Localization and Insulin-regulated Relocation of Phosphoinositide 5-Kinase PIKfyve in 3T3-L1 Adipocytes

Abstract

The mammalian phosphoinositide kinase PIKfyve catalyzes the synthesis of phosphatidylinositol 5-P and phosphatidylinositol 3,5-P 2 , thought essential in cellular functions, including membrane trafficking. To discern the intracellular loci of PIKfyve products' formation, we have examined the localization of PIKfyve protein versus enzymatic activity and a possible acutely regulated redistribution in 3T3-L1 adipocytes. Subcellular fractions of resting cells that were positive for immunoreactive PIKfyve, such as cytosol (ϳ76%), internal structures (low density microsomal fraction (LDM), composed of recycling endosomes, GLUT4 storage compartment, Golgi, and cytoskeletal elements) (ϳ20%), and plasma membrane ( ϳ 4%), expressed enzymatically active PIKfyve. While the presence of a FYVE finger in PIKfyve predicts early endosome targeting, density gradient sedimentation, immunoadsorption, and fluorescence microscopy analyses segregated the LDM-associated PIKfyve from the membranes of the recycling endosomes and GLUT4. PIKfyve fluorescence staining largely coincided with trans-Golgi network/multivesicular body markers, indicating PIKfyve's role in the late endocytic/ biosynthetic pathways. A subfraction of particulate PIKfyve resisted nonionic detergent treatment, implying association with cytoskeletal structures, previously found positive for key members of the insulin signaling cascade. Upon acute stimulation of 3T3-L1 adipocytes with insulin or pervanadate, a portion of the cytosolic PIKfyve was recruited onto LDM, which was coupled with a commensurate increase of PIKfyve lipid kinase activity and an electrophoretic mobility shift. We suggest the recruited PIKfyve specifies the site and timing of phosphoinositide signals that are relevant to the acute insulin action.

Core Protein Machinery for Mammalian Phosphatidylinositol 3,5-Bisphosphate Synthesis and Turnover That Regulates the Progression of Endosomal Transport

“…It should be noted that the in situ detection of endogenous Sac3 or ArPIKfyve in cells is currently precluded because of relatively low protein levels and inadequate antibodies for immunofluorescence microscopy. However, at least in the case of PIKfyve, the localization of ectopically expressed PIKfyve WT likely reflects that of the endogenous protein, as we have concluded previously based on data obtained in 3T3-L1 adipocytes, where the endogenous PIKfyve was successfully detected (46). Immunofluorescence microscopy with anti-Myc antibody revealed that the majority of COS7 cells expressing Myc-hSac3 alone displayed diffuse and perinuclear staining (80 -85%).…”

“…Polyclonal anti-HA (R4289), anti-IRAP, anti-GRP94, anti-Rab4, and monoclonal anti-transferrin receptor antibodies were gifts by Drs. Mike Czech, Paul Pilch, Steve Cala, Ira Melmann, and Ian Traubridge and used under previously specified conditions (38,(43)(44)(45)(46).…”

Phosphatidylinositol 3-Phosphate-interacting Domains in PIKfyve

“…The small 63-bp fragment was eliminated, whereas the remaining two fragments were ligated to give the pEGFP-PIKfyve S -(1-286)⌬177-198 construct. The generation of all other PIKfyve constructs in pEGFP that were used in this study has been described previously (12,21,23).…”

“…Fluorescence Microscopy Analysis-Subsequent to fixation in 4% formaldehyde and washings, transfected cells were processed for fluorescence microscopy analysis as described elsewhere (23). Coverslips were mounted on slides using the Slow Fade anti-fade kit (Molecular Probes).…”

“…22). Immunofluorescence microscopy studies detect PIKfyve, both endogenous and expressed in heterologous cell systems, associated with discrete vesicle structures, most of them positive for markers of the late endocytic pathway (23). Recent data demonstrating a dramatic endomembrane swelling and vacuolation upon expression of dominant-negative PIKfyve lipid kinase-deficient mutants and a subsequent correction of the abnormal phenotype upon microinjection of PtdIns 3,5-P 2 implicate PtdIns 3,5-P 2 production in the organization of a specialized membrane domain on late endocytic compartment (24,25).…”