title: The RLIP76 N-terminus binds ARNO to regulate PI 3-kinase, Arf6 and Rac signaling, cell spreading and migration.
abstract: RLIP76 is a multifunctional protein involved in tumor growth and angiogenesis, and a promising therapeutic target in many cancers. RLIP76 harbors docking sites for many proteins, and we have found that it interacts with ARNO, a guanine nucleotide exchange factor for Arf6, and that RLIP76 regulates activation of Rac1 via Arf6, and regulates cell spreading and migration in an ARNO and Arf6-dependent manner. Here we show that ARNO interacts with the RLIP76 N-terminal domain, and this domain was required for RLIP76-dependent cell spreading and migration. We identified two sites in the RLIP76 N-terminus with differential effects on ARNO binding and downstream signaling: Ser29/Ser30 and Ser62. Ser29/30 mutation to Alanine inhibited ARNO interaction and was sufficient to block RLIP76-dependent cell spreading and migration, as well as RLIP76-dependent Arf6 activation. In contrast, RLIP76(S62A) interacted with ARNO and supported Arf6 activation. However, both sets of mutations blocked Rac1 activation. RLIP76-mediated Rac and Arf6 activation required PI3K activity. S29/30A mutations inhibited RLIP76-dependent PI3K activation, but S62A mutation did not. Together these results show that ARNO interaction with the RLIP76 N-terminus regulates cell spreading and motility via PI3K and Arf6, independent of RLIP76 control of Rac.
authors.affiliation: Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
authors.forename: Seunghyung
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
authors.forename: Jeremy G T
authors.initials: JG
authors.lastname: Wurtzel
authors.affiliation: Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA; Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. Electronic address: goldfinger@temple.edu.
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Goldfinger
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002465:Cell Movement; D002478:Cells, Cultured; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D015398:Signal Transduction; D020744:rac GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D020690:GTPase-Activating Proteins; D019869:Phosphatidylinositol 3-Kinases; D020744:rac GTP-Binding Proteins; C490620:Ralbp1 protein, mouse; C102474:cytohesin-2; C000715727:Arf6 protein, mouse
keywords: ARF; Cell migration; Cell signaling; PI 3-kinase (PI3K); Rac