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 65alpha.
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.
Namchuk
M
M
Department of Medicine, University of California San Francisco, San Francisco, California 94143, USA.
Lindsay
L
L
Turck
C W
CW
Kanaani
J
J
Baekkeskov
S
S
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*
D012694:Serine; D005968:Glutamate Decarboxylase