title: VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking.
abstract: Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.
authors: Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016.
authors: Mo
authors: 0000-0002-0416-9132
authors: M
authors: Zhou
authors: Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016.
authors: Heidi
authors: H
authors: Wiener
authors: Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016.
authors: Wenjuan
authors: W
authors: Su
authors: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX 77030.
authors: Yong
authors: 0000-0002-0214-8151
authors: Y
authors: Zhou
authors: Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016.
authors: Caroline
authors: C
authors: Liot
authors: Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016.
authors: Ian
authors: 0000-0002-2275-137X
authors: I
authors: Ahearn
authors: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX 77030.
authors: John F
authors: JF
authors: Hancock
authors: Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016 philim01@nyumc.org.
authors: Mark R
authors: 0000-0002-1179-8156
authors: MR
authors: Philips
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism*; D004285:Dogs; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D054878:Lipoylation; D061985:Madin Darby Canine Kidney Cells; D008545:Melanoma / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D017368:Protein Prenylation; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D050505:Mutant Proteins; C419573:VPS35 protein, human; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human