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Keywords:

  • Kinase;
  • Protein array;
  • Selectivity screen;
  • Substrate screen

Abstract

Cyclin-dependent kinase (Cdk) 5 is a serine/threonine kinase that plays an important role during CNS development and its dysregulation is causally involved in the process of neuronal degeneration. To date more than 20 Cdk5 substrates have been identified and the number of Cdk5 substrates is still increasing. The different cellular functions of Cdk5 and its substrates are not completely known at present. High-throughput protein microarray technology is a powerful tool to identify a large number of potential kinase substrates in parallel under the same experimental conditions. Using Protoarray protein microarrays we identified protein phosphatase 1, regulatory subunit 14A (PPP1R14A) as a novel substrate of Cdk5/p25. Phosphorylation was confirmed in two secondary assays. Our findings may contribute to the elucidation of the physiological function of Cdk5 in synaptic signalling. Functional Kinome Arrays were validated in a second set of experiments to characterize the selectivity of the Cdk5 inhibitor indolinone A. This lead to the identification of two additional kinases that are targeted by this compound and may provide a deeper understanding of its neuroprotective mode of action. However, several false negative results possibly due to a denatured or inactive conformation of the arrayed proteins, sound a note of caution when using protein array techniques.