O-GlcNAcylation Of PKA Catalytic Subunits Enhances Its Activity:A Possible Mechanism Involved In Learning And Memory

Objective Protein kinase A(PKA) is a cyclic AMP(cAMP) dependent protein kinase that is involved in regulation of carbenhydrate, protein and lipid metabolism in the cell. It is well established that PKA/CREB signaling plays critical role in learning and memory. PKA holoenzyme consists of a dimeric regulatory subunits, with each regulatory subunit being bound to a catalytic subunit. PKA is activated by binding of cAMP to the regulatory subunits, which leads to the release of the catalytic subunits. In addition, studies have shown cAMP-independent activation of PKA. Protein O-GlcNAcylation is a unique posttranslational modification of protein and regulates multiple cellular functions. This process is catalyzed by O-linked N-acetylglucosamine transferase(O-GlcNActransferase, OGT). The O-GlcNAc group can be removed by ?-N-acetylglucosaminidase(O-GlcNAcase, OGA). In fasting rodents and individuals brains with Alzheimer's disease(AD), we observed a decreased O-GlcNAcylation level and hyperphosphorylation of tau at multiple sites, but not Serine 214 which is mainly phosphorylated by PKA. These observations implied that PKA activity may be regulated by O-GlcNAc. In this project, we determined the relationship between O-GlcNAcylation and the activity of PKA, and which may help us to understand the role of O-GlcNAcylation in learning and memory dysfunction in AD.Methods 1. O-GlcNAcylated proteins or PKA catalytic subunits were immunoprecipitated to study whether PKA catalytic subunits are modified by O-GlcNAc.2. We altered O-GlcNAcylation by overexpression of OGT or OGA or shOGT, and analyzed the phosphorylation of CREB, a substrate of PKA.3. We overexpressed PKA catalytic subunits in HEK-293 FT cells with OGT, immunopurified PKA and then measured kinase activity toward tau. The level of phosphorylation was analyzed by immuno-dot-blot.4. O-GlcNAcylation in rodent active metabolic brain slices was upregulated by PUGNAc, an OGA inhibitor. The phosphorylation of tau at Ser214 was analyzed by Western blots.5. OGT in mice brain was neuronal specifically knocked out. The O-GlcNAcylation, the phosphorylation levels of PKA substrates, and synaptic markers were analyzed by Western blots.6. O-GlcNAcylation in mouse brain was down-regulated by DON intracerebraventrical injection. Fear conditioning test was used to study the learning and memory 24 hr after injection. The O-GlcNAcylation and phosphorylation of PKA substrates were analyzed by Western blots after fear conditioning.7. Chronic ischemia was introduced by UCCAO(unilateral common carotid artery occlusion) in adult AEP knockout, wild-type and 3xTg-AD mice. Elevated plus maze,open field test, novel object recognition and Morris water maze were used to determine exploratory behavior, short term memory and spatial memory.Results 1. We found that PKA catalytic subunits(PKAc) ? and ? were modified by O-GlcNAc. Overexpression of OGT up-regulated the O-GlcNAcylation of PKAcs2. Up-regulation of O-GlcNAc caused an increase of CREB phosphorylation, while down-regulation of O-GlcNAc decreased the phosphorylation of CREB.3. O-GlcNAcylation enhanced the activities of both PKAc? and PKAc? toward tau.4. Up-regulation of O-GlcNAcylation by PUGNAc increased tau phosphorylation at Ser214, a PKA phosphorylating site, but not at Thr205, in active metabolic rat brain slides.5. Neuronal specific knockout of OGT caused decrease of O-GlcNAcylation, CREB phosphorylation and tau phosphorylation at PKA-site, but not at non-PKA-sites. The synaptic markers were dramatically declined in OGT knockout mouse brains.6. Intracerebroventricularly injection of DON led to decrease of O-GlcNAcylation level and phosphorylation of PKA substrate,and learning and memory impairment.7. Exploratory behavior was increased, and short-term memory and spatial memory were impaired in wild-type and 3xTg-AD mice with chronic ischemia.Conclusions1. PKA catalytic subunits ? and ? are modified by O-GlcNAc. O-GlcNAcylation enhances their kinase activity.2. Decrease in O-GlcNAcylation down-regulates PKA/CREB pathway, which may lead to learning and memory deficits.3. These results indicate that in addition to cAMP and phosphorylation, O-GlcNAcylation is a novel way to regulate PKA activity. Down regulation of O-GlcNAcylation in AD brain may contribute to deficits of learning and memory.