Structural and functional studies of the AMP-activated protein kinase γ2 isoform
File(s)
Author(s)
Chen, ShuYang
Type
Thesis
Abstract
AMPK is a kinase that plays a key role in controlling energy metabolism. AMPK is activated by upstream kinases in response to a fall in ATP levels. Once activated, AMPK promotes catabolic pathways and inhibits anabolic pathways. Dysregulation of energy homeostasis is associated with various human metabolic diseases, such as obesity, type 2 diabetes, cancer. Therefore, it is crucial to gain a better understanding of the regulation and role of AMPK.
AMPK is a heterotrimeric complex consisting of three subunits, a catalytic subunit α, and two regulatory subunits β and γ. In mammalian cells, each subunit exists as multiple isoforms encoded by separate genes. There are three γ isoforms, the γ1 isoform is ubiquitously expressed, while the γ2 and γ3 isoforms are more tissue specific. The γ isoforms contain highly conserved C-terminal regions that encompass 4 CBS (cystathionine-β-synthase) domains that are involved in nucleotide binding. The γ2 isoform is poorly studied functionally and structurally, in part due to technical difficulties of expression in recombinant systems. Mutations in human γ2 have been identified that lead to a cluster of severe cardiac abnormalities, including Wolff-Parkinson White syndrome and cardiac hypertrophy.
This study focuses on the γ2 isoform and its properties are investigated in comparison with the γ1 isoform. Several forms of γ2 have been reported with differing N-terminal extensions that are not present in γ1, and not conserved in γ3. A novel binding partner, 14-3-3ε has been identified which forms a stable co-complex with AMPK containing full-length γ2 but not the truncated form of γ2. I show that binding of 14-3-3ε to AMPK requires phosphorylation of sites within the N-terminal region of full-length γ2, and the interaction is characterised by X-ray crystallography. Stable recombinant expression of an AMPK γ2 containing complex with 14-3-3ε is presented, and nucleotide binding and AMPK activity of the complex were investigated.
AMPK is a heterotrimeric complex consisting of three subunits, a catalytic subunit α, and two regulatory subunits β and γ. In mammalian cells, each subunit exists as multiple isoforms encoded by separate genes. There are three γ isoforms, the γ1 isoform is ubiquitously expressed, while the γ2 and γ3 isoforms are more tissue specific. The γ isoforms contain highly conserved C-terminal regions that encompass 4 CBS (cystathionine-β-synthase) domains that are involved in nucleotide binding. The γ2 isoform is poorly studied functionally and structurally, in part due to technical difficulties of expression in recombinant systems. Mutations in human γ2 have been identified that lead to a cluster of severe cardiac abnormalities, including Wolff-Parkinson White syndrome and cardiac hypertrophy.
This study focuses on the γ2 isoform and its properties are investigated in comparison with the γ1 isoform. Several forms of γ2 have been reported with differing N-terminal extensions that are not present in γ1, and not conserved in γ3. A novel binding partner, 14-3-3ε has been identified which forms a stable co-complex with AMPK containing full-length γ2 but not the truncated form of γ2. I show that binding of 14-3-3ε to AMPK requires phosphorylation of sites within the N-terminal region of full-length γ2, and the interaction is characterised by X-ray crystallography. Stable recombinant expression of an AMPK γ2 containing complex with 14-3-3ε is presented, and nucleotide binding and AMPK activity of the complex were investigated.
Version
Open Access
Date Issued
2021-06
Date Awarded
2021-11
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Carling, David
Gamblin, Steven
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)