Our objectives are to define the role of the inositol kinase PPIP5K at the exocyst and determine if its activation impacts exocyst interaction with the GTPase RalA. This investigation is significant because RalA activation and its association with the exocyst complex are essential steps for Ras-driven cancer development and anchorage-independent growth. Diphosphoinositols are known to compete with inositol membrane lipids for binding to PH domains, and PIP2 inhibits RalA association with the exocyst. We propose to test the hypothesis that PPIP5K is activated by interaction with exocyst subunits and produces diphosphoinositols that promotes RalA interaction with the exocyst. This model will form the basis for a new strategy to target oncogenic Ras signaling through PPIP5K inhibition thereby preventing RalA action. This study builds on our existing work that identified PPIP5K as an exocyst-associated protein that desensitizes cells to apoptosis-inducing agents. We will accomplish these objectives through three overlapping aims: 1. Map interactions between PPIP5K and the exocyst complex. We have identified PPIP5K as an exocyst-associated enzyme and in this aim we will identify the direct interactions between PPIP5K and the exocyst complex. This will contribute to our understanding of the exocyst assembly by linking PPIP5K to one of the sub-complexes that interact during exocyst assembly. This would place the kinase activity on either the vesicular membrane or plasma membrane, which will be a key site for defining the role of the diphosphoinositols produced. 2. Identify diphosphoinositol-binding exocyst subunits and its impact on RalA binding. Diphosphoinositols are produced at the exocyst and we will test if they impact interaction of RalA with this complex. In this aim, we will identify diphosphoinositol binding exocyst subunits, determine if these interactions prevent exocyst association with PIP2, and determine if this relieves inhibition of RalA binding by PIP2. 3. Define activation mechanism and activating trigger for PPIP5K. Diphosphoinositols are short-lived signaling molecules, so they are synthesized at their sites of action. PPIP5K kinase activity is inhibited by another domain of PPIP5K, but which domain performs this action and how the inhibition is relieved remains unknown. In this aim, we will identify the mechanism for its inhibition and determine how it is activated at the exocyst complex.
Activating mutations in members of the Ras family drives over one third of human cancers. These Ras-driven cancers require the activation of RalA and its binding to a complex of proteins called the exocyst, which aids in the transport of proteins and lipids to the cell surface. We propose an enzyme called PPIP5K produces signaling molecules at the exocyst that promotes RalA binding. If accurate, inhibition of PPIP5K may attenuate the mutant Ras phenotype.
