Comprehensive Characterization of AMP-Activated Protein  Kinase Catalytic Domain by Top-Down Mass Spectrometry.
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.
Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53706, USA
Deyang
D
Yu
Ying
Y
Peng
Serife
S
Ayaz-Guner
Zachery R
ZR
Gregorich
Ying
Y
Ge
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013912:Threonine / Q000378:metabolism
D021122:Protein Subunits; D011994:Recombinant Proteins; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases