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Abstract
GAD65, the smaller isoform of the gamma-aminobutyric acid-synthesizing enzyme glutamic acid decarboxylase is detected as an alpha/beta 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 beta-cells. Here we demonstrate that GAD65alpha but not beta is phosphorylated in vivo and in vitro in several cell types. Phosphorylation is not the cause of the alpha/beta heterogeneity but represents a unique post-translational modification of GAD65alpha. Two-dimensional protein analyses identified five phosphorylated species of three different charges, which are likely to represent mono-, di-, and triphosphorylated GAD65alpha in different combinations of phosphorylated serines. Phosphorylation of GAD65alpha 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 Km or Vmax for glutamate. The results are consistent with a model in which GAD65alpha and -beta constitute the two subunits of the native GAD65 dimer, only one of which, alpha, undergoes phosphorylation following membrane anchoring, perhaps to regulate specific aspects of GAD65 function in the synaptic vesicle membrane.
Highlights
Glutamic acid decarboxylase (GAD)1 (EC 4.1.1.15) catalyzes the ␣-decarboxylation of glutamate generating ␥-aminobutyric acid (GABA)
GAD65 Is Phosphorylated in Vitro in Whole Cell Lysates—We initially analyzed whether human GAD65 (hGAD65) expressed in BHK-77-3 and COS-7 cells could be phosphorylated in cellular lysates
Analyses of 32P autoradiograms of the immunoblots revealed one major phosphorylated band which co-migrated with the GAD65␣ band, and in some experiments, a weaker band which co-migrated with the GAD65␣Ј band
Summary
Glutamic acid decarboxylase (GAD) (EC 4.1.1.15) catalyzes the ␣-decarboxylation of glutamate generating ␥-aminobutyric acid (GABA). GAD65 constitutes the majority of this enzyme reservoir [14], which can be activated by influx of co-factor, or perhaps by targeting the protein to compartments in the cell where PLP is available. Membrane anchoring is reversible, suggesting that trafficking of GAD65 between membranes and cytosol may regulate its proximity to synaptic vesicles and the efficacy by which its product GABA can be accumulated for secretion [16]. The native form of both GAD65 and GAD67 is a non-covalently associated homodimer detected by gel filtration and native gel electrophoresis.. The native form of both GAD65 and GAD67 is a non-covalently associated homodimer detected by gel filtration and native gel electrophoresis.2 Both the GAD67 and GAD65 dimers dissociate into monomers on SDS-PAGE under either reducing or nonreducing conditions. Reducing SDS-PAGE resolves GAD65 but not GAD67 into two
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