A New Dimension to Ras Function: A Novel Role for Nucleotide-Free Ras in Class II Phosphatidylinositol 3-Kinase Beta (PI3KC2 beta) Regulation
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Date
2012-09Author
Wong, Katy A.
Russo, Angela
Wang, Xuerong
Chen, Yun-Ju
Lavie, Arnon
O’Bryan, John P.
Publisher
Public Library of ScienceMetadata
Show full item recordAbstract
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, PI3KC2 beta, as an ITSN1 target on vesicles and the presence of a Ras binding domain (RBD) in PI3KC2 beta suggests a role for Ras in PI3KC2 beta activation. Here, we demonstrate that nucleotide-free Ras negatively regulates PI3KC2 beta activity. PI3KC2 beta preferentially interacts in vivo with dominant-negative (DN) Ras, which possesses a low affinity for nucleotides. PI3KC2 beta interaction with DN Ras is disrupted by switch 1 domain mutations in Ras as well as RBD mutations in PI3KC2 beta. Using purified proteins, we demonstrate that the PI3KC2 beta-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 PI3KC2 beta lipid kinase activity in vitro. Our findings indicate that PI3KC2 beta interacts with and is regulated by nucleotide-free Ras. These data suggest a novel role for nucleotide-free Ras in cell signaling in which PI3KC2 beta stabilizes nucleotide-free Ras and that interaction of Ras and PI3KC2 beta mutually inhibit one another.