权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

To clarify the mechanism of activation of PKN and the interaction with target molecules in vivo

阐明PKN的激活机制以及体内与靶分子的相互作用

基本信息

批准号：
15510175
负责人：
MUKAI Hideyuki
金额：
$ 2.18万
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：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15510175/
关键词：
PKN1 RhoA RhoB protein kinase FRET kinase activity CFP YFP substrate specificity CKAR NKAR

项目摘要

1.Construction of NCOP (PKN conformation probe).(1)The various expression vectors for fusion proteins of PKN1 with CFP and YFP were constructed.(2)The YFP/CFP FRET ratios of the proteins produced by some of these vectors were high and were dependent on the concentration of PKN activators such as unsaturated fatty acid and some detergents.(3)The localization of these recombinant proteins were homogeneous in the cytoplasm but were distributed in perinuclear region and plasma membrane when they were co-expressed with the active form of RhoB. However, the YFP/CFP FRET ratios were not significantly different from those when co-expressed with the inactive form of RhoB. These results suggest that the binding with RhoB is not sufficient for the active conformation change of PKN1.2.Construction of NKAR (PKN activation reporter)The specific phosphorylation motif of PKN was analyzed using biotin-tagged combinatorial peptide library. As a result, the phosphorylation motif was very similar to that of PKC family member. Several synthetic peptides were designed based on the information of the subtle difference between PKN and PKC and finally the peptide motif was elucidated which is selective for PKC but is not phosphorylated by PKN. This would help to brush up the present OKAR system. On the other hand, the peptide motif specific for PKN has not been elucidated yet. We are still trying to identify the specific substrate sequence for PKN and would like to setup the assay system for PKN in crude fraction and NKAR.

1.PKN构象探针NCOP的构建。(1)构建PKN1与CFP和YFP融合蛋白的表达载体。(2)部分载体产生的蛋白的YFP/CFP FRET比值较高，且与PKN激活剂（如不饱和脂肪酸和一些洗涤剂）的浓度有关。(3)这些重组蛋白在细胞质中定位均匀，但与RhoB活性形式共表达时分布于核周区和质膜。然而，YFP/CFP的FRET比率与与无活性形式的RhoB共表达时没有显著差异。这些结果表明，与RhoB的结合并不足以导致PKN1.2的主动构象改变。PKN激活报告基因NKAR的构建利用生物素标记组合肽库分析PKN的特异性磷酸化基序。结果表明，磷酸化基序与PKC家族成员的磷酸化基序非常相似。根据PKN和PKC之间的细微差别设计了几种合成肽，并最终阐明了PKC选择性而不被PKN磷酸化的肽基序。这将有助于刷新目前的OKAR系统。另一方面，PKN特异性肽基序尚未被阐明。我们仍在努力确定PKN的特定底物序列，并希望建立粗馏分和NKAR中PKN的测定系统。