title: Requirement for PIKfyve enzymatic activity in acute and long-term insulin cellular effects.
abstract: PIKfyve is a phosphoinositide 5-kinase that can also act as a protein kinase. PIKfyve's role in acute insulin action has been suggested on the basis of its association with the insulin stimulatable phosphatidylinositol-3-kinase and the ability of acute insulin to recruit and phosphorylate PIKfyve on intracellular membranes of 3T3-L1 adipocytes. Here we have examined several classical insulin-regulated long- and short-term responses in insulin-sensitive cells expressing high levels of either active PIKfyve or kinase-dead mutants with a dominant-negative effect. Up-regulation of PIKfyve protein expression was documented in the early stages of differentiation of cultured 3T3-L1 fibroblasts into adipocytes and a kinase-dead mutant, PIKfyveDeltaK, introduced into the preadipocyte stage profoundly delayed the hormone-induced adipogenesis. Next, insulin-induced mitogenesis was markedly inhibited in HEK293 stable cell lines, inducibly expressing the dominant-negative kinase-dead PIKfyve(K1831E) mutant but not in cells expressing PIKfyve(WT). Similarly, expression of the dominant negative mutants PIKfyve(K1831E) or PIKfyveDeltaK strongly inhibited insulin-stimulated translocation of GLUT4 in 3T3-L1 adipocytes, or GLUT1-mediated glucose uptake in Chinese hamster ovary T cells expressing the human insulin receptor. Expression of PIKfyveDeltaK and PIKfyve(WT) in Chinese hamster ovary T cells decreased or increased, respectively, insulin-stimulated Akt phosphorylation at Ser473 but not at Thr308. Furthermore, a powerful inhibition of PIKfyve was documented at a very low concentration (ID(50) = 6 micro M) of the cell-permeable kinase inhibitor curcumin. When introduced into 3T3-L1 adipocytes, curcumin markedly inhibited insulin-induced GLUT4 translocation and glucose transport. Together these data indicate that PIKfyve enzymatic activity functions as a positive regulatory intermediate in insulin acute and long-term biological responses and identify Ser473 in Akt as one potential PIKfyve downstream target.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Ognian C
authors.initials: OC
authors.lastname: Ikonomov
authors.forename: Diego
authors.initials: D
authors.lastname: Sbrissa
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Mlak
authors.forename: Assia
authors.initials: A
authors.lastname: Shisheva
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000201:enzymology / Q000648:ultrastructure; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002454:Cell Differentiation; D006224:Cricetinae; D003474:Curcumin / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008856:Microscopy, Fluorescence; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D014162:Transfection
chemical_list: D003474:Curcumin; D004247:DNA; D004791:Enzyme Inhibitors; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases; D010768:Phosphoserine; D010769:Phosphothreonine; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; C495678:Slc2a1 protein, mouse; C495693:Slc2a4 protein, mouse; C492139:PIKFYVE protein, human; C116930:Pikfyve protein, mouse; C494918:AKT1 protein, human