Scarpetta M A, Uhler M D
Mental Health Research Institute, University of Michigan, Ann Arbor 48109.
J Biol Chem. 1993 May 25;268(15):10927-31.
Oligonucleotides derived from the previously published rat testicular protein kinase inhibitor (PKI) sequence (Van Patten, S. M., Ng, D. C., Th'ng, J. P. H., Angelos, K. L., Smith, A. J., and Walsh, D. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5383-5387) were used to isolate a DNA fragment coding for a mouse homologue of the rat testicular PKI. This DNA fragment was then used to screen a mouse brain cDNA library, and two cDNA clones related to the testicular PKI (PKI beta) were isolated. Sequencing and comparison showed that the two cDNAs differ only in the presence of a 105-base pair insert, which results in an amino-terminal extension of the predicted PKI beta 2 protein by 20 residues relative to the PKI beta 1 protein. By using the appropriate primers, PCR was used to amplify specific regions of both of these clones from mouse brain cDNA. When both clones were expressed in vitro, the mRNA for PKI beta 2 produced a protein product that was larger and much more effectively translated, suggesting a functional role for the inserted sequence. Both isoforms were transiently expressed in COS-1 cells to evaluate their ability to inhibit the catalytic subunit of PKA in vivo. Extracts from cells expressing the PKI beta 2 isoform showed greater inhibition of catalytic subunit kinase activity than extracts expressing the PKI beta 1 isoform. Northern blot analysis of poly(A)+ RNA from various mouse tissues showed the presence of transcripts of 1.8 kilobases related to these cDNAs. Analysis of brain RNA from several species indicated that expression of these PKI mRNAs is evolutionarily conserved. Together with previous studies, these results indicate the presence of at least three PKI proteins in mouse. The skeletal muscle isoform has been designated PKI alpha, while the testicular isoform is PKI beta. We propose to designate the two testicular isoforms described here PKI beta 1 and PKI beta 2.
从先前发表的大鼠睾丸蛋白激酶抑制剂（PKI）序列（Van Patten, S. M., Ng, D. C., Th'ng, J. P. H., Angelos, K. L., Smith, A. J., and Walsh, D. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5383 - 5387）推导而来的寡核苷酸，被用于分离编码大鼠睾丸PKI小鼠同源物的DNA片段。然后，该DNA片段被用于筛选小鼠脑cDNA文库，并分离出两个与睾丸PKI（PKIβ）相关的cDNA克隆。测序和比较表明，这两个cDNA仅在存在一个105个碱基对的插入片段上有所不同，这导致预测的PKIβ2蛋白的氨基末端相对于PKIβ1蛋白延伸了20个残基。通过使用合适的引物，利用PCR从小鼠脑cDNA中扩增这两个克隆的特定区域。当这两个克隆在体外表达时，PKIβ2的mRNA产生了一个更大且翻译效率更高的蛋白质产物，表明插入序列具有功能作用。两种同工型都在COS - 1细胞中瞬时表达，以评估它们在体内抑制PKA催化亚基的能力。表达PKIβ2同工型的细胞提取物对催化亚基激酶活性的抑制作用，比表达PKIβ1同工型的提取物更强。对来自各种小鼠组织的聚腺苷酸加尾（poly(A)+）RNA进行的Northern印迹分析表明，存在与这些cDNA相关的1.8千碱基的转录本。对几种物种的脑RNA分析表明，这些PKI mRNA的表达在进化上是保守的。与先前的研究一起，这些结果表明小鼠中至少存在三种PKI蛋白。骨骼肌同工型已被命名为PKIα，而睾丸同工型为PKIβ。我们提议将这里描述的两种睾丸同工型命名为PKIβ1和PKIβ2。
