title: A new dimension to Ras function: a novel role for nucleotide-free Ras in Class II phosphatidylinositol 3-kinase beta (PI3KC2β) regulation.
abstract: The intersectin 1 (ITSN1) scaffold stimulates Ras activation on endocytic vesicles without activating classic Ras effectors. The identification of Class II phosphatidylinositol 3-kinase beta, PI3KC2β, as an ITSN1 target on vesicles and the presence of a Ras binding domain (RBD) in PI3KC2β suggests a role for Ras in PI3KC2β activation. Here, we demonstrate that nucleotide-free Ras negatively regulates PI3KC2β activity. PI3KC2β preferentially interacts in vivo with dominant-negative (DN) Ras, which possesses a low affinity for nucleotides. PI3KC2β interaction with DN Ras is disrupted by switch 1 domain mutations in Ras as well as RBD mutations in PI3KC2β. Using purified proteins, we demonstrate that the PI3KC2β-RBD directly binds nucleotide-free Ras in vitro and that this interaction is not disrupted by nucleotide addition. Finally, nucleotide-free Ras but not GTP-loaded Ras inhibits PI3KC2β lipid kinase activity in vitro. Our findings indicate that PI3KC2β interacts with and is regulated by nucleotide-free Ras. These data suggest a novel role for nucleotide-free Ras in cell signaling in which PI3KC2β stabilizes nucleotide-free Ras and that interaction of Ras and PI3KC2β mutually inhibit one another.
authors.affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois, United States of America.
authors.forename: Katy A
authors.initials: KA
authors.lastname: Wong
authors.forename: Angela
authors.initials: A
authors.lastname: Russo
authors.forename: Xuerong
authors.initials: X
authors.lastname: Wang
authors.forename: Yun-Ju
authors.initials: YJ
authors.lastname: Chen
authors.forename: Arnon
authors.initials: A
authors.lastname: Lavie
authors.forename: John P
authors.initials: JP
authors.lastname: O'Bryan
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D058541:Class II Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D058541:Class II Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D018631:ras Proteins