The class II phosphoinositide 3-kinase PI3K-C2 alpha is concentrated in the trans-Golgi network and present in clathrin-coated vesicles

In recent years, a large family of phosphoinositide 3-kinase (PI3K) isozyme s has been characterized and cloned. Several of these PI3K enzymes have ove rlapping tissue distributions and it remains unclear if and how their 3-pho sphoinositide products elicit differential, intracellular effects. One poss ibility is that the PI3K enzymes display a restricted distribution within t he cell to produce their 3-phospholipid products in specific, subcellular c ompartments. In the present study we characterize the subcellular distribut ion of the novel class II PI3K isozyme PI3K-C2 alpha in several mammalian c ell types. Differential centrifugation of COS-1 and U937 cells together wit h Western blot analysis demonstrated that PI3K-C2 alpha is constitutively a ssociated with phospholipid membranes. Centrifugation of rat brain homogena tes and Western blotting revealed that in contrast to the class IA PI3K enz ymes, PI3K-C2 alpha could be copurified with a population of clathrin-coate d vesicles (CCVs). Furthermore, a PI3K activity refractory to wortmannin tr eatment was detected in CCV preparations consistent with the presence of th e PI3K-C2 alpha isozyme. These biochemical observations were supported by i mmunofluorescence analysis that revealed PI3K-C2a to have a punctate distri bution and an enrichment of immunoreactivity within a perinuclear site cons istent with its presence in the endoplasmic reticulum or Golgi apparatus. D ual label immunofluorescence demonstrated that in this region, the distribu tion of PI3K-C2a closely paralleled that of gamma-adaptin, a component of t he AP-1 adaptor that is present in the trans-Golgi and the trans-Golgi netw ork (TGN) resident protein TGN-46. Neither the phospholipid association nor the subcellular localization of PI3K-C2a was dependent upon either its COO H-terminal PX or C2 domains. Mutants lacking these domains demonstrated a s imilar distribution to the wild type enzyme when expressed as recombinant p roteins. Treatment of cells with brefeldin A disrupted the perinuclear stai ning pattern of both PI3K-C2 alpha and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosyl ation factor GTPase activity.