Phosphorylation and modulation of brain glutamate transporters by protein kinase C.

M Casado,A Bendahan,F Zafra,N C Danbolt,C Aragón,C Giménez,B I Kanner

doi:10.1016/s0021-9258(19)74251-3

M Casado, A Bendahan + Show 5 more

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https://doi.org/10.1016/s0021-9258(19)74251-3

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Abstract

High affinity sodium- and potassium-coupled L-glutamate transport into presynaptic nerve terminals and fine glial processes removes the neurotransmitter from the synaptic cleft, thereby terminating glutamergic transmission. This report describes that the purified L-glutamate transporter from pig brain is phosphorylated by protein kinase C, predominantly at serine residues. Upon exposure of C6 cells, a cell line of glial origin, to 12-O-tetradecanoylphorbol-13-acetate, about a 2-fold stimulation of L-glutamate transport is observed within 30 min. Concomitantly, the level of phosphorylation increases with similar kinetics. The phorbol ester also stimulates L-glutamate transport in HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase and transfected with pT7-GLT-1. The latter is a recently cloned rat brain glutamate transporter of glial origin. Mutation of serine 113 to asparagine does not affect the levels of expressed transport but abolishes its stimulation by the phorbol ester. To our knowledge, this is the first direct demonstration of the regulation of a neurotransmitter transporter by phosphorylation.

Highlights

High affinity sodium- and potassium-coupled L-glu- and Kanner (1993))
Guasglutamate transporter from pig brain is phosphoryl- tella et al (1990) and Pacholczyk et al (1991)). ated by protein kinase Cypredominantlyat serine res- It is conceivable that the reuptake process is subject to ppioaHodbbrhheuiosooLgeeursiasrbptn.vocha,Ueelod2leprl-ssyotwftonloieainrlttdefhieaxois1clnpnst2tiooei-m3nsdOs0ucut-wrriltmmeeaieattoiuthisnforle.aanasCCtdreowe6oesfcncicLatoLchenom--logmglsbsylliu,uilmitntptaaaaaihnlmmncoatertlraaylbtltvk,eoetalilhincttn-ercre1eaiat3nninloc-iessafsavpp.vceogoiTelrrlrtihtuotaiaesfslitne,ppptsgehhhelmyocoyctwlssiiionpno(feaRltonshh(egeZaoiuAtcaaradaf2orlr,staarcreaecaegnthntuisadmlialano.l,tih.ni,t1oitibce9n1ri8.9at39cfHsu)i0ed,on)vi,wcnewatcerinaholvdulinecdsrhgi,ionslodugmruituuatradmhmykiemb-naceutoeoepwrnuettlp(alaeBelkrdeayeagds.reeusbIdyooptsfvuthtairetaakhmseepisbtshseoayafeoslssn.-r-, expressing T7 RNA polymerase and transfected with 1989)
The uptake measurements were expressed transport but abolishitessstimulation by thedone in tissue samples dissected out 20 min after the cessation phorbol ester

Summary

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Dishes and multiwell plates for cell culture were beads were washed five times in immunoprecipitation buffer. TLC celluloseplates for phosphoamino were diluted %fold by addition of an equal volume of 2 X reducing acid analysis were from Merck. Transfection reagent (DOTAP) was Laemmli sample buffer andthen resolved by SDS-PAGE. Brain lipids were prepared from bovine brain as published Cell Culture-C6 glioma cells, cloned originally from a rat glioma (Folch et al, 1957). Protein A-Sepharose CL-4B, asolectin (P-5638, (Benda et al, 1968) and obtained from the American Type Culture type I1 S),uridine, cholic acid, 12-0-tetradecanoylphorboll3-acetate Collection (Rockville,MD), were maintained inmonolayer culture in (TPA), 1-a-phosphatidyl-L-serinea,nd all other chemicals were pur- DMEM containing 10% (v/v) horse serum and 2.5% (v/v) fetal calf chased from Sigma

Methods

AND DISCUSSION

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