| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
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.
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