The regulation of AMP-activated protein kinase by phosphorylation.
The regulation of AMP-activated protein kinase by phosphorylation.
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DOI：
10.1042/bj3450437
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发表时间：
2000-02
期刊：
影响因子：
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通讯作者：
S. Stein;A. Woods;Neil A. Jones;M. Davison;D. Carling
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文献类型：
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作者：
S. Stein;A. Woods;Neil A. Jones;M. Davison;D. Carling
AMP激活的蛋白激酶（AMPK）级联是由细胞内AMP/ATP比率的增加而激活的。AMPK受AMP和可逆磷酸化的变构调节。在体外实验中，AMPK催化亚基（α）内的苏氨酸-172 （Thr(172)）被确定为amp活化蛋白激酶（AMPKK）磷酸化的主要位点。我们利用定点诱变技术研究了Thr（172）磷酸化对AMPK活性的作用。在alpha1或alpha2中，Thr（172）突变为天冬氨酸残基（T172D），导致一个激酶复合体具有大约。对应野生型复合物活性的50%。蛋白磷酸酶处理后，野生型AMPK的活性下降了90%以上，而T172D突变体复合物的活性仅下降了10-15%。Thr（172）突变为丙氨酸残基（T172A）几乎完全消除了激酶活性。这些结果表明，Thr（172）的磷酸化占AMPKK激活的大部分，但其他位点也参与其中。为了支持这一点，我们已经证明AMPKK磷酸化了α亚基上的至少两个其他位点和β亚基上的一个位点。此外，我们提供的证据表明，Thr（172）的磷酸化可能与AMPK复合物对AMP的敏感性有关。
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 alpha1 or alpha2 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 Thr(172) 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.