RLIP76 regulates Arf6-dependent cell spreading and migration by linking ARNO with activated R-Ras at recycling endosomes.
R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(Δ1-180) or RLIP76(Δ180-192). Spreading and migration required RLIP76(1-180), and RLIP76(Δ1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(Δ1-180). Similarly, RLIP76(Δ1-180) or RLIP76(Δ180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.
Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, United States.
Jeremy G T
JG
Wurtzel
Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, United States.
Seunghyung
S
Lee
Department of Diabetes and Metabolic Diseases Research, Division of Diabetes, Endocrinology & Metabolism, Beckman Research Institute, City of Hope, National Medical Center, Duarte, CA, United States.
Sharad S
SS
Singhal
Department of Diabetes and Metabolic Diseases Research, Division of Diabetes, Endocrinology & Metabolism, Beckman Research Institute, City of Hope, National Medical Center, Duarte, CA, United States.
Sanjay
S
Awasthi
Department of Medicine, University of California San Diego, La Jolla, CA, United States.
Mark H
MH
Ginsberg
Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, United States; Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, United States. Electronic address: goldfinger@temple.edu.
Lawrence E
LE
Goldfinger
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002465:Cell Movement / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D046912:Multiprotein Complexes; C490620:Ralbp1 protein, mouse; C102474:cytohesin-2; C513254:Rras protein, mouse; D020727:ADP-Ribosylation Factors; C000715727:Arf6 protein, mouse; D018631:ras Proteins
Migration; Ras; Recycling endosome; Small GTPase; Spreading