Proteomic screen identifies novel phosphatidylinositol(3,4)bisphosphate binding protein

Phosphoinositides represent a unique family of seven minor membrane lipids that play key roles in a variety of fundamental processes as diverse as vesicle trafficking, cytoskeletal rearrangements, and signal transduction. Phosphoinositides regulate the reversible binding of proteins to cellular membranes and recruit target proteins through specific and direct interactions mediated by one of many types of known phosphoinositide binding domains. We have developed a proteomic approach to identify novel targets of phosphoinositides by combining lipid blotting techniques with small pool screening. In screening through the Drosophila proteome, we found that many proteins bind to phosphoinositides nonspecifically; however we were successful at isolating a new phosphatidylinositol(3,4)bisphosphate (PI(3,4)P 2 ) binding protein. We have named this protein PHATE (pronounced "fate") for PH domain adaptor and phosphatidylinositol-( t hree,four)bisphosphate e ffector. In order to gain insights into the biological role of PHATE we explored the effects of ectopic expression on Drosophila imaginal disc development. Overexpression in fly wings generates ectopic vein and extra bristle phenotypes that are ameliorated by single allelic deletion of PI3K family members, especially the class II PI3K, PI3K_68D. The effect of PHATE on wing vein malformation is dependent on the ability of the PH domain to bind to PI(3,4)P 2 , but also appears to require regions C-terminal of the PH domain that include potential sites for interaction with SH3-domain-containing proteins. These results suggests that PHATE participates in recruiting SH3 domain-containing proteins to a membrane location where PI(3,4)P 2 is generated and that enhanced recruitment of these complexes may contribute to the vein malformation. The extra-vein phenotype exhibited by PHATE indicated a link to PI3K_68D and EGFR signaling. Genetic interactions were indeed observed in that PHATE enhanced the phenotypes of both KD-PI3K_68D and Ellipse. We therefore propose a model that PHATE may facilitate EGFR trafficking to the endosome and help maintain signaling at that location.