Activation of Brain L-glutamate Decarboxylase 65 Isoform (GAD65) by Phosphorylation at Threonine 95 (T95)

Mol Neurobiol. 2017 Mar;54(2):866-873. doi: 10.1007/s12035-015-9633-0. Epub 2016 Jan 16.


Protein phosphorylation plays an important role in regulating soluble L-glutamic acid decarboxylase (GAD) and membrane-associated GAD activity. Previously, we reported the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 (hGAD65) 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 demonstrated that protein kinase A (PKA) and protein kinase C isoform ε were the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. In the current study, using MALDI-TOF, a total of four potential phosphorylation sites were identified in GAD65, two of which (threonine-95 (T-95) and Ser-417) were not reported previously. We have identified one specific phosphorylation site, (T95), in hGAD65 that can be phosphorylated by kinase C ε (PKCε) using MALDITOF. When T95 is mutated to alanine, hGAD65 could no longer be phosphorylated by PKCε, and the effect of PKC-mediated activation on hGAD65 is abolished. However, when T95 is mutated to glutamic acid, which mimics the phosphorylation status of hGAD65, the activity was greatly increased. An increase of GAD65 activity by 55 % compared to the wild type hGAD65 was observed indicating that mutation of T95 to glutamic acid mimics the effect of phosphorylation. A model depicting the role of phosphorylation of GAD65 in regulation of GABA neurotransmission is presented.

Keywords: GABA; GAD65; Glutamate decarboxylase (GAD); Phosphorylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / enzymology*
  • Brain / pathology
  • Enzyme Activation / physiology
  • Glutamate Decarboxylase / genetics*
  • Glutamate Decarboxylase / metabolism*
  • Humans
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Male
  • Phosphorylation / physiology
  • Rats
  • Rats, Sprague-Dawley
  • Threonine / genetics*
  • Threonine / metabolism*


  • Isoenzymes
  • Threonine
  • Glutamate Decarboxylase