Fatal congenital heart glycogenosis caused by a recurrent activating R531Q mutation in the gamma 2-subunit of AMP-activated protein kinase (PRKAG2), not by phosphorylase kinase deficiency

Fatal congenital nonlysosomal cardiac glycogenosis has been attributed to a subtype of phosphorylase kinase deficiency, but the underlying genes and mutations have not been identified. Analyzing four sporadic, unrelated patients, we found no mutations either in the eight genes encoding phosphorylase kinase subunits or in the two genes encoding the muscle and brain isoforms of glycogen phosphorylase. However, in three of five patients, we identified identical heterozygous R531Q missense mutations of the PRKAG2 gene, which encodes the gamma 2-subunit of AMP-activated protein kinase, a key regulator of energy balance. Biochemical characterization of the recombinant R531Q mutant protein showed >100-fold reduction of binding affinities for the regulatory nucleotides AMP and ATP but an enhanced basal activity and increased phosphorylation of the alpha -subunit. Other PRKAG2 missense mutations were previously identified in patients with autosomal dominant hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome, characterized by juvenile-to-adult clinical onset, moderate cardiac glycogenosis, disturbed excitation conduction, risk of sudden cardiac death in midlife, and molecular perturbations that are similar to--but less severe than--those observed for the R531Q mutation. Thus, recurrent heterozygous R531Q missense mutations in PRKAG2 give rise to a massive nonlysosomal cardiac glycogenosis of fetal symptomatic onset and rapidly fatal course, constituting a genotypically and clinically distinct variant of hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome. R531Q and other PRKAG2 mutations enhance the basal activity and alpha -subunit phosphorylation of AMP-activated protein kinase, explaining the dominant nature of PRKAG2 disease mutations. Since not all cases displayed PRKAG2 mutations, fatal congenital nonlysosomal cardiac glycogenosis seems to be genetically heterogeneous. However, the existence of a heart-specific primary phosphorylase kinase deficiency is questionable, because no phosphorylase kinase mutations were found.     

Prion protein interacts with BACE1 protein and differentially regulates its activity toward wild type and Swedish mutant amyloid precursor protein

In Alzheimer disease amyloid-β (Aβ) peptides derived from the amyloid precursor protein (APP) accumulate in the brain. Cleavage of APP by the β-secretase BACE1 is the rate-limiting step in the production of Aβ. We have reported previously that the cellular prion protein (PrP(C)) inhibited the action of BACE1 toward human wild type APP (APP(WT)) in cellular models and that the levels of endogenous murine Aβ were significantly increased in PrP(C)-null mouse brain. Here we investigated the molecular and cellular mechanisms underlying this observation. PrP(C) interacted directly with the prodomain of the immature Golgi-localized form of BACE1. This interaction decreased BACE1 at the cell surface and in endosomes where it preferentially cleaves APP(WT) but increased it in the Golgi where it preferentially cleaves APP with the Swedish mutation (APP(Swe)). In transgenic mice expressing human APP with the Swedish and Indiana familial mutations (APP(Swe,Ind)), PrP(C) deletion had no influence on APP proteolytic processing, Aβ plaque deposition, or levels of soluble Aβ or Aβ oligomers. In cells, although PrP(C) inhibited the action of BACE1 on APP(WT), it did not inhibit BACE1 activity toward APP(Swe). The differential subcellular location of the BACE1 cleavage of APP(Swe) relative to APP(WT) provides an explanation for the failure of PrP(C) deletion to affect Aβ accumulation in APP(Swe,Ind) mice. Thus, although PrP(C) exerts no control on cleavage of APP(Swe) by BACE1, it has a profound influence on the cleavage of APP(WT), suggesting that PrP(C) may be a key protective player against sporadic Alzheimer disease.     

Genetic model for the chronic activation of skeletal muscle AMP-activated protein kinase leads to glycogen accumulation

The AMP-activated protein kinase (AMPK) is an important metabolic sensor/effector that coordinates many of the changes in mammalian tissues during variations in energy availability. We have sought to create an in vivo genetic model of chronic AMPK activation, selecting murine skeletal muscle as a representative tissue where AMPK plays important roles. Muscle-selective expression of a mutant noncatalytic gamma1 subunit (R70Qgamma) of AMPK activates AMPK and increases muscle glycogen content. The increase in glycogen content requires the presence of the endogenous AMPK catalytic alpha-subunit, since the offspring of cross-breeding of these mice with mice expressing a dominant negative AMPKalpha subunit have normal glycogen content. In R70Qgamma1-expressing mice, there is a small, but significant, increase in muscle glycogen synthase (GSY) activity associated with an increase in the muscle expression of the liver isoform GSY2. The increase in glycogen content is accompanied, as might be expected, by an increase in exercise capacity. Transgene expression of this mutant AMPKgamma1 subunit may provide a useful model for the chronic activation of AMPK in other tissues to clarify its multiple roles in the regulation of metabolism and other physiological processes.     

