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 (S422D) SGK1, inactive (K127N) SGK1, wild-type SGK3 and/or constitutively active (T308D,S473D) PKB. 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 (S422D) SGK1, SGK3 and (T308D,S473D) PKB, but not (K127N) SGK1. Site-directed mutagenesis of the SGK1 phosphorylation sites in the Nedd4-2 protein ((S382A,S468A) Nedd4-2) and in the EAAT1 protein ((T482A) EAAT1, (T482D) EAAT1) significantly blunts the effect of (S422D) SGK1. Moreover, the current is significantly larger in (T482D) EAAT1- than in (T482A) EAAT1-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.