Protein Phosphorylation of Human Brain Glutamic Acid Decarboxylase (GAD)65 and GAD67 and Its Physiological Implications

Wei, Jianning; Davis, Kathleen; Wu, Heng; Wu, Jang‐Yen

doi:10.1021/bi0496992

Cited by 54 publications

(68 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on these observations together with our previous observation of regulation of GAD by protein phosphorylation (18), a model leading from neuronal stimulation to regulation of GABA neurotransmission is proposed: When GABAergic neurons are stimulated, there is an increase in calcium influx (step 1). Elevation of calcium activates calpain (step 2).…”

Section: Discussionmentioning

confidence: 92%

“…65 and hGAD 67 -Previously we have shown that recombinant hGAD 65 could be phosphorylated by PKCε, and hGAD 67 could be phosphorylated by PKA in vitro (18). In order to test whether GAD phosphorylation may have any effect on cleavage by μ-calpain, phos-phorylated GAD was prepared as described (18). Briefly, highly purified hGAD 67 and hGAD 65 in standard GAD buffer (50 mM KP, 1mM AET, 0.2 mM PLP, pH 7.2) were first extensively dialyzed against dialysis buffer (20 mM MOPS, 1mM AET, 0.2 mM PLP, pH 7.2).…”

Section: Calpain Cleavage Assay In Vitro-recombinant Hgadmentioning

confidence: 97%

“…Previously we have reported that recombinant hGAD 67 was phosphorylated by PKA and recombinant hGAD 65 was phosphorylated by PKCε (18). In order to determine role of GAD phosphorylation on calpain cleave, we performed calpain cleavage assay following phosphorylation reaction.…”

Section: Effect Of Gad Phosphorylation On Calpain Cleavagementioning

confidence: 99%

“…The supernatant was collected and referred to as cell culture extract. 65 and hGAD 67 -Previously we have shown that recombinant hGAD 65 could be phosphorylated by PKCε, and hGAD 67 could be phosphorylated by PKA in vitro (18). In order to test whether GAD phosphorylation may have any effect on cleavage by μ-calpain, phos-phorylated GAD was prepared as described (18).…”

Section: Calpain Cleavage Assay In Vitro-recombinant Hgadmentioning

confidence: 99%

See 3 more Smart Citations

Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

Sha

Jin

et al. 2008

Brain Research

Self Cite

View full text Add to dashboard Cite

Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for γ-aminobutyric acid (GABA) biosynthesis. Previously, we reported the presence of truncated forms of GAD in vivo and in vitro. In addition, an unidentified endogenous protease responsible for proteolytic cleavage of full-length GAD (fGAD) to its truncated form (tGAD) was also observed. In this communication, we report that μ-calpain is a good candidate for conversion of fGAD 67 to tGAD 67 . This conclusion is based on the following observations: 1. Purified recombinant GAD 67 is cleaved by μ-calpain at specific sites; 2. In brain synaptosomal preparation, GAD 67 is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. In μ-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when μ-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; 5. μ-calpain is activated by neuronal stimulation and Ca 2+ -influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed. KeywordsCalpain; GABA synthesis; GAD; Proteolytic CleavageIn the central nervous system (CNS), γ-aminobutyric acid (GABA) is synthesized by a single enzymatic reaction catalyzed by L-glutamic decarboxylase (EC 4.1.1.15; GAD) (1,2). Mammalian species express two isoforms of GAD, namely, GAD 65 and GAD 67 , referring to GAD with a molecular weight of 65 kDa and 67 kDa, respectively. Previously, we reported **Address correspondence and reprint requests to Dr. Jang-Yen Wu, Florida Atlantic University, Department of Biomedical Science, 777 Glades Road, Boca Raton, FL 33431-0991, U.S. ; Email: jwu@fau.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the presence of the truncated GAD (tGAD) derived from proteolytic cleavage of the full-length (fGAD) in vivo as well as in vitro (3,4). The presence of smaller forms of GAD was also observed from other laboratories (5-8). However, no information was reported regarding the identity of the endogenous proteases responsible for GAD cleavage and their physiological significance. Recently, we found that recombinant human brain GAD 65 is specifically cleaved at arginine 69 from the N-terminal of fGAD 65 to produce tGAD 65 , which is ∼2-3 fold more active than fGAD 65 (3). In addition, GAD 67 was found to be cleaved at two specific sites, one at arginine 70 and another at arginine 90, to produ...