Reelin-mediated signaling locally regulates protein kinase B/Akt and glycogen synthase kinase 3beta

Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation in vivo. Here we show that binding of Reelin to these receptors stimulates phosphatidylinositol 3-kinase, resulting in activation of protein kinase B and inhibition of glycogen synthase kinase 3beta. We present genetic evidence that this cascade is dependent on apolipoprotein E receptor 2, very low density lipoprotein receptor, and Dab1. Reelin-signaling components are enriched in axonal growth cones, where tyrosine phosphorylation of Dab1 is increased in response to Reelin. These findings suggest that Reelin-mediated phosphatidylinositol 3-kinase signaling in neuronal growth cones contributes to final neuron positioning in the mammalian brain by local modulation of protein kinase B and glycogen synthase kinase 3beta kinase activities.     

The AMPK gamma1 R70Q mutant regulates multiple metabolic and growth pathways in neonatal cardiac myocytes

Although mutations in the gamma-subunit of AMP-activated protein kinase (AMPK) can result in excessive glycogen accumulation and cardiac hypertrophy, the mechanisms by which this occurs have not been well defined. Because >65% of cardiac AMPK activity is associated with the gamma1-subunit of AMPK, we investigated the effects of expression of an AMPK-activating gamma1-subunit mutant (gamma1 R70Q) on regulatory pathways controlling glycogen accumulation and cardiac hypertrophy in neonatal rat cardiac myocytes. Whereas expression of gamma1 R70Q displayed the expected increase in palmitate oxidation rates, rates of glycolysis were significantly depressed. In addition, glycogen synthase activity was increased in cardiac myocytes expressing gamma1 R70Q, due to both increased expression and decreased phosphorylation of glycogen synthase. The inhibition of glycolysis and increased glycogen synthase activity were correlated with elevated glycogen levels in gamma1 R70Q-expressing myocytes. In association with the reduced phosphorylation of glycogen synthase, glycogen synthase kinase (GSK)-3beta protein and mRNA levels were profoundly decreased in the gamma1 R70Q-expressing myocytes. Consistent with GSK-3beta negatively regulating hypertrophy via inhibition of nuclear factor of activated T cells (NFAT), the dramatic downregulation of GSK-3beta was associated with increased nuclear activity of NFAT. Together, these data provide important new information about the mechanisms by which a mutation in the gamma-subunit of AMPK causes altered AMPK signaling and identify multiple pathways involved in regulating both cardiac myocyte metabolism and growth that may contribute to the development of the gamma mutant-associated cardiomyopathy.     

Regulation of the muscle-specific AMP-activated protein kinase alpha2beta2gamma3 complexes by AMP and implications of the mutations in the gamma3-subunit for the AMP dependence of the enzyme

The AMP-activated protein kinase is an evolutionarily conserved heterotrimer that is important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory gamma-subunit of the kinase, AMP-activated protein kinase gamma3, has a key role in glucose and fat metabolism in skeletal muscle, as suggested by metabolic characterization of humans, pigs and mice harboring substitutions in the AMP-binding Bateman domains of gamma3. We demonstrate that AMP-activated protein kinase alpha2beta2gamma3 trimers are allosterically activated approximately three-fold by AMP with a half-maximal stimulation (A(0.5)) at 1.9 +/- 0.5 or 2.6 +/- 0.3 microm, as measured for complexes expressed in Escherichia coli or mammalian cells, respectively. We show that mutations in the N-terminal Bateman domain of gamma3 (R225Q, H306R and R307G) increased the A(0.5) values for AMP, whereas the fold activation of the enzyme by 200 microm AMP remained unchanged in comparison to the wild-type complex. The mutations in the C-terminal Bateman domain of gamma3 (H453R and R454G), on the other hand, substantially reduced the fold stimulation of the complex by 200 microm AMP, and resulted in AMP dependence curves similar to those of the double mutant, R225Q/R454G. A V224I mutation in gamma3, known to result in a reduced glycogen content in pigs, did not affect the fold activation or the A(0.5) values for AMP. Importantly, we did not detect any increase in phosphorylation of Thr172 of alpha2 by the upstream kinases in the presence of increasing concentrations of AMP. Taken together, the data show that different mutations in gamma3 exert different effects on the allosteric regulation of the alpha2beta2gamma3 complex by AMP, whereas we find no evidence for their role in regulating the level of phosphorylation of alpha2 by upstream kinases.     

