Requirement for PIKfyve Enzymatic Activity in Acute and Long-Term Insulin Cellular Effects

• Published in: Endocrinology (Philadelphia) 2002-12, Vol.143 (12), p.4742-4754
• Main Authors: Ikonomov, Ognian C, Sbrissa, Diego, Mlak, Krzysztof, Shisheva, Assia
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; 3T3 Cells; Adipocytes; Adipocytes - enzymology; Adipocytes - ultrastructure; Adipogenesis; AKT protein; Animals; Biological activity; Biological Transport; Cell Differentiation; Cell lines; CHO Cells; Cricetinae; Curcumin; Curcumin - pharmacology; DNA - biosynthesis; Enzymatic activity; Enzyme Inhibitors - pharmacology; Glucose; Glucose transport; Glucose Transporter Type 1; Glucose Transporter Type 4; Humans; Insulin; Insulin - pharmacology; Kinases; Lymphocytes; Lymphocytes T; Mice; Microscopy, Fluorescence; Monosaccharide Transport Proteins - metabolism; Muscle Proteins; Mutants; Mutation; Ovaries; Phosphatidylinositol 3-Kinases - genetics; Phosphatidylinositol 3-Kinases - metabolism; Phosphatidylinositol 3-Kinases - physiology; Phosphorylation; Phosphoserine - metabolism; Phosphothreonine - metabolism; Protein-Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-akt; Transfection; Translocation
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2002-220615

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, PIKfyveΔK, 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 PIKfyveK1831E mutant but not in cells expressing PIKfyveWT. Similarly, expression of the dominant negative mutants PIKfyveK1831E or PIKfyveΔK 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 PIKfyveΔK and PIKfyveWT 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 (ID50 = 6 μm) 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.