2004 Fiscal Year Final Research Report Summary
To clarify the mechanism of activation of PKN and the interaction with target molecules in vivo
| Project/Area Number |
15510175
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Living organism molecular science
|
|---|
| Research Institution | Kobe University |
|---|
Principal Investigator |
MUKAI Hideyuki Kobe University, Biosignal Research Center, Associate professor, バイオシグナル研究センター, 助教授 (80252758)
|
|---|
| Project Period (FY) |
2003 – 2004
|
| Keywords | PKN1 / RhoA / RhoB / protein kinase / FRET / kinase activity / CFP / YFP |
|---|
| Research Abstract |
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.
|
