Localization and insulin-regulated relocation of phosphoinositide 5-kinasePIKfyve in 3T3-L1 adipocytes

The mammalian phosphoinositide kinase PIKfyve catalyzes the synthesis of ph osphatidylinositol 5-P and phosphatidylinositol 3,5-P-2, thought essential in cellular functions, including membrane trafficking. To discern the intra cellular loci of PIKfyve products' formation, we have examined the localiza tion of PIKfyve protein versus enzymatic activity and a possible acutely re gulated redistribution in 3T3-L1 adipocytes. Subcellular fractions of resti ng cells that were positive for immunoreactive PIKfyve, such as cytosol (si milar to 76%), internal structures (low density microsomal fraction (LDM), composed of recycling endosomes, GLUT4 storage compartment, Golgi, and cyto skeletal elements) (similar to 20%), and plasma membrane ((similar to)4%), expressed enzymatically active PIKfyve. While the presence of a FYVE finger in PIKfyve predicts early endosome targeting, density gradient sedimentati on, immunoadsorption, and fluorescence microscopy analyses segregated the L DM-associated PIKfyve from the membranes of the recycling endosomes and GLU T4. PIKfyve fluorescence staining largely coincided with trans-Gels network /multivesicular body markers, indicating PIKfyve's role in the late endocyt ic/biosynthetic pathways. A subfraction of particulate PIKfyve resisted non ionic detergent treatment, implying association with cytoskeletal structure s, previously found positive for key members of the insulin signaling casca de. Upon acute stimulation of 3T3-L1 adipocytes with insulin or pervanadate , a portion of the cytosolic PIKfyve was recruited onto LDM, which was coup led with a commensurate increase of PIKfyve lipid kinase activity and an el ectrophoretic mobility shift. We suggest the recruited PIKfyve specifies th e site and timing of phosphoinositide signals that are relevant to the acut e insulin action.