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 5318, 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 PH 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. (C) 2010 Elsevier Inc. All rights reserved.