Role for a novel signaling intermediate, phosphatidylinositol 5-phosphate, in insulin-regulated F-actin stress fiber breakdown and GLUT4 translocation.
The cellular functions and regulation of phosphatidylinositol (PtdIns) 5-phosphate (5-P), the newest addition to the family of phosphoinositides (PIs), are still elusive. Here we have examined a plausible role of PtdIns 5-P as a signaling intermediate in acute insulin action. A wortmannin-insensitive transient increase of PtdIns 5-P mass levels that peaked at 10 min, and declined 20-30 min after insulin stimulation, was observed in both Chinese hamster ovary (CHO)-T cells stably expressing the insulin receptor and 3T3-L1 adipocytes. Similarly to insulin, found to induce a rapid disassembly of Texas-Red phalloidin-labeled actin stress fibers in CHO-T cells, microinjected PtdIns 5-P, but not other PIs, decreased the number and length of F-actin stress fibers in this cell type to a magnitude seen in response to insulin. Likewise, increases of PtdIns 5-P by ectopic expression of the PtdIns 5-P-producing enzyme PIKfyve yielded a similar effect. As with insulin, the PtdIns 5-P-induced loss of actin stress fibers was independent of PI 3-kinase activation. Furthermore, sequestration of functional PtdIns 5-P, either by ectopic expression of 3xPHD domains that bind selectively PtdIns 5-P or by microinjecting the GST-3xPHD fusion peptide, abrogated insulin-induced F-actin stress fiber disassembly in CHO-T cells. In 3T3-L1 adipocytes, microinjected PtdIns 5-P, but not other PIs, partially mimicked insulin's effect of translocating enhanced green fluorescent protein-GLUT4 to the cell surface. Conversely, insulin-induced myc-GLUT4 vesicle dynamics was arrested in the presence of coexpressed enhanced green fluorescent protein-3xPHD. Involvement of PIKfyve membrane recruitment, but not activation, and/or a decrease in PtdIns 4,5-bisphosphate levels are likely to be among the mechanisms underlying the insulin-induced PtdIns 5-P increase. Together, these results identify PtdIns 5-P as a novel key intermediate for insulin signaling in F-actin remodeling and GLUT4 translocation.
Department of Physiology, Wayne State University School of Medicine, 540 East Canfield, Detroit, Michigan 48201, USA.
Diego
D
Sbrissa
Ognian C
OC
Ikonomov
Jana
J
Strakova
Assia
A
Shisheva
D041721:3T3-L1 Cells; D000199:Actins / Q000378:metabolism*; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008845:Microinjections; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism*
D000199:Actins; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; C495693:Slc2a4 protein, mouse; C113567:phosphatidylinositol 5-phosphate; D019869:Phosphatidylinositol 3-Kinases; C116930:Pikfyve protein, mouse