CATALYTIC SUBUNITS OF THE PORCINE AND RAT 5'-AMP-ACTIVATED PROTEIN-KINASE ARE MEMBERS OF THE SNF1 PROTEIN-KINASE FAMILY
CATALYTIC SUBUNITS OF THE PORCINE AND RAT 5'-AMP-ACTIVATED PROTEIN-KINASE ARE MEMBERS OF THE SNF1 PROTEIN-KINASE FAMILY
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DOI：
10.1016/0167-4889(94)00222-z
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发表时间：
1995-04-06
影响因子：
5.1
通讯作者：
WITTERS, LA
中科院分区：
文献类型：
--
作者：
GAO, G;WIDMER, J;WITTERS, LA
5'- amp激活的蛋白激酶（AMPK）分别通过乙酰辅酶a羧化酶和hmg -辅酶a还原酶的磷酸化来调节脂肪酸和甾醇合成途径。高纯度猪肝激酶含有三个明显的亚基，分子量分别为63 kDa、40 kDa和38 kDa。63 kDa蛋白（AMPK63(cat)）的肽测序显示，该多肽是该激酶的催化亚基。利用猪多肽序列克隆了猪AMPK63（cat）催化结构域的部分长度cdna及其大鼠同源物，推导出的氨基酸序列几乎相同。用这些部分长度cDNA和退化寡核苷酸筛选大鼠肝脏cDNA文库，得到了几个独特的克隆，其中一些在激酶的催化结构域有142 bp的缺失。利用重叠文库和pcr克隆构建了全长一致的1.7 kb开放阅读框序列。大鼠AMPK63（cat）的大mRNA （8.5 kb）几乎在所有大鼠组织中表达，在心脏和骨骼肌中可检测到最高水平。使用PCR，在所有检查的大鼠组织中都发现了催化结构域存在或不存在142 bp缺失的两种mRNA。比较推断的AMPK63（cat）蛋白序列，发现其在催化和非催化结构域与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线虫激酶。激酶结构和功能（代谢物感知）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。
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 (AMPK63(cat)) 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 AMPK63(cat), 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 AMPK63(cat) (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 AMPK63(cat) 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.