Research ArticleBiochemistry

Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety

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Science Signaling  31 Mar 2020:
Vol. 13, Issue 625, eaaz0344
DOI: 10.1126/scisignal.aaz0344

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How Ca2+ inhibits KRAS

The RAS protein family regulates cell proliferation, differentiation, and survival. Mutations in RAS proteins are frequently implicated in driving tumor growth. Grant et al. resolved the structure of the C terminus of the RAS isoform KRAS4b in complex with the Ca2+-sensing protein calmodulin (CaM) and developed FRET sensors to explore the interaction in cells. They found that a farnesylated region of KRAS4b was bound in a hydrophobic pocket of CaM in response to increased intracellular Ca2+, thereby sequestering KRAS4b away from the membrane and dampening its output. Exploiting this sequestration mechanism might yield a new way to inhibit KRAS signaling.

Abstract

KRAS4b is a small guanosine triphosphatase (GTPase) protein that regulates several signal transduction pathways that underlie cell proliferation, differentiation, and survival. KRAS4b function requires prenylation of its C terminus and recruitment to the plasma membrane, where KRAS4b activates effector proteins including the RAF family of kinases. The Ca2+-sensing protein calmodulin (CaM) has been suggested to regulate the localization of KRAS4b through direct, Ca2+-dependent interaction, but how CaM and KRAS4b functionally interact is controversial. Here, we determined a crystal structure, which was supported by solution nuclear magnetic resonance (NMR), that revealed the sequestration of the prenyl moiety of KRAS4b in the hydrophobic pocket of the C-terminal lobe of Ca2+-bound CaM. Our engineered fluorescence resonance energy transfer (FRET)–based biosensor probes (CaMeRAS) showed that, upon stimulation of Ca2+ influx by extracellular ligands, KRAS4b reversibly translocated in a Ca2+-CaM–dependent manner from the plasma membrane to the cytoplasm in live HeLa and HEK293 cells. These results reveal a mechanism underlying the inhibition of KRAS4b activity by Ca2+ signaling pathways.

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