PHOSPHORYLATION OF SERINE RESIDUE-3, RESIDUE-6, RESIDUE-10, AND RESIDUE-13 DISTINGUISHES MEMBRANE-ANCHORED FROM SOLUBLE GLUTAMIC-ACID DECARBOXYLASE-65 AND IS RESTRICTED TO GLUTAMIC-ACID DECARBOXYLASE 65-ALPHA

GAD65, the smaller isoform of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase is detected as an alpha/beta doubl et of distinct mobility on SDS-polyacrylamide gel electrophoresis. Glu tamic acid decarboxylase (GAD) 65 is reversibly anchored to the membra ne of synaptic vesicles in neurons and synaptic like microvesicles in pancreatic beta-cells. Here we demonstrate that GAD65 alpha but not be ta is phosphorylated in vivo and in vitro in several cell types. Phosp horylation is not the cause of the alpha/beta heterogeneity but repres ents a unique post-translational modification of GAD65 alpha. Two-dime nsional protein analyses identified five phosphorylated species of thr ee different charges, which are likely to represent mono-, di-, and tr iphosphorylated GAD65 alpha in different combinations of phosphorylate d serines. Phosphorylation of GAD65 alpha was located at serine residu es 3, 6, 10, and 13, shown to be mediated by a membrane bound kinase, and distinguish the membrane anchored, and soluble forms of the enzyme . Phosphorylation status does not affect membrane anchoring of GAD65, nor its K-m or V-max for glutamate. The results are consistent with a model in which GAD65 alpha and -beta constitute the two subunits of th e native GAD65 dimer, only one of which, alpha, undergoes phosphorylat ion following membrane anchoring, perhaps to regulate specific aspects of GAD65 function in the synaptic vesicle membrane.