Protein phosphorylation of human brain glutamic acid decarboxylase (GAD)65 and GAD67 and its physiological implications.
Previously, we reported that protein phosphorylation plays an important role in regulating soluble l-glutamic acid decarboxylase (GAD) [Bao, J. (1995) J. Biol. Chem. 270, 6464-6467] and membrane-associated GAD activity [Hsu, C. C. (1999) J. Biol. Chem. 274, 24366-24371]. Here, we report the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrate that protein kinase A (PKA) and protein kinase C isoform epsilon are the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. Direct phosphorylation of GAD65 and GAD67 was demonstrated by incorporation of [(32)P] from [gamma-(32)P]ATP into purified GAD65 and GAD67 and immunoblotting assay using anti-phosphoserine/threonine antibodies. We have identified one specific phosphorylation site, threonine 91 (T91), in hGAD67 that can be phosphorylated by PKA using MALDI-TOF. Site-directed mutation of T91 to alanine abolished PKA-mediated phosphorylation and inhibition of GAD activity. Furthermore, mutation of T91 to aspartic acid or glutamic acid mimics the effect of phosphorylation. A model depicting the effect of phosphorylation on GAD activity upon neuronal stimulation is also proposed.
Wei
Jianning
J
Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, Florida 33431, USA.
Davis
Kathleen M
KM
Wu
Heng
H
Wu
Jang-Yen
JY
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001921:Brain / Q000201:enzymology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051744:Protein Kinase C-epsilon; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007527:Isoenzymes; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D010749:Phosphoprotein Phosphatases; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2