title: Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening.
abstract: 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.
authors.affiliation: AstraZeneca R&D, Södertälje, Sweden. ajamsa1@hotmail.com
authors.forename: Anne
authors.initials: A
authors.lastname: Jämsä
authors.forename: Karin
authors.initials: K
authors.lastname: Agerman
authors.forename: Ann-Cathrin
authors.initials: AC
authors.lastname: Radesäter
authors.forename: Jan
authors.initials: J
authors.lastname: Ottervald
authors.forename: Jonas
authors.initials: J
authors.lastname: Malmström
authors.forename: Gösta
authors.initials: G
authors.lastname: Hiller
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
authors.forename: Mervi
authors.initials: M
authors.lastname: Vasänge
mesh_terms: 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
chemical_list: 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