Evidence that Akt mediates platelet-derived growth factor-dependent increases in activity and surface expression of the neuronal glutamate transporter, EAAC1.
Previously we have shown that platelet-derived growth factor (PDGF) rapidly increases the activity of the neuronal glutamate transporter, EAAC1. This increase in activity is associated with a rapid (within minutes) redistribution of transporter from a subcellular compartment to the plasma membrane and is blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K). Similar effects of PI3K inhibitors have been observed for insulin-dependent up-regulation of the GLUT4 subtype of glucose transporter. Although GLUT4 regulation also depends on the serine-threonine kinase (Akt/protein kinase B), a downstream target of PI3K, the downstream effectors responsible of PDGF-dependent regulation of EAAC1 have not been identified. In the present study, PDGF increased the level of Akt phosphorylation (Ser 473) in C6 glioma cells, a cell line that has been used to study regulated trafficking of endogenous EAAC1. Two inhibitors of PI3K blocked this effect. In transient transfection studies, a dominant negative mutant of Akt-1 blocked PDGF-induced redistribution of epitope-tagged EAAC1 (myc-EAAC1). Conversely, constitutively active Akt-1 (CA Akt-1) increased the cell surface expression of myc-EAAC1. A lentiviral vector engineered to express CA Akt-1 increased Akt activation, cell surface expression of endogenous EAAC1, and Na(+)-dependent glutamate transport activity. Together, these studies suggest that Akt is required for PDGF-induced regulation of EAAC1.
Krizman-Genda
Elizabeth
E
Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104, USA.
González
Marco I
MI
Zelenaia
Olga
O
Robinson
Michael B
MB
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D019921:Biotinylation / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050580:Excitatory Amino Acid Transporter 3 / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D005818:Genetic Engineering / Q000379:methods; D005910:Glioma; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014162:Transfection / Q000379:methods
D004791:Enzyme Inhibitors; D050580:Excitatory Amino Acid Transporter 3; C489428:Myc protein, mouse; D010982:Platelet-Derived Growth Factor; D016271:Proto-Oncogene Proteins c-myc; C493399:Slc1a1 protein, mouse; D018698:Glutamic Acid; D012694:Serine; D012964:Sodium; D051057:Proto-Oncogene Proteins c-akt