NDLI: Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

Content Provider	Springer Nature Link
Author	Yu, Deyang Peng, Ying Ayaz Guner, Serife Gregorich, Zachery R. Ge, Ying
Copyright Year	2015
Abstract	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 AMPK$^{Δ}$) in E. coli, comprehensively characterized AMPK$^{Δ}$ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPK$^{Δ}$ affects its activity. Unexpectedly, we found that bacterially-expressed AMPK$^{Δ}$ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPK$^{Δ}$ 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 AMPK$^{Δ}$. 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. Graphical Abstract ᅟ
Starting Page	220
Ending Page	232
Page Count	13
File Format	PDF
ISSN	10440305
Journal	Journal of The American Society for Mass Spectrometry
Volume Number	27
Issue Number	2
e-ISSN	18791123
Language	English
Publisher	Springer US
Publisher Date	2015-10-21
Publisher Institution	The American Society for Mass Spectrometry
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	AMPK Phosphorylation Activity Mass spectrometry Electron capture dissociation Analytical Chemistry Biotechnology Organic Chemistry Proteomics Bioinformatics
Content Type	Text
Resource Type	Article
Subject	Spectroscopy Structural Biology

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