|Title:||Mechanistic studies of Ras nanoclusters and Ras effectors interacting with the lipid membrane|
|Abstract:||Ras proteins are oncoproteins and play a major role in human cancers where they fail to switch off the signal for cell growth. More than 30% of human cancers are related to mutations of Ras proteins with K-Ras4B being the most frequently mutated isoform. They are plasma membrane localized molecular switches that function by shuttling between inactive GDP-bound and active GTP-bound forms. Signaling of K-Ras4B strongly depends on its correct localization in the plasma membrane. C6-ceramide has been shown to inhibit the growth activity of K-Ras4B mutated cells. However, the mechanism underlying this inhibition remains elusive. We established a heterogeneous model biomembrane containing C6-cermaide and used this membrane to demonstrate that the addition of K-Ras4B leads to drastic changes in the lateral membrane organization. In contrast to the partitioning behavior in other membranes, K-Ras4B forms small, monodisperse nanoclusters dispersed in a fluid-like environment, in all likelihood induced by some kind of lipid sorting mechanism. Fluorescence cross-correlation data indicate no direct interaction among C6-ceramide and K-Ras4B, suggesting that K-Ras4B essentially recruits other lipids. Moreover, a FRET-based binding assay reveals that the stability of K-Ras4B inserted in the membranes containing C6-ceramide is significantly reduced. A molecular mechanism for the inhibition of K-Ras4B mutated cells’ activity through C6-ceramide is postulated from the results of this study. In vivo studies suggest that intermolecular interactions foster the self-association of both N-Ras and K-Ras4B and the formation of nanoclusters in the cell membrane. As sites for effector binding, nanocluster formation is thought to be essential for effective signal transmission of both N-Ras and K-Ras4B. To shed more light on the spatial arrangement and mechanism underlying the proposed crosstalk between spatially segregated Ras proteins, we studied the simultaneous localization of N-Ras and K-Ras4B and their effect on the lateral organization of a heterogeneous model biomembrane using AFM and FRET methodology. We show that, owing to the different nature of their membrane anchor system, N-Ras and K-Ras4B not only avoid assembly in bulk solution, they also do not colocalize, but rather form individual nanoclusters which diffuse independently in the fluid membrane plane. Development of effective K-Ras4B inhibitors has been challenging, hence new approaches to inhibit this oncogenic protein are urgently required. The polybasic domain of K-Ras4B with its stretch of lysine residues is essential for its plasma membrane targeting and localization. Employing CD and fluorescence spectroscopy, confocal fluorescence and atomic force microscopy we show that the molecular tweezer CLR01 is able to efficiently bind to the lysine stretch in the polybasic domain of K-Ras4B, resulting in dissociation of the K-Ras4B protein from the lipid membrane and disintegration of K-Ras4B nanoclusters in the lipid bilayer. It was shown that targeting of the polybasic domain of K-Ras4B by properly designed tweezers might represent an effective strategy for inactivation of K-Ras4B signaling. Raf interaction with K-Ras4B is of great significance, which is the key step of MAPK signaling. Since B- and C-Raf proteins are autoinhibited in the cytosol with a closed conformation by binding of 14-3-3 and its inhibitory domains, they have to be recruited by GTP-bound, active K-Ras4B protein and dephosphorylated in order to be an open conformation. We found that A-, B- and C-Raf-RBD are all able to recognize the K-Ras4B in the lipid membrane. Interestingly, B and C-Raf-RBD directly bind to the lipid membrane, which was unexpected through the current understanding of Ras binding domain. A-Raf-RBD has a different effect on the lipid membrane. It was shown that the height of A-Raf-RBD in the lipid membrane is far beyond the size of A-Raf-RBD proteins, which indicates that A-Raf-RBD preferentially forms nanoclusters in the lipid membrane. Importantly, RBDs of A-, B- and C-Raf are proved to determine the population of Raf/K-Ras4B complex formation in the lipid membrane. Considering the formation of Raf/K-Ras4B is crucial for Ras-Raf activation, the RBD of Raf may plays a role in regulating Raf activation.|
|Subject Headings (RSWK):||Protein|
|Appears in Collections:||Physikalische Chemie|
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