The regulation of AMP-activated protein kinase by phosphorylation

被引:493
|
作者
Stein, SC
Woods, A
Jones, NA
Davison, MD
Carling, D
机构
[1] Hammersmith Hosp, Imperial Coll, Sch Med, Cellular Stress Grp,MRC,Clin Sci Ctr, London W12 0NN, England
[2] AstraZeneca Pharmaceut, Proteom Grp, EST Biol, Macclesfield SK10 2TG, Cheshire, England
关键词
cell signalling; protein kinase cascade; site-directed mutagenesis; two-dimensional gel electrophoresis;
D O I
10.1042/0264-6021:3450437
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) On AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha 1 or alpha 2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr172 accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP.
引用
收藏
页码:437 / 443
页数:7
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