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EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase
Author(s) -
Rajamanickam Jeyaganesh,
Palmada Monica,
Lang Florian,
Boehmer Christoph
Publication year - 2007
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2007.04585.x
Subject(s) - sgk1 , microbiology and biotechnology , biology , phosphorylation , biochemistry , kinase
EAAT4 (SLC1A6) is a Purkinje‐Cell‐specific post‐synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4‐mediated [ 3 H] glutamate uptake and cell surface abundance are enhanced by co‐expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 ( T40A EAAT4) or of both phosphorylation sites ( T40AT504A EAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4‐2. Co‐expression of Nedd4‐2 inhibited wild‐type EAAT4 but not the T40AT504A EAAT4 mutant. Besides, RNA interference‐mediated reduction of endogenous Nedd4‐2 (xNedd4‐2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4‐2.