Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro
Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro
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DOI：
10.1074/jbc.m604399200
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发表时间：
2006-09-01
影响因子：
4.8
通讯作者：
Carlson, Marian
中科院分区：
文献类型：
--
作者：
Momcilovic, Milica;Hong, Seung-Pyo;Carlson, Marian
Snf1/AMP激活的蛋白激酶(AMPK)家族在代谢调节中起重要作用，从酵母到哺乳动物都高度保守。上游的激酶在功能上也是保守的，AMPK激酶LKB1和钙/钙调节非依赖性蛋白激酶在缺乏天然Snf1激活激酶Sak1、Tos3和Elm1的突变酵母细胞中激活Snf1。在这里，我们利用酵母遗传系统来识别哺乳动物AMPK激酶家族的成员，根据他们作为Snf1激活激酶的功能。利用酵母表达载体中的小鼠胚胎cDNA文库，将SAK1 Delta转化为3Delta elm1 Delta酵母细胞。对SNF(+)生长表型的选择产生了表达LKB1、钙/钙调素依赖的蛋白激酶和丝裂原活化蛋白激酶家族成员之一的转化生长因子-β-激活蛋白激酶(TAK1)的cDNA质粒。我们提出了TAK1在体内和体外激活Snf1蛋白激酶的遗传和生化证据。我们进一步证明，融合到其结合伙伴TAB1的激活域的重组TAK1在AMPK催化域的激活环中磷酸化Thr-172。最后，TAK1和TAB1在HeLa细胞中的表达或用细胞因子处理细胞可刺激AMPK苏氨酸-172的磷酸化。这些发现表明，TAK1是Snf1/AMPK激酶家族的一个功能成员，并支持TAK1作为哺乳动物细胞中真正的AMPK激酶的候选者。
The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation and is highly conserved from yeast to mammals. The upstream kinases are also functionally conserved, and the AMPK kinases LKB1 and Ca2+/calmodulindependent protein kinase kinase activate Snf1 in mutant yeast cells lacking the native Snf1-activating kinases, Sak1, Tos3, and Elm1. Here, we exploited the yeast genetic system to identify members of the mammalian AMPK kinase family by their function as Snf1-activating kinases. A mouse embryo cDNA library in a yeast expression vector was used to transform sak1 Delta tos3 Delta elm1 Delta yeast cells. Selection for a Snf(+) growth phenotype yielded cDNA plasmids expressing LKB1, Ca2+/calmodulin-dependent protein kinase kinase, and transforming growth factor-beta-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase family. We present genetic and biochemical evidence that TAK1 activates Snf1 protein kinase in vivo and in vitro. We further show that recombinant TAK1, fused to the activation domain of its binding partner TAB1, phosphorylates Thr-172 in the activation loop of the AMPK catalytic domain. Finally, expression of TAK1 and TAB1 in HeLa cells or treatment of cells with cytokines stimulated phosphorylation of Thr-172 of AMPK. These findings indicate that TAK1 is a functional member of the Snf1/AMPK kinase family and support TAK1 as a candidate for an authentic AMPK kinase in mammalian cells.