Phosphorylation Weakens but Does Not Inhibit Membrane Binding and Clustering of K-Ras4B

Abstract

K-Ras4B is one of the most frequently mutated Ras isoforms in cancer. The signaling activity of K-Ras4B depends on its localization to the plasma membrane (PM), which is mainly mediated by its polybasic farnesylated C-terminus. On top of the constitutive cycles that maintain the PM enrichment of K-Ras4B, conditional phosphorylation at Ser181 located within this motif has been found to be involved in regulating K-Ras4B's cell distribution and signaling activity. However, discordant observations have undermined our understanding of the role this phosphorylation plays. Here, we report an efficient strategy for producing K-Ras4B simultaneously bearing phosphate, farnesyl, and methyl modifications on a preparative scale, a very useful in vitro system when used in concert with model biomembranes. By using this system, we determined that phosphorylation at Ser181 does not fully inhibit membrane binding and clustering of K-Ras4B but reduces its membrane binding affinity, depending on membrane fluidity. In addition, phosphorylated K-Ras4B maintains tight association with its cytosolic shuttle protein PDEδ. After delivering K-Ras4B containing nonhydrolyzable phosphoserine mimetic into cells, the protein displayed a decreasing PM distribution compared with nonphosphorylable K-Ras4B, implying that phosphorylation might facilitate the dissociation of K-Ras4B from the PM. In addition, phosphorylation does not alter the localization of K-Ras4B in the liquid-disordered lipid subdomains of the membrane but slightly alters the thermotropic properties of K-Ras4B-incorporated membranes probably due to minor differences in membrane partitioning and dynamics. These results provide novel mechanistic insights into the role that phosphorylation at Ser181 plays in regulating K-Ras4B's distribution and activity.

Raf-1 Cysteine-Rich Domain Increases the Affinity of K-Ras/Raf at the Membrane, Promoting MAPK Signaling

“…In the oncogenic state, the active conformation of K-Ras4B can be extracted from the membrane by calmodulin (Jang et al, 2017;Sperlich et al, 2016). Of note, phosphorylation of Ser-181 weakens membrane attachment, although may not abolish it (Zhang et al, 2017). Previously we suggested that, regardless of the nucleotide-binding states, active K-Ras4B may liberate the HVR from the catalytic domain exposing the effector binding site, while inactive K-Ras4B may sequester the membrane-interacting HVR burying the effector binding site .…”

RAS Proteins and Their Regulators in Human Disease

“…The polybasic KRAS4b HVR can be phosphorylated at serine 181 by protein kinase C (Ballester et al, 1987) resulting in a decreased net charge, reduction in affinity for the PM, and accumulation of KRAS on endo-membranes (Zhang et al, 2017; Bivona et al, 2006; Sung et al, 2013). Ca 2+ /calmodulin is able to “extract” KRAS4b from the PM and redistribute it to endo-membranes (Fivaz and Meyer, 2005).…”

K-Ras prenylation as a potential anticancer target

“…However, K-Ras4b contains an isoform-specific polybasic sequence in the HVR, which fosters association to the acidic regions of the plasma membrane (PM) [23]. Of note, phosphorylation of S181 within the polybasic sequence can further modulate K-Ras4b-PM interaction [24].…”

“…It has been shown that PKC can phosphorylate K-Ras4B on S181, reducing the strong positive charge of the HVR's polybasic sequence, thereby weakening interaction with the negatively charged PM, potentially fostering membrane dissociation [24]. Interestingly, Ca2+-binding calmodulin was found to bind K-Ras through its farnesylated C-termini in a hydrophobic pocket [38].…”