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Volume 272, Number 3, Issue of January 17, 1997 pp. 1548-1557
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Phosphorylation of Serine Residues 3, 6, 10, and 13 Distinguishes Membrane Anchored from Soluble Glutamic Acid Decarboxylase 65 and Is Restricted to Glutamic Acid Decarboxylase 65

(Received for publication, August 1, 1996, and in revised form, October 25, 1996)

Mark Namchuk , LeAnn Lindsay , Christoph W. Turck , Jamil Kanaani and Steinunn Baekkeskov

From the  Department of Medicine, Department of Microbiology and Immunology, and Hormone Research Institute and the ^¶ Howard Hughes Medical Institute, and Department of Medicine, University of California San Francisco, San Francisco, California 94143

GAD65, the smaller isoform of the -aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase is detected as an / doublet of distinct mobility on SDS-polyacrylamide gel electrophoresis. Glutamic acid decarboxylase (GAD) 65 is reversibly anchored to the membrane of synaptic vesicles in neurons and synaptic-like microvesicles in pancreatic -cells. Here we demonstrate that GAD65 but not  is phosphorylated in vivo and in vitro in several cell types. Phosphorylation is not the cause of the / heterogeneity but represents a unique post-translational modification of GAD65. Two-dimensional protein analyses identified five phosphorylated species of three different charges, which are likely to represent mono-, di-, and triphosphorylated GAD65 in different combinations of phosphorylated serines. Phosphorylation of GAD65 was located at serine residues 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 and - constitute the two subunits of the native GAD65 dimer, only one of which, , undergoes phosphorylation following membrane anchoring, perhaps to regulate specific aspects of GAD65 function in the synaptic vesicle membrane.

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