Characterization of PKI gamma, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase
Characterization of PKI gamma, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase
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DOI：
10.1074/jbc.272.29.18169
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发表时间：
1997-07-18
影响因子：
4.8
通讯作者：
Uhler, MD
中科院分区：
文献类型：
--
作者：
Collins, SP;Uhler, MD
试图了解的蛋白激酶抑制剂（PKI）蛋白的生理作用受到阻碍的知识缺乏有关的PKI家族的分子异质性。这里确定的PKI γ cDNA序列预测了75个氨基酸的开放阅读框，与PKI α显示35%的同一性，与PKI β 1显示30%的同一性。发现对于PKI α和PKI β 1的高亲和力以及cAMP依赖性蛋白激酶的催化（C）亚基的核输出重要的残基在PKI γ中是保守的。北方印迹分析表明，1.3-脱氢酶PKI γ信息广泛表达，在心脏、骨骼肌和睾丸中表达水平最高。RNA酶保护分析表明，在大多数组织检查PKI γ的表达水平等于或高于其他已知的PKI亚型，并在几个小鼠来源的细胞系，PKI γ是主要的PKI信息。通过DEAE离子交换层析从小鼠心脏部分纯化PKI活性，解析了两个主要抑制峰，并且针对重组PKI α和PKI γ产生的异构体特异性多克隆抗体鉴定了这些抑制活性为PKI α和PKI γ。在大肠杆菌中表达的PKI α和PKI γ的抑制效力的比较显示，PKI γ是体外C α磷酸转移酶活性的有效竞争性抑制剂（Ki = 0.44 nM），但效力比PKI α（Ki = 0.073 nM）低6倍。与PKI α一样，PKI γ能够阻断Flag标记的C亚基在瞬时转染的哺乳动物细胞中的核积累。最后，发现鼠PKI γ基因与小鼠2号染色体上的鼠腺苷脱氨酶基因重叠。这些结果表明，PKI γ是一种新的，功能性的PKI亚型，占以前观察到的PKI活性和PKI mRNA水平在几种哺乳动物组织之间的差异。
Attempts to understand the physiological roles of the protein kinase inhibitor (PKI) proteins have been hampered by a lack of knowledge concerning the molecular heterogeneity of the PKI family. The PKI gamma cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKI alpha and 30% identity to PKI beta 1. Residues important for the high affinity of PKI alpha and PKI beta 1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKI gamma. Northern blot analysis showed that a 1.3-kilobase PKI gamma message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKI gamma is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKI gamma is the predominant PKI message. Partial purification of PKI activities from mouse heart by DEAE ion exchange chromatography resolved two major inhibitory peaks, and isoform-specific polyclonal antibodies raised against recombinant PKI alpha and PKI gamma identified these inhibitory activities to be PKI alpha and PKI gamma. A comparison of inhibitory potencies of PKI alpha and PKI gamma expressed in Escherichia coli revealed that PKI gamma was a potent competitive inhibitor of C alpha phosphotransferase activity in vitro (K-i = 0.44 nM) but is 6-fold less potent than PKI alpha (K-i = 0.073 nM). Like PKI alpha, PKI gamma was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKI gamma gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKI gamma is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.