Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis.
Bacterially expressed heterotrimeric (alpha1, beta1, and gamma1) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the alpha-subunit, viz. Thr258 and Ser485 (Ser491 in the alpha2-subunit) by mass spectrometry, in addition to the previously characterized Thr172 site. Also, autophosphorylation sites in the beta1-subunit were identified as Ser96, Ser101, and Ser108. Mutagenesis of Thr172, Thr258, and Ser485 to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr172 was involved in AMPK activation, whereas Thr258 and Ser485 were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the alpha-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser485 phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition.
Woods
Angela
A
Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology and Université de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
Vertommen
Didier
D
Neumann
Dietbert
D
Turk
Roland
R
Bayliss
Jayne
J
Schlattner
Uwe
U
Wallimann
Theo
T
Carling
David
D
Rider
Mark H
MH
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D013058:Mass Spectrometry; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry / Q000378:metabolism; D010769:Phosphothreonine / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014162:Transfection
D009097:Multienzyme Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases