A new dimension to Ras function: a novel role for nucleotide-free Ras in Class II phosphatidylinositol 3-kinase beta (PI3KC2β) regulation

PLoS One. 2012;7(9):e45360. doi: 10.1371/journal.pone.0045360. Epub 2012 Sep 13.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • COS Cells
  • Cell Line
  • Chlorocebus aethiops
  • Class II Phosphatidylinositol 3-Kinases / genetics
  • Class II Phosphatidylinositol 3-Kinases / metabolism*
  • Humans
  • Point Mutation / genetics
  • Protein Binding / genetics
  • Protein Binding / physiology
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-akt / metabolism
  • ras Proteins / genetics
  • ras Proteins / metabolism*


  • Class II Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins c-akt
  • ras Proteins