dependent MAPK signaling 12–14 . Presence of saturated lipids in the nanoclusters of KRAS mutants also compromises recruitment of effector CRAF 17,20 . Thus, the opposing effects of LPCAT1 on EGFR and KRAS are consistent with the opposing lipid preferences of these two membrane proteins.…”

“…It was then reported that KRAS signaling nanoclusters selectively enrich an anionic phospholipid, phosphatidylserine (PS), more specifically PS species with unsaturated sn-2 acyl chains 15–24 . Depletion of endogenous PS disrupts the nanoclustering, signaling and oncogenic activities of KRAS mutants 15–20,25–30 . Acute addback of PS species with unsaturated sn-2 acyl chains, but not the fully saturated PS, effectively restores the nanoclustering and effector recruitment of mutant KRAS in the PS-depleted cells 17–21 .…”