Regulation of the glutamate transporter EAAT1 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid-inducible kinase isoforms SGK1/3 and protein kinase B.
Surface expression of the glial glutamate transporter EAAT1 is stimulated by insulin-like growth factor 1 through activation of phosphatidylinositol-3-kinase. Downstream targets include serum and glucocorticoid-sensitive kinase isoforms SGK1, SGK2 and SGK3, and protein kinase B. SGK1 regulates Nedd4-2, a ubiquitin ligase that prepares cell membrane proteins for degradation. To test whether Nedd4-2, SGK1, SGK3 and protein kinase B regulate EAAT1, cRNA encoding EAAT1 was injected into Xenopus oocytes with or without additional injection of wild-type Nedd4-2, constitutively active S422DSGK1, inactive K127NSGK1, wild-type SGK3 and/or constitutively active T308D,S473DPKB. Glutamate induces a current in Xenopus oocytes expressing EAAT1, but not in water-injected oocytes, which is decreased by co-expression of Nedd4-2, an effect reversed by additional co-expression of S422DSGK1, SGK3 and T308D,S473DPKB, but not K127NSGK1. Site-directed mutagenesis of the SGK1 phosphorylation sites in the Nedd4-2 protein (S382A,S468ANedd4-2) and in the EAAT1 protein (T482AEAAT1, T482DEAAT1) significantly blunts the effect of S422DSGK1. Moreover, the current is significantly larger in T482DEAAT1- than in T482AEAAT1-expressing oocytes, indicating that a negative charge mimicking phosphorylation at T482 increases transport. The experiments reveal a powerful novel mechanism that regulates the activity of EAAT1. This mechanism might participate in the regulation of neuronal excitability and glutamate transport in other tissues.
Boehmer
Christoph
C
Department of Physiology I, University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.
Henke
Guido
G
Schniepp
Roman
R
Palmada
Monica
M
Rothstein
Jeffrey D
JD
Bröer
Stefan
S
Lang
Florian
F
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D004594:Electrophysiology; D056827:Endosomal Sorting Complexes Required for Transport; D027341:Excitatory Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D009865:Oocytes / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D018075:RNA, Complementary / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D027341:Excitatory Amino Acid Transporter 1; D017874:Immediate-Early Proteins; D007527:Isoenzymes; D008025:Ligases; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D018075:RNA, Complementary; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus