Study on the stress-induced signaling pathway of PKN

为了阐明应激作用下PKN从细胞质向细胞核易位的机制，我构建了PKN HA-和GFP（绿色荧光蛋白）标记缺失突变体的表达载体。这些实验结果表明，部分氨基末端区域对PKN的易位起重要作用。为了阐明PKN在热应激下的作用，我们研究了PKN是否调节热休克蛋白的表达。热休克转录因子1（HSF1）和PKN催化活性片段的go表达诱导了α -结晶蛋白的积累。HSF1和PKN催化活性片段的共同表达激活了α -结晶蛋白启动子报告基因的表达。这一结果表明PKN可能参与了应激信号通路的基因表达，并已在《生物化学》杂志上报道。Biophys。在缺血应激动物模型中，如短暂缺血后沙鼠海马CA1锥体细胞层延迟性神经元死亡，在受影响组织中观察到PKN的碎片化。在Fas结扎和staurosporine和依托泊苷处理下，凋亡的Jurkat和U937细胞的PKN也分别在特定位点被切割。这种蛋白水解被一种caspase抑制剂acetyl-Asp-Glu-Val-Asp-aldehyde抑制，当PKN与重组caspase-3体外孵育时，也观察到类似的断裂现象，这表明PKN是被caspase-3或相关蛋白酶切割的。PKN的位点定向诱变研究表明，主要的蛋白水解发生在氨基末端调节结构域和羧基末端催化结构域之间，无论是否存在花生四烯酸，它都会产生一个组成性的活性激酶片段。PKN的断裂可能参与信号转导，最终导致细胞凋亡。这些结果已在《国家科学进展》杂志上报道。学会科学。美国。

In order to clarify the mechanism of translocation of PKN from cytosol to the nucleus by stresses, I constructed expression vector for HA- and GFP (green fluorescent protein)- tagged-deletion mutants of PKN.The results of these experiments indicate that some part of amino-terminal region is important for translocation of PKN.To clarify the role of PKN under heat stress, we examined whether PKN regulates the expression of heat shock proteins. Go-expression of heat shock transcription factor 1(HSF1) and the catalytically active fragment of PKN induced the accumulation of alphaB-crystallin. The expression of the reporter gene for alphaB-crystallin promoter was activated by co- expression of HSF1 and the catalytically active fragment of PKN.This result suggests that PKN may be involved in the stress-signaling pathway to gene expression, and has been reported in Biochem. Biophys. Res. Comm. In animal models of ischemic stress, such as delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia, fragmentation of PKN was observed in the affected tissues. PKN was also cleaved at specific sites in apoptotic Jurkat and U937 cells on Fas ligation and treatment of staurosporine and etoposide, respectively. This proteolysis was inhibited by a caspase inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde, and the similar fragmentation was observed when PKN was incubated with recombinant caspase-3 in vitro, suggesting that PKN is cleaved by caspase-3 or related protease. Study using site directed mutagenesis of PKN revealed that the major proteolysis took place between the amino-terminal regulatory domain and carboxyl-terminal catalytic domain, and it generated a constitutive active kinase fragment irrespective of the presence of arachidonic acid. The cleavage of PKN may contribute to signal transduction eventually leading to apoptosis. These results have been reported in Proc. Natl. Acad. Sci. USA.