Synthesis And Functional Study Of Ras Proteins With Multi-post-translational Modifications

Ras proteins are membrane-bound small GTPases that act as molecular switches by cycling between the active GTP-bound state and the inactive GDP-bound state,resulting in the regulation of a variety of cellular processes.K-Ras4 B is one of the most frequently mutated Ras isoforms in cancers.The signaling activity of K-Ras4 B 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 steady-state PM enrichment of K-Ras4 B,conditional phosphorylation at Ser181 located within the polybasic 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-Ras4 B protein 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 inhibit membrane binding and clustering of K-Ras4 B but reduces its membrane binding affinity,depending on membrane fluidity.In addition,phosphorylation does not affect K-Ras4B’s association with its shuttle protein,PDEδ,that sustains the constitutive spatial cycle of K-Ras4 B.After delivering K-Ras4 B containing non-hydrolyzable phosphonomethylene alanine(Pma)into cells,the protein displayed a decreasing PM distribution compared with non-phosphorylable K-Ras4 B,implying that phosphorylation might facilitate the dissociation of K-Ras4 B from the PM due to its reduced membrane binding ability.In addition,phosphorylation does not alter the localization of K-Ras4 B in the liquid-disordered lipid subdomains of the membrane,but slightly alters the thermotropic properties of K-Ras4B-incorporated membranes 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 the distribution and activity of K-Ras4 B.Rheb(Ras homolog enriched in brain)is another member of the Ras superfamily of small GTPases,and is conserved from yeast to mammals.We firstly found that Rheb protein can be uibiquitinated in cells and tried to synthesize the Rheb-Ub conjugates.This may lay a foundation for the further investigation.