Related Experiment Videos
PKN interacts with a paraneoplastic cerebellar degeneration-associated antigen, which is a potential transcription
H Takanaga1, H Mukai, H Shibata
1Faculty of Science, Kobe University, Kobe, 657, Japan.
Abstract:
PKN is a fatty acid-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. PKN has been recently reported to interact with a small GTP-binding protein Rho and cytoskeletal proteins such as neurofilament and alpha-actinin. To identify the new components of the PKN-signaling pathway, the yeast two-hybrid system was employed. Using the amino-terminal regulatory domain of PKN as a bait, cDNA encoding a neural antigen PCD17, which is recognized by characteristic antibodies of patients with paraneoplastic cerebellar degeneration, was isolated from a human brain cDNA library. The interaction between PKN and PCD17 was also determined by the in vitro binding analysis. PCD17 was coimmunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and the amino-terminal region of PCD17. PCD17 was phosphorylated by PKN, and the extent of this phosphorylation was enhanced by addition of 40 microM arachidonic acid. The amino-terminal region of PCD17 could form a homodimer in vitro, and PCD17 fused to the Gal4 DNA binding domain showed the transcriptional transactivation of the chloramphenicol acetyltransferase reporter gene linked to 5 Gal4 binding sites and minimal promoter in rat C6 glioma cells. These results suggest the participation of PCD17 in gene expression and lead to a clue for elucidating the PKN signaling pathway from the cytosol to the nucleus.
Insights
Protein kinase N (PKN) interacts with neural antigen PCD17, identified via yeast two-hybrid screening. PKN phosphorylates PCD17, suggesting PCD17
Area of Science:
- Molecular Biology
- Cell Signaling
- Neuroscience
Background:
- Protein kinase N (PKN) is a serine/threonine kinase activated by fatty acids.
- PKN interacts with Rho GTPase and cytoskeletal proteins.
- Components of the PKN signaling pathway remain largely uncharacterized.
Purpose of the Study:
- To identify novel components of the PKN signaling pathway.
- To investigate the interaction between PKN and the neural antigen PCD17.
- To elucidate the functional role of PCD17 in cellular processes.
Main Methods:
- Yeast two-hybrid system for identifying interacting proteins.
- In vitro binding assays to confirm PKN-PCD17 interaction.
- Co-immunoprecipitation to validate interaction in mammalian cells.
- In vitro phosphorylation assays to assess PKN's activity on PCD17.
- Reporter gene assays to evaluate PCD17's transcriptional activity.
Main Results:
- The neural antigen PCD17 was identified as a binding partner of PKN.
- PKN directly interacts with and phosphorylates PCD17.
- PCD17 phosphorylation by PKN is enhanced by arachidonic acid.
- PCD17 exhibits homodimerization and transcriptional transactivation capabilities.
- PCD17's function suggests a role in gene expression regulation.
Conclusions:
- PCD17 is a novel substrate and interaction partner of PKN.
- PCD17 participates in gene expression, potentially linking PKN signaling to nuclear events.
- This discovery provides insights into the PKN signaling pathway, extending from the cytosol to the nucleus.