Regulation of PIKfyve phosphorylation by insulin and osmotic stress.
PIKfyve is a protein and lipid kinase that plays an important role in membrane trafficking, including TGN to endosome retrograde sorting and in insulin-stimulated translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the plasma membrane. We have previously demonstrated that PIKfyve is phosphorylated in response to insulin in a PI3-kinase and protein kinase B (PKB)-dependent manner. However, it has been implied that this was not due to direct phosphorylation of PIKfyve by PKB, but as a result of an insulin-induced PIKfyve autophosphorylation event. Here we demonstrate that purified PIKfyve is phosphorylated in vitro by a recombinant active PKB on two separate serine residues, S318 and S105, which flank the N-terminal FYVE domain of the protein. Only S318, however, becomes phosphorylated in intact cells stimulated with insulin. We further demonstrate that S318 is phosphorylated in response to hyperosmotic stress in a PI3-kinase- and PKB-independent manner. Importantly, the effects of insulin and sorbitol were not prevented by the presence of an ATP-competitive PIKfyve inhibitor (YM20163) or in a mutant PIKfyve lacking both lipid and protein kinase activity. Our results confirm, therefore, that PIKfyve is directly phosphorylated by PKB on a single serine residue in response to insulin and are not due to autophosphorylation of the enzyme. We further reveal that two stimuli known to promote glucose uptake in cells, both stimulate phosphorylation of PIKfyve on S318 but via distinct signal transduction pathways.
Hill
Elaine V
EV
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 ITD, UK.
Hudson
Claire A
CA
Vertommen
Didier
D
Rider
Mark H
MH
Tavaré
Jeremy M
JM
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D009997:Osmotic Pressure; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013012:Sorbitol / Q000494:pharmacology
D064413:3-Phosphoinositide-Dependent Protein Kinases; D007328:Insulin; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D012694:Serine; D013012:Sorbitol; C116930:Pikfyve protein, mouse