Phosphoinositide 3-kinase C2α links clathrin to microtubule-dependent movement

Phosphoinositide 3-kinase C2 alpha(PI3K-C2 alpha) is a type II PI-3-kinase that has been implicated in several important membrane transport and signaling processes. We previously found that overexpression of PI3K-C2 alpha inhibits clathrin-mediated membrane trafficking and induces proliferation of novel clathrin-coated structures within the cytoplasm. Using fluorescently tagged fusions of PI3K-C2 alpha and clathrin, we explored the behavior of these structures in intact cells. Both proteins are present in the structures, and using rapid image acquisition and fluorescence photoactivation probes, we find that they exhibit localized, rapid mobility (5-20 mu m/s). The movement is microtubule-based as revealed by use of inhibitors, and PI3K-C2 alpha accumulates on microtubules rapidly and reversibly following cytoplasmic acidification, which also blocks movement. Dynactin mediates the movement of these clathrin-PI3K-C2 alpha structures, since disruption of dynactin function by overexpression of its p50 subunit also inhibits movement. Finally, immunoprecipitation experiments reveal an interaction between endogenous PI3K-C2 alpha and dynactin subunits. Together, these results reveal a molecular linkage between PI3K-C2 alpha and the microtubule motor machinery, with implications for membrane trafficking in intact cells.