A. Shisheva et al., Localization and insulin-regulated relocation of phosphoinositide 5-kinasePIKfyve in 3T3-L1 adipocytes, J BIOL CHEM, 276(15), 2001, pp. 11859-11869
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.