Compartmentalization of phosphatidylinositol 4,5-bisphosphate signaling evidenced using targeted phosphatases.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a prevalent phosphoinositide in cell membranes, with important functions in cell signaling and activation. A large fraction of PIP(2) associates with the detergent-resistant membrane "raft" fraction, but the functional significance of this association remains controversial. To measure the properties of raft and nonraft PIP(2) in cell signaling, we targeted the PIP(2)-specific phosphatase Inp54p to either the raft or nonraft membrane fraction using minimal membrane anchors. Interestingly, we observed that targeting Inp54p to the nonraft fraction resulted in an enrichment of raft-associated PIP(2) and striking changes in cell morphology, including a wortmannin-sensitive increase in cell filopodia and cell spreading. In contrast, raft-targeted Inp54p depleted the raft pool of PIP(2) and produced smooth T cells void of membrane ruffling and filopodia. Furthermore, raft-targeted Inp54p inhibited capping in T cells stimulated by cross-linking the T cell receptor, but without affecting the T cell receptor-dependent Ca(2+) flux. Altogether, these results provide evidence of compartmentalization of PIP(2)-dependent signaling in cell membranes such as predicted by the membrane raft model.