Jiang Kun, Apostolatos André H, Ghansah Tomar, Watson James E, Vickers Timothy, Cooper Denise R, Epling-Burnette P K, Patel Niketa A
Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612, USA.
Biochemistry. 2008 Jan 15;47(2):787-97. doi: 10.1021/bi7019782. Epub 2007 Dec 20.
Protein kinase C (PKC) delta plays an important role in cellular proliferation and apoptosis where it is involved in the caspase-3 mediated apoptotic pathway. Cleavage of PKCdeltaI by caspase-3 releases a catalytically active C-terminal fragment that is sufficient to induce apoptosis. In this paper, we identified a novel human PKCdelta isozyme, PKCdeltaVIII (Genbank accession number DQ516383) in human teratocarcinoma (NT2) cells that differentiate into hNT neurons upon retinoic acid (RA) treatment. Expression of PKCdeltaVIII was confirmed by real-time RT-PCR analysis, and we observed that after an initial peak at 24 h following RA treatment, its expression gradually declined with prolonged RA treatment. PKCdeltaVIII is generated via the utilization of an alternative 5' splice site, and this results in an insertion of 31 amino acids in the caspase-3 recognition sequence DMQD. The function of PKCdeltaVIII was examined by subcloning it into an expression vector and raising an antibody specific to PKCdeltaVIII. Using in vivo and in vitro assays, we demonstrated that PKCdeltaVIII is resistant to caspase-3 cleavage. Next, we sought to determine the role of PKCdeltaVIII in apoptosis in NT2 cells. Overexpression of PKCdeltaVIII and knockdown using PKCdeltaVIII siRNA suggest an antiapoptotic function for the PKCdeltaVIII isozyme. We demonstrate that antisense oligonucleotides (ASO) directed toward the 5' splice site I promote the expression of the PKCdeltaVIII isozyme. Our results indicated that ASO mediated PKCdeltaVIII expression rescued NT2 cells from etoposide-induced apoptosis. We conclude that the novel human PKCdeltaVIII splice variant functions as an antiapoptotic protein in NT2 cells.
蛋白激酶C（PKC）δ在细胞增殖和凋亡中发挥重要作用，它参与半胱天冬酶-3介导的凋亡途径。半胱天冬酶-3对PKCδI的切割会释放出一个具有催化活性的C端片段，该片段足以诱导细胞凋亡。在本文中，我们在人畸胎瘤（NT2）细胞中鉴定出一种新型的人PKCδ同工酶PKCδVIII（基因库登录号DQ516383），该细胞在视黄酸（RA）处理后可分化为hNT神经元。通过实时RT-PCR分析证实了PKCδVIII的表达，并且我们观察到在RA处理后24小时出现初始峰值后，其表达随着RA处理时间的延长而逐渐下降。PKCδVIII是通过利用一个替代的5'剪接位点产生的，这导致在半胱天冬酶-3识别序列DMQD中插入31个氨基酸。通过将PKCδVIII亚克隆到表达载体中并制备针对PKCδVIII的特异性抗体来检测其功能。使用体内和体外试验，我们证明PKCδVIII对半胱天冬酶-3的切割具有抗性。接下来，我们试图确定PKCδVIII在NT2细胞凋亡中的作用。PKCδVIII的过表达和使用PKCδVIII siRNA的敲低表明PKCδVIII同工酶具有抗凋亡功能。我们证明针对5'剪接位点I的反义寡核苷酸（ASO）促进了PKCδVIII同工酶的表达。我们的结果表明，ASO介导的PKCδVIII表达使NT2细胞从依托泊苷诱导的凋亡中获救。我们得出结论，新型的人PKCδVIII剪接变体在NT2细胞中作为一种抗凋亡蛋白发挥作用。
