Cell migration and proliferation are just two of the processes regulated by Ras proteins, through Raf-mediated activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Rajalingam and colleagues, reporting in Nature Cell Biology, now show that the evolutionarily conserved and ubiquitously expressed protein prohibitin certainly doesn't live up to its name, instead 'activating' the Ras-induced ERK/MAPK pathway and 'promoting' epithelial cell migration.

The authors had previously identified prohibitin in an RNA interference (RNAi)-based screen, and, in the present study, they showed that suppressing prohibitin expression by RNAi reduced cell spreading and increased cell–cell adhesion in treated HeLa cells and in other cancer cell lines. The RNAi-treated cells, when viewed by scanning and transmission electron microscopy, had almost no intercellular spaces and lacked any membrane protrusions. Confocal microscopy showed that this resulted from increased levels of the adherens junction proteins cadherin and -catenin at regions of cell–cell contact, which stabilized the junctions.

When epidermal growth factor (EGF) was added to prohibitin-depleted HeLa cells — to stimulate the EGF receptor (EGFR) and its relative HER2 — these cells could not migrate efficiently on collagen as they normally do. The authors subsequently found a similar phenotype when cells were depleted of EGFR or HER2.

Members of the EGFR family signal through Ras proteins to ERK/MAPK (through Raf and MEK (MAPK and ERK kinase)) and to Akt (through phosphatidylinositol 3-kinase). Inhibiting prohibitin expression reduced basal and EGF-induced phosphorylation of ERK/MAPK, but had no effect on Akt, nor on the protein levels of Ras, C-Raf, MEK1/2 or ERK/MAPK1/2. However, prohibitin reduction did block the EGF-induced phosphorylation of C-Raf on Ser338. Phosphorylation on Ser338 is required for C-Raf activation after Ser259 is dephosphorylated, and high levels of Ser259-phosphorylated C-Raf were seen in the absence of prohibitin. So EGF-induced C-Raf activation requires prohibitin.

This being the case, Rajalingam and colleagues surmised that direct C-Raf activation should overcome the formation of increased intercellular adhesion and the reduced ERK/MAPK phosphorylation that occurred in the absence of prohibitin — which it did, when indirectly activated, constitutively expressed or artificially targeted to the membrane. At the membrane, Raf is usually recruited by Ras to caveolae, and the authors managed to detect prohibitin and C-Raf in the caveolin-1-rich fractions of HeLa cells and other cancer cell lines. Despite EGF stimulation, C-Raf could not be detected in caveolae in prohibitin-depleted cells. Prohibitin and C-Raf interacted in vitro and endogenously in cells, and this direct interaction was necessary for EGF to activate C-Raf. Prohibitin was also needed for Ras to interact with, and to activate, C-Raf.

C-Raf activation by Ras requires, among many events, a 14-3-3 protein to be displaced from Ser259-phosphorylated C-Raf. After this Ras-mediated event, Ser259 is dephosphorylated and C-Raf can associate with the membrane, where it is phosphorylated on Ser338 and Tyr341 for full activation. Adding EGF resulted in the dephosphorylation of Ser259 on C-Raf and its subsequent membrane localization only when prohibitin was present. Even Ras activation by EGF couldn't promote 14-3-3 displacement from C-Raf if prohibitin was missing. Prohibitin is therefore needed for Ras to displace 14-3-3. As a Ser259Ala mutant of C-Raf, which cannot bind 14-3-3 and binds to Ras with a high affinity, could rescue C-Raf activation in prohibitin-depleted cells, Rajalingam and colleagues ended their report by suggesting that Ser259 is the site from which 14-3-3 is displaced by Ras and prohibitin.

Author: Katrin Bussell - Copyright © 2005 Nature Publishing Group, a division of MacMillan Publishers Limited; used with permission