Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening

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.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.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.Cyclin-dependent kinase-5 (Cdk5) is a member of the cyclin-dependent kinase (Cdk) family of serine/threonine kinases [1]. Unlike other Cdk's, Cdk5 is not regulated by cyclins and is not involved in cell cycle control. The activity of Cdk5 is regulated by its binding to neuron-specific activator proteins, p35 and p39, [2,3] and by phosphorylation [4]. Although Cdk5 is widely expressed, its kinase activity is detected primarily in the nervous system, mainly because highest expression of its activators is restricted to post-mitotic neurons [5].Although Cdk5 activity is necessary for many physiological functions and development of the nervous system, deregulated Cdk5 activity is neurotoxic and has been linked to neurodegenerative diseases such as Alzheimer's disease (AD). Conversion of p35 to p25 by the calcium activated protease calpain, is thought to cause deregulation of Cdk5 activity in AD brain [6,7]. The dimeric Cdk5/p25 has been shown to possess prolonged enzymatic activity and potentially alter its