Modulation of D-Serine Levels via Ubiquitin-dependent Proteasomal Degradation of Serine Racemase

Elena Dumin,Inna Bendikov,Veronika N Foltyn,Yoshio Misumi,Yukio Ikehara,Elena Kartvelishvily,Herman Wolosker

doi:10.1074/jbc.m601971200

Abstract

Mammalian serine racemase is a brain-enriched enzyme that converts L- into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the "glycine site" of N-methyl D-aspartate (NMDA) receptors that is required for the receptor/channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels. We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine racemase and D-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain d-serine levels and NMDA receptor activity.

Highlights

Stroke and neurodegenerative diseases [2]
We found that serine racemase is degraded by the ubiquitin-proteasome system and that Golga3 greatly increases serine racemase levels by decreasing its ubiq
Golga3 associates with the cytosolic surface of the Golgi apparatus, where it may be involved in vesicular trafficking [31, 32]

Summary

Introduction

Stroke and neurodegenerative diseases [2]. An interesting feature of NMDA receptors is the requirement of simultaneous binding of two agonists for channel opening, i.e. the NMDA channel only operates when both a glutamate site and a coagonist site are occupied [1]. In Vitro Binding Assays—Extracts of transfected HEK293 cells were incubated with 5 ␮g/ml GST or GST-serine racemase [22] bound to glutathione-Sepharose beads for 1 h at 4 °C in buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.1% Triton X-100, and protease inhibitors (Complete, Roche Diagnostics). Co-localization of SR and Golga3—In co-transfected HEK293 cells, serine racemase and Golga3 exhibited co-localization both at the cytosol and perinuclear region (Fig. 3, A–C).

Results

Conclusion