Differential modulation of late sodium current by protein kinase A in R1623Q mutant of LQT3

AimsIn the type 3 long QT syndrome (LQT3), shortening of the QT interval by overdrive pacing is used to prevent life-threatening arrhythmias. However, it is unclear whether accelerated heart rate induced by β-adrenergic agents produces similar effects on the late sodium current (I_Na) to those by overdrive pacing therapy. We analyzed the β-adrenergic-like effects of protein kinase A and fluoride on I_Na in R1623Q mutant channels.Main methodscDNA encoding either wild-type (WT) or R1623Q mutant of hNa_v1.5 was stably transfected into HEK293 cells. I_Na was recorded using a whole-cell patch-clamp technique at 23 °C.Key findingsIn R1623Q channels, 2 mM pCPT-AMP and 120 mM fluoride significantly delayed macroscopic current decay and increased relative amplitude of the late I_Na in a time-dependent manner. Modulations of peak I_Na gating kinetics (activation, inactivation, recovery from inactivation) by fluoride were similar in WT and R1623Q channels. The effects of fluoride were almost completely abolished by concomitant dialysis with a protein kinase inhibitor. We also compared the effect of pacing with that of β-adrenergic stimulation by analyzing the frequency-dependence of the late I_Na. Fluoride augmented frequency-dependent reduction of the late I_Na, which was due to preferential delay of recovery of late I_Na. However, the increase in late I_Na by fluoride at steady-state was more potent than the frequency-dependent reduction of late I_Na.SignificanceDifferent basic mechanisms participate in the QT interval shortening by pacing and β-adrenergic stimulation in the LQT3.     

The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase

In addition to acetyl-CoA carboxylase and HMG-CoA reductase, the AMP-activated protein kinase phosphorylates glycogen synthase, phosphorylase kinase, hormone-sensitive lipase and casein. A number of other substrates for the cyclic AMP-dependent protein kinase, e.g., L-pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, are not phosphorylated at significant rates. Examination of the sites phosphorylated on acetyl-CoA carboxylase, hormone-sensitive lipase, glycogen synthase and phosphorylase kinase suggests a consensus recognition sequence in which the serine residue phosphorylated by the AMP-activated protein kinase has a hydrophobic residue on the N-terminal side (i.e., at -1) and at least one arginine residue at -2, -3 or -4. Substrates for cyclic AMP-dependent protein kinase which lack the hydrophobic residue at -1 are not substrates for the AMP-activated protein kinase.     

Characterization of the role of gamma2 R531G mutation in AMP-activated protein kinase in cardiac hypertrophy and Wolff-Parkinson-White syndrome

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a key role in the regulation of energy metabolism. In humans, mutations in the gamma2-subunit of AMPK cause cardiac hypertrophy associated with Wolff-Parkinson-White syndrome, characterized by ventricular preexcitation. The effect of these mutations on AMPK activity and in development of the disease is enigmatic. Here we report that transgenic mice with cardiac-specific expression of gamma2 harboring a mutation of arginine residue 531 to glycine (RG-TG) develop a striking cardiac phenotype by 4 wk of age, including hypertrophy, impaired contractile function, electrical conduction abnormalities, and marked glycogen accumulation. At this stage, AMPK activity isolated from hearts of RG-TG mice was almost completely abolished but could be restored after phosphorylation by an upstream AMPK kinase. At 1 wk of age, there was no detectable evidence of a cardiac phenotype, and AMPK activity in RG-TG hearts was similar to that in nontransgenic, control mice. We propose that mutations in gamma2 lead to suppression of total cardiac AMPK activity secondary to increased glycogen accumulation. The subsequent decrease in AMPK activity provides a mechanism that may explain the development of cardiac hypertrophy in this model.     

Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p

In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.     

Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP.

A mutant clone resistant to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP-dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expressed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and express two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.     

