Role of Intersectin in Cell Signaling

Role of Intersectin in Cell Signaling Katy Ann Wong, Ph.D. Department of Pharmacology University of Illinois at Chicago Chicago, Illinois (2012) Dissertation Chairperson: (John P. O’Bryan) Cell communication is imperative to biological development and growth. The temporal and spatial manner in which proteins come into contact with each other regulates when pathways are activated and inactivated. My lab studies Intersectin (ITSN), a multi-domain scaffolding protein that facilitates protein:protein interactions and regulates multiple signal transduction pathways. ITSN has been linked to endocytosis, transcription, receptor down regulation, and GTPase regulation. My dissertation work has focused on the role of ITSN in GTPase regulation as well as compartmentalized signaling. Previously, our lab has shown that ITSN activates the monomeric GTPase, Ras, on a pool of vesicles. Additionally, ITSN activates phosphoinositide-3-kinase Class II beta (PI3K-C2β), which has a Ras binding domain (RBD). These observations lead to the hypothesis that Ras is involved in ITSN activation of PI3K-C2β. Indeed my research shows that nucleotide-free Ras binds to and inhibits PI3K-C2β activity. Furthermore I show that point mutations in the effector region of Ras as well as in the RBD of PI3K-C2β disrupt binding indicating that PI3K-C2β is a novel Ras effector. These findings give new insight into GTPase regulation, and suggest that the nucleotide-free state of GTPases may not be as short lived as previously indicated. The subcellular localization of a protein determines what effectors it is exposed to. Previous yeast-two hybrid data suggested a role for ITSN in compartmentalized signaling through endosomal trafficking. Indeed I have seen ITSN associated with early and late endosomes as well as localize with the early endosomal marker EEA1. Interestingly, PI3K-C2β is also on early and late endosomes, but does not localize with EEA1, indicating that there are different pools of ITSN associated with PI3K-C2β and with the EEA1 positive early endosomes.