show abstract

Section: Discussionmentioning

confidence: 92%

Section: Calpain Cleavage Assay In Vitro-recombinant Hgadmentioning

confidence: 97%

Section: Effect Of Gad Phosphorylation On Calpain Cleavagementioning

confidence: 99%

Section: Calpain Cleavage Assay In Vitro-recombinant Hgadmentioning

confidence: 99%

See 2 more Smart Citations

Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

Sha

Jin

et al. 2008

Brain Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…Glutamate decarboxylase 1 (GAD1) is inhibited upon phosphorylation at Thr91 residue by catalytic subunit of protein kinase A while glutamate decarboxylase 2 (GAD2), is activated on phosphorylation by protein kinase C, epsilon (PRKCE). Similarly, GAD1 and GAD2 are activated by protein phosphatases which include PP1, PP2A and PPP3CA (Wei et al 2004). …”

Section: Signaling Pathways Regulating Glutamate Metabolismmentioning

confidence: 99%

A pathway map of glutamate metabolism

Yelamanchi

Jayaram

Thomas

et al. 2015

J. Cell Commun. Signal.

131

100

View full text Add to dashboard Cite

Glutamate metabolism plays a vital role in biosynthesis of nucleic acids and proteins. It is also associated with a number of different stress responses. Deficiency of enzymes involved in glutamate metabolism is associated with various disorders including gyrate atrophy, hyperammonemia, hemolytic anemia, γ-hydoxybutyric aciduria and 5-oxoprolinuria. Here, we present a pathway map of glutamate metabolism representing metabolic intermediates in the pathway, 107 regulator molecules, 9 interactors and 3 types of post-translational modifications. This pathway map provides detailed information about enzyme regulation, protein-enzyme interactions, post-translational modifications of enzymes and disorders due to enzyme deficiency. The information included in the map was based on published experimental evidence reported from mammalian systems.

show abstract

Phosphorylation‐induced transient intrinsic structure in the kinase‐inducible domain of CREB facilitates its recognition by the KIX domain of CBP

et al. 2006

View full text Add to dashboard Cite

Phosphorylation at Ser-133 of the kinase inducible domain of CREB (KID) triggers its binding to the KIX domain of CBP via a concomitant coil-to-helix transition. The exact role of this key event is still puzzling: it does not switch between disordered and ordered states, nor its direct interactions fully account for selectivity. Hence, we reasoned that phosphorylation may shift the conformational preferences of KID towards a binding-competent state. To this end we investigated the intrinsic structural properties of the unbound KID in phosphorylated and unphosphorylated forms by simulated annealing and molecular dynamics simulations. Although helical populations show subtle differences, phosphorylation reduces the flexibility of the turn segment connecting the two helices in the complexed structure and induces a transient structural element that corresponds to its bound conformation. It is stabilized by the pSer-133-Arg-131 interaction, which is absent from the unphosphorylated KID. Diminishing this coupling decreases the 3.1 kcal/mol contribution of pSer-133 to the binding free energy (DeltaGbind) of the phosphorylated KID to KIX by 1.1 kcal/mol, as computed in reference to Ser-133. In a binding competent form of the S133E KID mutant, the contribution of Glu-133 to DeltaGbind is by 1.5 kcal/mol smaller than that of pSer, suggesting that altered structural properties due to pSer --> Glu replacement impair the binding affinity. Thus, we propose that phoshorylation contributes to selectivity not merely by the direct interactions of the phosphate group with KIX, but also by promoting the formation of a transient structural element in the highly conserved turn segment.

show abstract

Protein Phosphorylation of Human Brain Glutamic Acid Decarboxylase (GAD)65 and GAD67 and Its Physiological Implications

Cited by 54 publications

References 23 publications

Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

A pathway map of glutamate metabolism

Phosphorylation‐induced transient intrinsic structure in the kinase‐inducible domain of CREB facilitates its recognition by the KIX domain of CBP

Contact Info

Product

Resources

About