Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane

Abstract

Prohibitin (PHB) is indispensable for Ras-induced Raf-1 activation, cell migration and growth; however, the exact role of PHB in the molecular pathogenesis of cancer metastasis remains largely unexamined. Here, we found a positive correlation between plasma membrane-associated PHB and the clinical stages of cancer. The level of PHB phosphorylated at threonine 258 (T258) and tyrosine 259 (Y259) in human cancer-cell membranes correlated with the invasiveness of cancer cells. Overexpression of phosphorylated PHB (phospho-PHB) in the lipid-raft domain of the cell membrane enhanced cell migration/invasion through PI3K/Akt and Raf-1/ERK activation. It also enhanced epithelial-mesenchymal transition, matrix metalloproteinase-2 activity and invasiveness of cancer cells in vitro. Immunoprecipitation analysis demonstrated that phospho-PHB associated with Raf-1, Akt and Ras in the membrane and was essential for the activation of Raf-1 signaling by Ras. Mice implanted with cancer cells stably overexpressing PHB in the plasma membrane showed enlarged cervical tumors, enhanced metastasis and shorter survival time compared with mice implanted with cancer cells without PHB overexpression. Dephosphorylation of PHB at T258 by site-directed mutagenesis diminished the in vitro and in vivo effects of PHB. These results suggest that increase in phospho-PHB T258 in the raft domain of the plasma membrane has a role in the Ras-driven activation of PI3K/Akt and Raf-1/ERK-signaling cascades and results in the promotion of cancer metastasis.

Function of Flotillins in Receptor Tyrosine Kinase Signaling and Endocytosis: Role of Tyrosine Phosphorylation and Oligomerization

“…Thus, at least a fraction of prohibitin must be found in another cellular localization such as the plasma membrane. Importantly, prohibitin has been shown to be essential for cell migration that is induced by the Ras-Raf-MAPK pathway (115), and very recent findings show that phosphorylation of prohibitin, which takes place in a raft domain, is necessary for the Ras-dependent enhancement of cellular metastasis (118), verifying the results of Rajalingam et al (115). Thus, there are several parallels in prohibitin and flotillin function, including MAPK signaling and cell migration, although they appear to be involved in different steps of the signaling.…”

Prohibitin-2 Binding Modulates Insulin-like Growth Factor-binding Protein-6 (IGFBP-6)-induced Rhabdomyosarcoma Cell Migration

“…On the other hand, Akt-induced threonine 258 phosphorylation of PHB1 inhibits its interactions with PIP3 and Shp2, which may facilitate insulin-induced PIP3/Akt signaling in 3T3-L1 adipocytes (50). Increased threonine 258 phosphorylation of PHB1 in lipid rafts is associated with enhanced cancer cell migration/invasion (23). Consistent with this, viral capsid protein VP1 decreased both cervical cancer migration/invasion and threonine 258 phosphorylation of PHB1 in lipid rafts (51).…”

“…Overexpression of PHB1 in 3T3-L1 fibroblasts up-regulates basal ERK MAPK signaling, leading to adipogenesis (58). In contrast, increased PHB1 phosphorylation, rather than changed PHB1 protein levels in lipid rafts, plays an important role in the Ras-driven activation of PIP3/AKT and Raf-1/ERK signaling cascades, resulting in the promotion of cancer cell metastasis (23). Our results differ from these findings, showing that reduced PHB levels in RMS cells had no effect on basal or IGFBP-6-induced ERK MAPK activation.…”

“…Knockdown of PHB2 Does Not Affect Basal or IGFBP-6-induced MAPK Activation-PHB1 has been reported to regulate the ERK MAPK pathway in both normal and cancer cells (11,19,23), and we have shown that IGFBP-6-induced ERK and JNK MAPK phosphorylation contributes to IGFBP-6-induced Rh30 cell migration (4). We therefore investigated if PHB2 knockdown affected MAPK signaling.…”

MIG-7 Controls COX-2/PGE2-Mediated Lung Cancer Metastasis

“…Immunohistochemistry and histopathology analyses were conducted as described previously (22,24). The images were scanned into a digital format by Scanscope XT system (Aperio Technologies) and analyzed using Aperio ImageScope 9.1 software (Aperio Technologies).…”