title: Secreted Glioblastoma Nanovesicles Contain Intracellular Signaling Proteins and Active Ras Incorporated in a Farnesylation-dependent Manner.
abstract: Glioblastomas (GBMs) are malignant brain tumors with a median survival of less than 18 months. Redundancy of signaling pathways represented within GBMs contributes to their therapeutic resistance. Exosomes are extracellular nanovesicles released from cells and present in human biofluids that represent a possible biomarker of tumor signaling state that could aid in personalized treatment. Herein, we demonstrate that mouse GBM cell-derived extracellular nanovesicles resembling exosomes from an H-RasV12 myr-Akt mouse model for GBM are enriched for intracellular signaling cascade proteins (GO: 0007242) and Ras protein signal transduction (GO: 0007265), and contain active Ras. Active Ras isolated from human and mouse GBM extracellular nanovesicles lysates using the Ras-binding domain of Raf also coprecipitates with ESCRT (endosomal sorting complex required for transport)-associated exosome proteins Vps4a and Alix. Although we initially hypothesized a role for active Ras protein signaling in exosome biogenesis, we found that GTP binding of K-Ras was dispensable for its packaging within extracellular nanovesicles and for the release of Alix. By contrast, farnesylation of K-Ras was required for its packaging within extracellular nanovesicles, yet expressing a K-Ras farnesylation mutant did not decrease the number of nanovesicles or the amount of Alix protein released per cell. Overall, these results emphasize the primary importance of membrane association in packaging of extracellular nanovesicle factors and indicate that screening nanovesicles within human fluids could provide insight into tissue origin and the wiring of signaling proteins at membranes to predict onset and behavior of cancer and other diseases linked to deregulated membrane signaling states.
authors.affiliation: From the Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037 and.
authors.forename: Natalie
authors.initials: N
authors.lastname: Luhtala
authors.affiliation: From the Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037 and.|the Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Aaron
authors.initials: A
authors.lastname: Aslanian
authors.affiliation: the Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.affiliation: From the Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037 and hunter@salk.edu.
authors.forename: Tony
authors.initials: T
authors.lastname: Hunter
mesh_terms: D000818:Animals; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D055252:Cell-Derived Microparticles / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D051379:Mice; D054876:Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D048490:raf Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C117307:KRAS protein, human; C117020:PDCD6IP protein, human; C492144:Pdcd6ip protein, mouse; D048490:raf Kinases; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Ras protein; cell signaling; endosomal sorting complexes required for transport (ESCRT); exosome (vesicle); glioblastoma; proteomics