The cost of publication in Journal of Biomedical Science is borne by the National Science Council, Taiwan.
Research
Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening
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* Corresponding author: Anne Jämsä ajamsa1@hotmail.com
- † Equal contributors
1 AstraZeneca R&D, Forskargatan 20, Building 212, S-151 85 Södertälje, Sweden
2 Karolinska Institutet, NVS, KI-ADRC, Novum, 5th floor, S-141 57 Huddinge, Sweden
Journal of Biomedical Science 2009, 16:55 doi:10.1186/1423-0127-16-55
Published: 17 June 2009Abstract
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.
Conclusion
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 read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.