Functional analysis of mutations in the gamma 2 subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff-Parkinson-White syndrome.
Mutations in the gene encoding the gamma(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 gamma(2) polypeptides are all able to form functional complexes following co-expression with either alpha(1)beta(1) or alpha(2)beta(1) in mammalian cells. None of the mutations caused any detectable change in the phosphorylation of threonine 172 within the alpha 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 beta-synthase (CBS) domains of gamma(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 gamma(2) have different effects on AMPK function, suggesting that they may lead to abnormal development of the heart through distinct mechanisms.
Daniel
Tyrone
T
Cellular Stress Group, MRC Clinical Sciences Centre, Hammersmith Hospital, DuCane Road, London W12 0NN, United Kingdom.
Carling
David
D
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D006332:Cardiomegaly / Q000201:enzymology / Q000235:genetics*; D002352:Carrier Proteins; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013314:Stress, Mechanical; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014927:Wolff-Parkinson-White Syndrome / Q000201:enzymology / Q000235:genetics*
D002352:Carrier Proteins; D009097:Multienzyme Complexes; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases; C526181:PRKAA2 protein, human; C526182:PRKAB1 protein, human; D017346:Protein Serine-Threonine Kinases; C078651:SNF4 protein, S cerevisiae; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human