Regulation of D-serine Neuromodulation by PKCalpha and PICK1

Johns Hopkins University, Baltimore MD

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The long term objective of this project is to understand the signaling pathways which regulate the activity of serine racemase (SR), a glial enzyme that catalyzes the conversion of L-serine to D-serine. D-serine formed by the action of SR has been proposed to function as a neuromodulator. Activation of the alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA)-type glutamate receptors on glial cells releases D-serine from astrocytic cultures. D- serine then enters the synaptic cleft and activates post-synaptic glutamate N-methyl-D-aspartate (NMDA) receptors in conjunction with L-glutamate. The D-serine regulatory pathway could therefore represent therapeutic targets for diseases involving glutamate excitotoxicity such as Alzheimer's, stroke, epilepsy and ALS among others as well as for conditions involving hypofunction of NMDA receptors such as Schizophrenia. The specific aims of this project are twofold: 1) I wish to determine the functional effects of protein kinase C-alpha (PKCalpha) phosphorylation on the enzymatic activity of SR and examine the role of the protein interactor of C kinase-1 (PICK1) in this regulatory process. 2) I will develop a novel assay to rapidly and accurately measure D-Serine levels in vitro and from intact cells and monitor SR activity specifically with respect to phosphorylation by PKCa and interaction with PICK1. The assay will be done using D-serine deaminase (DsdA), a bacterial enzyme that specifically converts D-serine to pyruvate, in conjunction with the lactate dehydrogenase (LDH) fluorescence shift assay. All this will primarily be accomplished by a combination of radiolabeling experiments, the use of pharmacological agents, mass spectrometry analysis, site-directed mutagenesis and the DsdA-LDH assay. [unreadable] [unreadable] [unreadable]