Study on the stress-induced signaling pathway of PKN

应激诱导的PKN信号通路研究

• 批准号: 09680695
• 负责人: MUKAI Hideyuki
• 金额: $ 1.98万
• 依托单位: KOBE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680695/
• 关键词: PKN; translocation; stress; caspase; apoptosis

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.

为了阐明PKN在逆境条件下从胞浆向核转位的机制，构建了HA-和GFP的表达载体（绿色荧光蛋白）标记的PKN缺失突变体，这些实验结果表明PKN氨基末端的某些区域对于PKN的转运是重要的。我们检测PKN是否调节热休克蛋白的表达。热休克转录因子1（HSF 1）和PKN的催化活性片段的GO表达诱导α B-晶体蛋白的积累。共表达HSF 1和PKN的催化活性片段激活了α B-晶状体蛋白启动子的报告基因的表达。在缺血性应激的动物模型中，如短暂缺血后沙鼠海马CA 1锥体细胞层中的迟发性神经元死亡，在受影响的组织中观察到PKN的碎片化。凋亡的Jurkat和U937细胞在Fas连接和星形孢菌素和依托泊苷处理后，PKN也分别在特定位点被切割。这种蛋白水解被caspase抑制剂乙酰-Asp-Glu-Val-Asp-aldehyde所抑制，并且当PKN与重组caspase-3体外孵育时观察到类似的断裂，表明PKN被caspase-3或相关蛋白酶切割。利用PKN的定点突变的研究表明，主要的蛋白水解发生在氨基端的调节结构域和羧基端的催化结构域之间，它产生了一个组成性的活性激酶片段，而不管花生四烯酸的存在。PKN的裂解可能有助于信号转导，最终导致细胞凋亡。这些结果已在Proc. Natl. Acad. Sci. USA.

项目成果

Kitagawa, M.: "Molecular cloning and characterization of a novel mitochondrial phosphoprotein,MIPP65,from rat liver." Exp.Cell Res.235. 71-78 (1997)

Kitakawa, M.：“来自大鼠肝脏的新型线粒体磷蛋白 MIPP65 的分子克隆和表征。”

Matsuzawa,K.: "Domain-specific phosphorylation of vimentin and glial fibrillary acidic protein by PKN." Biochem.Biophys.Res.Commun.234. 621-625 (1997)

Matsuzawa,K.：“PKN 对波形蛋白和胶质纤维酸性蛋白进行域特异性磷酸化。”

Takanaga, H., Mukai, H., Shibata, H., Toshimori, M., and Ono.Y.: "PKN Interacts with a Paraneoplastic Cerebellar Degeneration-Associated Antigen, Which Is a Potential Transcription Factor" Exp.Cell.Res. 241. 363-372 (1998)

Takanaga, H.、Mukai, H.、Shibata, H.、Toshimori, M. 和 Ono.Y.：“PKN 与副肿瘤性小脑变性相关抗原相互作用，该抗原是一种潜在的转录因子”Exp.Cell.Res

Mukai,H.: "Interaction of PKN with α-actinin." J.Biol.Chem.272. 4740-4746 (1997)

Mukai，H.：“PKN 与 α-辅肌动蛋白的相互作用。”J.Biol.Chem.272-4746（1997）。

Kitagawa, M., Mukai, H., and Ono, Y.: "Molecular Cloning and Characterization of a Novel Mitochondrial Phosphoprotein, MIPP65, from Rat Liver" Exp.Cell.Res. 235. 71-78 (1997)

Kitakawa, M.、Mukai, H. 和 Ono, Y.：“来自大鼠肝脏的新型线粒体磷蛋白 MIPP65 的分子克隆和表征”Exp.Cell.Res。