PDF(Pubmed)
Abstract：
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.
摘要：
AMP激活的蛋白激酶（AMPK）是细胞能量状态的传感器。当ADP：ATP和/或AMP：ATP比率（信号能量不足）增加激活时，AMPK用于恢复能量平衡。AMP与AMPK-γ亚基上的三个CBS重复序列（CBS1，CBS3，CBS4）中的一个或多个的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）保护免受Thr172去磷酸化；（iii）变构激活。令人惊讶的是,据报道，ADP的结合模拟了前两种效应，但不是第三个。我们现在表明，在生理相关浓度的Mg。ATP2-(高于标准测定中使用的那些)ADP结合确实引起变构激活。然而,ADP仅引起适度的激活，因为（与AMP不同），浓度略高于激活变得明显的浓度，ADP开始在催化位点引起竞争性抑制。我们的结果对ADP作用的生理相关性表示怀疑，并表明AMP是体内的主要激活剂。我们还在人α2β2γ1复合物的三个γ亚基CBS重复序列中的每一个上对与腺嘌呤核苷酸结合的疏水残基进行了突变，并检查了它们对AMP和ADP调节的影响。CBS3位点的突变对AMP激活的所有三种机制具有最大的影响，特别是在较低的ATP浓度下，而CBS4的突变降低了对AMP的敏感性。ADP的变构激活似乎需要所有三个位点。
