Functional Analysis of Mutations in the γ2 Subunit of AMP-activated Protein Kinase Associated with Cardiac Hypertrophy and Wolff-Parkinson-White Syndrome

Abstract

Mutations in the gene encoding the ␥ 2 subunit of the AMP-activated protein kinase (AMPK) have recently been shown to cause cardiac hypertrophy and ventricular pre-excitation (Wolff-Parkinson-White syndrome). We have examined the effect of four of these mutations on AMPK activity. The mutant ␥ 2 polypeptides are all able to form functional complexes following co-expression with either ␣ 1 ␤ 1 or ␣ 2 ␤ 1 in mammalian cells. None of the mutations caused any detectable change in the phosphorylation of threonine 172 within the ␣ subunit of AMPK. Consequently, in the absence of an appropriate stimulus the mutant complexes, like the wild-type complex, exist in an inactive form demonstrating that the mutations do not lead to constitutive activation of the kinase. Three of the mutations we studied occur within the cystathionine ␤-synthase (CBS) domains of ␥ 2 . Two of these mutations lead to a marked decrease in AMP dependence, whereas the third reduces AMP sensitivity. These findings suggest that the CBS domains play an important role in AMP-binding within the complex. In contrast, a fourth mutation, which lies between adjacent CBS domains, has no significant effect on AMPK activity in vitro. These results indicate that mutations in ␥ 2 have different effects on AMPK function, suggesting that they may lead to abnormal development of the heart through distinct mechanisms.The AMP-activated protein kinase (AMPK) 1 is the central component of a protein kinase cascade that plays a pivotal role in the regulation of energy metabolism (1). In response to activation following an increase in the AMP/ATP ratio, AMPK phosphorylates a number of downstream targets culminating in the switching off of energy (ATP)-utilizing pathways and the switching on of energy-generating pathways (1, 2). Activation of AMPK is complex and involves direct allosteric activation of the enzyme by AMP as well as phosphorylation, catalyzed by an upstream kinase, AMPK kinase (AMPKK) (3, 4). AMPK is a heterotrimeric complex, consisting of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥) (5, 6) and isoforms of all three subunits have been identified (7-9). Although there is compelling evidence indicating that formation of the heterotrimeric complex is necessary for significant kinase activity (6, 10) the precise role of the regulatory subunits remains unclear. In addition, at present our understanding of the physiological relevance of the different subunit isoforms is very limited.Several groups have reported recently the identification of six different mutations in the ␥2 subunit from patients with cardiac hypertrophy and associated electrophysiologic abnormalties (11-14). All six mutations result in amino acid substitutions and are located within the C-terminal half of the protein. The mutations lead to the development of aberrant conduction systems, including pre-excitation, characteristic of Wolff-Parkinson-White syndrome (15) and in all but one case, the mutations also result in severe cardiac hypertrophy. No evidence of cardiac hypertro...

Transgenic Mouse Model of Ventricular Preexcitation and Atrioventricular Reentrant Tachycardia Induced by an AMP-Activated Protein Kinase Loss-of-Function Mutation Responsible for Wolff-Parkinson-White Syndrome

“…In assays for cardiac AMPK activity, increasing concentrations of AMP were not accompanied by a corresponding increase in enzymatic activity, perhaps in keeping with previous studies that showed that mutations within the CBS domains of ␥-2 interfere with AMP binding and activation of AMPK. 18,19 It is also significant that 3 other AMPK mutations known to cause WPW have also been shown in vitro to be associated with loss of enzymatic activity. 19 Thus, the mutation R302Q occurring in a CBS motif prevents binding of AMP to the ␥-2 subunit and is responsible for the phenotype of the transgenic animal model and the human phenotype in the families we have studied.…”

“…The present in vivo studies showed minimal cardiac AMPK activity in the transgenic mutant mice, consistent with results of in vitro studies. In vitro studies by Daniel and Carling 18 showed marked attenuation or absent enzymatic activity associated with the R302Q mutation. Further in vitro studies by Scott et al 19 confirm the mutation R302Q to be associated with loss of enzymatic activity and Simultaneous ECG and intracardiac electrograms during atrial stimulation in TG R302Q mouse with ventricular preexcitation.…”

Adenosine Monophosphate-Activated Protein Kinase: A Central Regulator of Metabolism with Roles in Diabetes, Cancer, and Viral Infection

“…Additional clues came from observations that dominant mutations in the gene encoding the human g2 isoform (expressed at high levels in cardiac muscle), which were associated with a heart disease characterized by premature excitation of the large chambers (Blair et al 2001;Gollob et al 2001), were shown to interfere with binding of, and activation by, AMP (Daniel and Carling 2002;Adams et al 2004;Scott et al 2004). The g subunits in all AMPK orthologs contain four tandem repeats of a sequence known as a CBS repeat, so called because they are also found in the enzyme cystathionine b-synthase (Bateman 1997).…”

Role of AMP-activated protein kinase gamma 3 genetic variability in glucose and lipid metabolism in non-diabetic whites

“…The only mutations at the protein level of human AMPK subunit isoforms that are known to confer functional effects on AMPK activity are localised in the C-terminus of PRKAG2. Most of these polymorphisms impair or even abolish AMPK activation due to a hampered binding affinity of the regulatory PRKAG2 subunit to AMP or ATP [16,17]. Carriers of these PRKAG2 variants predispose to hypertrophic cardiomyopathy owing to cardiac glycogenosis [18] and often suffer from the WolffParkinson-White syndrome [19].…”