- 作者: Anne Jamsa; Karin Agerman; Ann-Cathrin Radesater; Jan Ottervald; Jonas Malmstrom; Gosta Hiller; Gang Liu; Mervi Vasange
- 作者服務機構: AstraZeneca R&D, Forskargatan 20, Building 212, S-151 85 Sodertalje, Sweden
- 中文摘要: --
- 英文摘要:
Background :
Deregulated activation of cyclin-dependent kinase-5 (Cdk5) is implicated in
neurodegenerative disorders such as Alzheimer’s disease. One of the restricting factors
for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized
cellular in vitro assay systems.
Methods :
HEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an
assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we
utilized an antibody that recognizes phospho serine in a consensus motif for Cdk
substrates.
Results :
Western blot analysis of transfected cells detected a 200 kDa band that was identified, by
mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B).
Phosphorylation of NMHC-B was evident only in cells that were double transfected with
Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors.
Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y
cell line.
Conclusions :
A novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for
screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a readout
in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also
pharmacologically characterized in this assay system. - 中文關鍵字: --
- 英文關鍵字: --