K-Ras resides on the Arf6-mediated CIE system and its active type interacted with Arf6T27N

Ras is known as an oncogene transferring signals from the plasma membrane. Recent studies have demonstrated that plasma membrane was not the unique platform for Ras signaling. Ras could also be endocytosed and transported to different endomembrane compartments, evoking different signal pathways there. It is of great significance to exploit the unique intracellular trafficking features of different Ras isoforms to develop new anti-Ras drugs. ADP-ribosylation factor 6 (Arf6) was known to mediate one of the clathrin-independent endocytosis (CIE) pathways. The role of Arf6 in K-Ras dynamic remains largely unknown. In this study, we showed that K-RasG12V co-localized with Arf6 at the plasma membrane, and entered the tubular endosomes or protrusions induced by cytochalasin D or aluminum fluoride in the same way as H-RasG12V does. A subcellular fractionation experiment demonstrated that Arf6 siRNA treatment reduced the plasma membrane presence of both endogenous Ras isoforms and inhibited the phosphorylation of Erk triggered by EGF. When co-expressed with Arf6Q67L, both isoforms were sequestered into the large phosphatidylinositol 4,5-biphosphate [PI(4,5)P2]-enriched vacuoles. However, when co-expressed with Arf6T27N, K-RasG12V co-localized with Arf6T27N at the tubular endosomes significantly than H-RasG12V. Immunoprecipitation and GST fusion protein pull-down studies found out for the first time that K-RasG12V interacted with Arf6T27N. Swapping mutation study showed that the above difference was due to different C-termini. Our study indicated that Arf6 was involved in the dynamic regulation of both Ras isoforms.

Graphical abstractFig. A showed K-RasG12V colocalized with Arf6T27N at the tubular endosome significantly than H-RasG12V. Fig. B showed the percentage of co-localization analyzed with Metamorph Software. Fig. C showed an interaction between K-RasG12V and Arf6T27N.Figure optionsDownload full-size imageDownload high-quality image (126 K)Download as PowerPoint slideHighlights► K-RasG12V co-localized with Arf6T27N significantly than H-RasG12V. ► Not H-RasG12V but K-RasG12V interacted with Arf6T27N. ► C-terminus of K-Ras was responsible for the above interaction. ► The interaction was GTP-dependent and could be enhanced by EGF stimulation.