AMP 激活的蛋白激酶可被 ADP 异源激活,但 AMP 仍是关键的激活配体

IF 4.4 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Biochemical Journal Pub Date : 2024-04-24 DOI:10.1042/bcj20240082
Hawley, Simon A., Russell, Fiona M., Hardie, D. Grahame
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引用次数: 0

摘要

AMP激活蛋白激酶(AMPK)是细胞能量状态的传感器。当ADP:ATP和/或AMP:ATP比率增加时(能量不足信号),AMPK被激活,从而恢复能量平衡。AMPK-γ 亚基上的三个 CBS 重复序列(CBS1、CBS3、CBS4)中的一个或多个与 AMPK 结合,通过三种互补机制激活激酶复合物:(i) 通过上游激酶 LKB1 促进 α 亚基 Thr172 磷酸化;(ii) 防止 Thr172 去磷酸化;(iii) 异位激活。令人惊讶的是,据报道结合 ADP 可模拟前两种效应,但不能模拟第三种效应。我们现在的研究表明,在 Mg.ATP2- 的生理相关浓度下(高于标准测定中使用的浓度),ADP 结合确实会导致异源活化。然而,ADP 只引起适度的活化,因为(与 AMP 不同)在浓度刚刚超过活化变得明显时,ADP 开始在催化位点引起竞争性抑制。我们的结果使人对 ADP 作用的生理相关性产生怀疑,并表明 AMP 才是体内的主要激活剂。我们还对人α2β2γ1复合体的三个γ亚基 CBS 重复位点上参与结合腺嘌呤核苷酸的疏水残基进行了突变,并研究了它们对 AMP 和 ADP 调节的影响。CBS3 位点的突变对所有三种 AMP 激活机制的影响最大,尤其是在较低的 ATP 浓度下,而 CBS4 的突变则降低了对 AMP 的敏感性。这三个位点似乎都是 ADP 异源激活所必需的。
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AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand
The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2− (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2β2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.
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来源期刊
Biochemical Journal
Biochemical Journal 生物-生化与分子生物学
CiteScore
8.00
自引率
0.00%
发文量
255
审稿时长
1 months
期刊介绍: Exploring the molecular mechanisms that underpin key biological processes, the Biochemical Journal is a leading bioscience journal publishing high-impact scientific research papers and reviews on the latest advances and new mechanistic concepts in the fields of biochemistry, cellular biosciences and molecular biology. The Journal and its Editorial Board are committed to publishing work that provides a significant advance to current understanding or mechanistic insights; studies that go beyond observational work using in vitro and/or in vivo approaches are welcomed. Painless publishing: All papers undergo a rigorous peer review process; however, the Editorial Board is committed to ensuring that, if revisions are recommended, extra experiments not necessary to the paper will not be asked for. Areas covered in the journal include: Cell biology Chemical biology Energy processes Gene expression and regulation Mechanisms of disease Metabolism Molecular structure and function Plant biology Signalling
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