Endomembrane association of activated H‐Ras, but not K‐Ras, causes autophagic cell death in human glioblastoma

Abstract

Activating mutations in H-Ras or K-Ras are oncogenic in many types of cells. However, in U-251 glioblastoma cells expression of activated H-Ras (G12→V) induces a novel phenotype, characterized by accumulation of cytoplasmic vacuoles and non-apoptotic death. Activated H-Ras produces a similar phenotype in nine different glioblastoma cell lines. These effects are not observed with the GDP-locked H-Ras (S17 →N). Electron microscopy and immunofluorescence microscopy indicate that the H-Ras-induced vacuoles exhibit properties of enlarged autophagosomes and autolysosomes. Most notably, the membranes surrounding the vacuoles incorporate GFP-LC3, an autophagosome marker. As vacuoles accumulate there is a marked increase in conversion of endogenous LC3 to the lipid-conjugated form (LC3-II) required for autophagosome biogenesis. Myc-tagged H-Ras(G12V) localizes to the limiting membranes surrounding the vacuoles. When membrane association of H-Ras(G12V) is prevented by a serine substitution at the COOH-terminal cysteine farnesylation site (C186), the induction of vacuoles is abolished. In contrast to the effects of H-Ras(G12V), the activated form of K-Ras4B does not induce the vacuolar phenotype. This suggests that induction of autophagic cell death in glioblastoma cells is related to the activation of unique H-Ras effector pathways. Domain swapping experiments are underway to further explore the features of H-Ras versus K-Ras required to induce autophagic cell death. Supported by a grant from the NIH (R01CA34569) to W.A.M.