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

1.PKN构象探针的构建(1)构建了PKN1与CFP、YFP融合蛋白的表达载体。(2)部分表达载体表达的蛋白YFP/CFP FRET比值较高，且与不饱和脂肪酸和某些去污剂等PKN激活剂的浓度有关。(3)这些重组蛋白在细胞质中定位均一，但与RhoB活性形式共表达时分布于核周和质膜。然而，YFP/CFP FRET比率与非活性形式的RhoB共表达时没有显著差异。这些结果表明，与RhoB的结合不足以引起PKN1.2的活性构象变化。2.构建PKN激活报告基因(Nkar)利用生物素标记的组合肽文库分析PKN的特异性磷酸化基序。结果表明，该磷酸化基序与PKC家族成员的磷酸化基序非常相似。根据PKN和PKC的细微差别，设计了几种合成肽，最终确定了对PKC具有选择性但不被PKN磷酸化的多肽基序。这将有助于改善目前的奥卡尔制度。另一方面，PKN的特异性多肽基序还没有被阐明。我们仍在努力确定PKN的特定底物序列，并希望建立粗提物和NKAR中PKN的检测系统。

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.