Cocaine regulates protein kinase B and glycogen synthase kinase-3 activity in selective regions of rat brain

Protein kinase B (also known as Akt) signaling regulates dopamine-mediated locomotor behaviors. Here the ability of cocaine to regulate Akt and glycogen synthase kinase 3 (GSK3) was studied. Rats were injected with cocaine or saline in a binge-pattern, which consisted of three daily injections of 15 mg/kg cocaine or 1 mL/kg saline spaced 1 h apart for 1, 3, or 14 days. Amygdala, nucleus accumbens, caudate putamen, and hippocampus tissues were dissected 30 min following the last injection and analyzed for phosphorylated and total Akt and GSK3(alpha and beta) protein levels using western blot analysis. Phosphorylation of Akt on the threonine-308 (Thr308) residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 was significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure.     

Evidence for new alleles in the protein kinase adenosine monophosphate-activated gamma(3)-subunit gene associated with low glycogen content in pig skeletal muscle and improved meat quality.

Several quantitative trait loci (QTL) affecting muscle glycogen content and related traits were mapped to pig chromosome 15 using a three-generation intercross between Berkshire x Yorkshire pigs. On the basis of the QTL location the PRKAG3 (protein kinase, AMP-activated, gamma(3)-subunit) gene was considered to be a good candidate for the observed effects. Differences in the PRKAG3 gene sequences of the founder animals of the intercross were analyzed. The RN(-) mutation previously reported was not present in the cross but three missense substitutions and a polymorphic short interspersed element (SINE) were identified. To confirm the hypothesis that at least one of these mutations was associated with differences in meat quality, >1800 animals from several unrelated commercial lines were genotyped for the candidate substitutions and an association study was performed. The results demonstrate the presence of new economically important alleles of the PRKAG3 gene affecting the glycogen content in the muscle and the resulting meat quality. Haplotype analysis was shown to resolve the effects of PRKAG3 more clearly than analysis of individual polymorphisms. Because of their prevalence in the more common commercial breeds, the potential implications for the pig industry and consumers are considerably greater than the original discovery of the RN(-) mutation. Furthermore, these results illustrate that additional alleles of genes involved in major mutations may play a significant role in quantitative trait variation.     

Dopamine differentially induces aggregation of A53T mutant and wild type alpha-synuclein: insights into the protein chemistry of Parkinson's disease

Aggregation of α-synuclein is known to be a causal factor in the genesis of Parkinson’s disease and Dementia with Lewy bodies. Duplication and/or triplication and mutation of the α-synuclein gene are associated with sporadic and familial Parkinson’s disease. Synucleinopathies appear to primarily affect dopaminergic neurons. The present studies investigate the role of dopamine in α-synuclein aggregation through NMR. Dopamine causes aggregation of both wild type and A53T mutant α-synuclein in a temperature-dependent manner, but the mutant A53T shows a greater propensity to aggregate in the presence of dopamine only at 37 °C. A single point mutation in the α-synuclein A53T mutant gene results in a structural change in the protein and drastically increases its propensity to aggregate in the presence of dopamine. The present data indicate that mutation in the α-synuclein gene may predispose the protein to dopamine-induced aggregation, thereby contributing to disease pathogenesis.     

Characterization of cyanobacterial glycogen isolated from the wild type and from a mutant lacking of branching enzyme

Cyanobacteria produce glycogen as their primary form of carbohydrate storage. The genomic DNA sequence of Synechocystis sp. PCC6803 indicates that this strain encodes one glycogen-branching enzyme (GBE) and two isoforms of glycogen synthase (GS). To confirm the putative GBE and to demonstrate the presence of only one GBE gene, we generated a mutant lacking the putative GBE gene, sll0158, by replacing it with a kanamycin resistance gene through homologous recombination. GBE in sll0158(-) mutant was eliminated; the mutant strain produced less glucan, equivalent to 48% of that produced by the wild type. In contrast to the wild-type strain that had 74% of the glucan being water-soluble, the mutant had only 14% of the glucan water-soluble. Molecular structures of glucans produced by the mutant and the wild type were characterized by using high-performance size-exclusion and anion-exchange chromatography. The glycogen produced by the wild type displayed a molecular mass of 6.6 x 10(7) daltons (degree of polymerization (DP) 40700) and 10% branch linkages, and the alpha-D-glucan produced by the mutant displayed a molecular mass of 4.7-5.6 x 10(3) daltons (DP 29-35) with slight branch linkages. The results indicated that sll0158 was the major functional GBE gene in Synechocystis sp. PCC6803.