Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening.
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 read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.
Jämsä
Anne
A
AstraZeneca R&D, Södertälje, Sweden. ajamsa1@hotmail.com
Agerman
Karin
K
Radesäter
Ann-Cathrin
AC
Ottervald
Jan
J
Malmström
Jonas
J
Hiller
Gösta
G
Liu
Gang
G
Vasänge
Mervi
M
D002478:Cells, Cultured; D051360:Cyclin-Dependent Kinase 5 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D006801:Humans; D018995:Myosin Heavy Chains / Q000378:metabolism*; D024501:Nonmuscle Myosin Type IIB / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D013379:Substrate Specificity; D014162:Transfection
D047428:Protein Kinase Inhibitors; D051360:Cyclin-Dependent Kinase 5; D024501:Nonmuscle Myosin Type IIB; C103815:nonmuscle myosin type IIB heavy chain; D018995:Myosin Heavy Chains