The class II phosphoinositide 3-kinase PI3K-C2alpha is concentrated in the trans-Golgi network and present in clathrin-coated vesicles.
In recent years, a large family of phosphoinositide 3-kinase (PI3K) isozymes has been characterized and cloned. Several of these PI3K enzymes have overlapping tissue distributions and it remains unclear if and how their 3-phosphoinositide products elicit differential, intracellular effects. One possibility is that the PI3K enzymes display a restricted distribution within the cell to produce their 3-phospholipid products in specific, subcellular compartments. In the present study we characterize the subcellular distribution of the novel class II PI3K isozyme PI3K-C2alpha in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2alpha is constitutively associated with phospholipid membranes. Centrifugation of rat brain homogenates and Western blotting revealed that in contrast to the class IA PI3K enzymes, PI3K-C2alpha could be co-purified with a population of clathrin-coated vesicles (CCVs). Furthermore, a PI3K activity refractory to wortmannin treatment was detected in CCV preparations consistent with the presence of the PI3K-C2alpha isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2alpha to have a punctate distribution and an enrichment of immunoreactivity within a perinuclear site consistent with its presence in the endoplasmic reticulum or Golgi apparatus. Dual label immunofluorescence demonstrated that in this region, the distribution of PI3K-C2alpha closely paralleled that of gamma-adaptin, a component of the AP-1 adaptor that is present in the trans-Golgi and the trans-Golgi network (TGN) resident protein TGN-46. Neither the phospholipid association nor the subcellular localization of PI3K-C2alpha was dependent upon either its COOH-terminal PX or C2 domains. Mutants lacking these domains demonstrated a similar distribution to the wild type enzyme when expressed as recombinant proteins. Treatment of cells with brefeldin A disrupted the perinuclear staining pattern of both PI3K-C2alpha and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.[1]References
- The class II phosphoinositide 3-kinase PI3K-C2alpha is concentrated in the trans-Golgi network and present in clathrin-coated vesicles. Domin, J., Gaidarov, I., Smith, M.E., Keen, J.H., Waterfield, M.D. J. Biol. Chem. (2000) [Pubmed]
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