Gao G, Widmer J, Stapleton D, Teh T, Cox T, Kemp B E, Witters L A
Endocrine-Metabolism Division, Dartmouth Medical School, Hanover, New Hampshire 03755-3833, USA.
Biochim Biophys Acta. 1995 Apr 6;1266(1):73-82. doi: 10.1016/0167-4889(94)00222-z.
The 5'-AMP-activated protein kinase (AMPK) regulates the fatty acid and sterol synthesizing pathways via phosphorylation of acetyl-CoA carboxylase and HMG-CoA reductase, respectively. Highly purified kinase from porcine liver contains three apparent subunits of molecular mass 63 kDa, 40 kDa and 38 kDa. Peptide sequencing of the 63 kDa protein (AMPK63cat) revealed that this polypeptide is the catalytic subunit of the kinase. Porcine peptide sequences were used to clone by RT-PCR partial length cDNAs for the catalytic domains of the porcine AMPK63cat, and its rat homolog, which were virtually identical in deduced amino acid sequence. Screening of a rat liver cDNA library with these partial length cDNAs and with degenerate oligonucleotides yielded several unique clones, some of which had a 142 bp deletion in the catalytic domain of the kinase. A consensus full-length sequence with a 1.7 kb open reading frame has been constructed from overlapping library and PCR-derived clones. A large mRNA for rat AMPK63cat (8.5 kb) is expressed in nearly all rat tissues, with highest levels detectable in heart and skeletal muscle. Using PCR, the presence of two mRNA species with or without the 142 bp deletion in the catalytic domain was noted in all rat tissues examined. Comparison of the deduced protein sequence of AMPK63cat reveals highly conserved homologies in both the catalytic and non-catalytic domains to several members of the SNF1 kinase family, including kinases from Arabidopsis, barley, rye, and S. cerevesiae, as well as to other mammalian kinases and to a C. elegans kinase. The high evolutionary conservation of both kinase structure and function (metabolite sensing) coupled with their pattern of tissue/organism expression suggest that the mammalian members of this kinase family likely play wider roles than the regulation of cellular lipid metabolism.
5'-AMP激活蛋白激酶（AMPK）分别通过磷酸化乙酰辅酶A羧化酶和3-羟基-3-甲基戊二酰辅酶A还原酶来调节脂肪酸和固醇合成途径。从猪肝中高度纯化得到的激酶含有分子量分别为63 kDa、40 kDa和38 kDa的三个明显亚基。对63 kDa蛋白（AMPK63cat）进行肽序列分析表明，该多肽是激酶的催化亚基。利用猪的肽序列通过RT-PCR克隆了猪AMPK63cat及其大鼠同源物催化结构域的部分长度cDNA，其推导的氨基酸序列几乎相同。用这些部分长度cDNA和简并寡核苷酸筛选大鼠肝脏cDNA文库，得到了几个独特的克隆，其中一些克隆在激酶的催化结构域中有142 bp的缺失。通过重叠文库和PCR衍生克隆构建了一个具有1.7 kb开放阅读框的共有全长序列。大鼠AMPK63cat的一个大mRNA（8.5 kb）在几乎所有大鼠组织中都有表达，在心脏和骨骼肌中检测到的水平最高。通过PCR发现在所有检测的大鼠组织中都存在两种mRNA，一种在催化结构域中有142 bp缺失，另一种没有。AMPK63cat推导的蛋白质序列比较显示，其催化结构域和非催化结构域与SNF1激酶家族的几个成员高度保守同源，包括来自拟南芥、大麦、黑麦和酿酒酵母的激酶，以及其他哺乳动物激酶和秀丽隐杆线虫激酶。激酶结构和功能（代谢物传感）的高度进化保守性及其组织/生物体表达模式表明，该激酶家族的哺乳动物成员可能发挥比调节细胞脂质代谢更广泛的作用。
