Collins S P, Uhler M D
Department of Biological Chemistry and the Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1997 Jul 18;272(29):18169-78. doi: 10.1074/jbc.272.29.18169.
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 PKIgamma cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKIalpha and 30% identity to PKIbeta1. Residues important for the high affinity of PKIalpha and PKIbeta1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKIgamma. Northern blot analysis showed that a 1.3-kilobase PKIgamma message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKIgamma is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKIgamma 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 PKIalpha and PKIgamma identified these inhibitory activities to be PKIalpha and PKIgamma. A comparison of inhibitory potencies of PKIalpha and PKIgamma expressed in Escherichia coli revealed that PKIgamma was a potent competitive inhibitor of Calpha phosphotransferase activity in vitro (Ki = 0.44 nM) but is 6-fold less potent than PKIalpha (Ki = 0.073 nM). Like PKIalpha, PKIgamma was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKIgamma gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKIgamma is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.
由于对蛋白激酶抑制剂（PKI）家族分子异质性缺乏了解，人们在试图理解PKI蛋白的生理作用时遇到了阻碍。此处测定的PKIγ cDNA序列预测有一个75个氨基酸的开放阅读框，与PKIα的同一性为35%，与PKIβ1的同一性为30%。发现PKIα和PKIβ1高亲和力以及环磷酸腺苷依赖性蛋白激酶催化（C）亚基核输出所必需的残基在PKIγ中是保守的。Northern印迹分析表明，一条1.3千碱基的PKIγ信息广泛表达，在心脏、骨骼肌和睾丸中水平最高。核糖核酸酶保护分析显示，在大多数检测的组织中，PKIγ的表达水平等于或高于其他已知的PKI同工型，并且在几种小鼠来源的细胞系中，PKIγ是主要的PKI信息。通过DEAE离子交换色谱法从小鼠心脏中部分纯化PKI活性，分离出两个主要的抑制峰，针对重组PKIα和PKIγ产生的同工型特异性多克隆抗体确定这些抑制活性分别为PKIα和PKIγ。对在大肠杆菌中表达的PKIα和PKIγ抑制效力的比较表明，PKIγ在体外是Ca磷酸转移酶活性的有效竞争性抑制剂（Ki = 0.44 nM），但效力比PKIα低6倍（Ki = 0.073 nM）。与PKIα一样，PKIγ能够在瞬时转染的哺乳动物细胞中阻断带有Flag标签的C亚基的核积累。最后，发现小鼠PKIγ基因与小鼠2号染色体上的小鼠腺苷脱氨酶基因重叠。这些结果表明，PKIγ是一种新的、有功能的PKI同工型，解释了之前在几种哺乳动物组织中观察到的PKI活性与PKI mRNA水平之间的差异。
