title: OSBP-related protein 3 (ORP3) coupling with VAMP-associated protein A regulates R-Ras activity.
abstract: ORP3 is an R-Ras interacting oxysterol-binding protein homolog that regulates cell adhesion and is overexpressed in several cancers. We investigated here a novel function of ORP3 dependent on its targeting to both the endoplasmic reticulum (ER) and the plasma membrane (PM). Using biochemical and cell imaging techniques we demonstrate the mechanistic requirements for the subcellular targeting and function of ORP3 in control of R-Ras activity. We show that hyperphosphorylated ORP3 (ORP3-P) selectively interacts with the ER membrane protein VAPA, and ORP3-VAPA complexes are targeted to PM sites via the ORP3 pleckstrin homology (PH) domain. A novel FFAT (two phenylalanines in an acidic tract)-like motif was identified in ORP3; only disruption of both the FFAT-like and canonical FFAT motif abolished the phorbol-12-myristate-13-acetate (PMA) stimulated interaction of ORP3-P with VAPA. Co-expression of ORP3 and VAPA induced R-Ras activation, dependent on the interactions of ORP3 with VAPA and the PM. Consistently, downstream AktS473 phosphorylation and β1-integrin activity were enhanced by ORP3-VAPA. To conclude, phosphorylation of ORP3 controls its association with VAPA. Furthermore, we present evidence that ORP3-VAPA complexes stimulate R-Ras signaling.
authors.affiliation: Minerva Foundation Institute for Medical Research, Biomedicum 2U, FI-00290 Helsinki, Finland.
authors.forename: Marion
authors.initials: M
authors.lastname: Weber-Boyvat
authors.affiliation: Minerva Foundation Institute for Medical Research, Biomedicum 2U, FI-00290 Helsinki, Finland.
authors.forename: Henriikka
authors.initials: H
authors.lastname: Kentala
authors.affiliation: VTT Medical Biotechnology and Turku Centre for Biotechnology, University of Turku, FI-20520 Turku, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Lilja
authors.affiliation: Minerva Foundation Institute for Medical Research, Biomedicum 2U, FI-00290 Helsinki, Finland.
authors.forename: Terhi
authors.initials: T
authors.lastname: Vihervaara
authors.affiliation: National Institute for Health and Welfare, Biomedicum 1, FI-00290 Helsinki, Finland.
authors.forename: Raisa
authors.initials: R
authors.lastname: Hanninen
authors.affiliation: Minerva Foundation Institute for Medical Research, Biomedicum 2U, FI-00290 Helsinki, Finland.
authors.forename: You
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Institute of Biotechnology, FI-00014 University of Helsinki, Finland.
authors.forename: Johan
authors.initials: J
authors.lastname: Peränen
authors.affiliation: Institute of Biotechnology, FI-00014 University of Helsinki, Finland.
authors.forename: Tuula A
authors.initials: TA
authors.lastname: Nyman
authors.affiliation: VTT Medical Biotechnology and Turku Centre for Biotechnology, University of Turku, FI-20520 Turku, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Ivaska
authors.affiliation: Minerva Foundation Institute for Medical Research, Biomedicum 2U, FI-00290 Helsinki, Finland; Institute of Biomedicine/Anatomy, FI-00014 University of Helsinki, Finland. Electronic address: vesa.olkkonen@helsinki.fi.
authors.forename: Vesa M
authors.initials: VM
authors.lastname: Olkkonen
mesh_terms: D020816:Amino Acid Motifs; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004789:Enzyme Activation; D050556:Fatty Acid-Binding Proteins; D057809:HEK293 Cells; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000096:biosynthesis / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
chemical_list: D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D019012:Integrin beta1; D013755:Tetradecanoylphorbol Acetate; D033921:Vesicular Transport Proteins; D018631:ras Proteins; C471934:OSBPL3 protein, human; D000098825:Oxysterol Binding Proteins; C114051:VAPA protein, human; C016658:phorbolol myristate acetate; C085929:RRAS protein, human
keywords: FFAT motif; Integrin; ORP3; Phosphorylation; R-Ras; VAPA