Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity.
AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the beta1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the beta1-subunit of AMPK revealed amino acids 68-123 of the beta1-subunit to be sufficient for GDE binding. W100G and K128Q, both beta1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68-123 of the beta1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.
Sakoda
Hideyuki
H
Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
Fujishiro
Midori
M
Fujio
Junko
J
Shojima
Nobuhiro
N
Ogihara
Takehide
T
Kushiyama
Akifumi
A
Fukushima
Yasushi
Y
Anai
Motonobu
M
Ono
Hiraku
H
Kikuchi
Masatoshi
M
Horike
Nanao
N
Viana
Amelia Y I
AY
Uchijima
Yasunobu
Y
Kurihara
Hiroki
H
Asano
Tomoichiro
T
D055372:AMP-Activated Protein Kinases; D000818:Animals; D000906:Antibodies; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000502:physiology; D005982:Glutathione Transferase / Q000235:genetics; D006003:Glycogen / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009097:Multienzyme Complexes; D011485:Protein Binding / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D006004:Glycogen Debranching Enzyme System; D009097:Multienzyme Complexes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006003:Glycogen; D005982:Glutathione Transferase; D011494:Protein Kinases; C526182:PRKAB1 protein, human; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases