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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Identification of novel substrates for Cdk5 and new targets for Cdk5 inhibitors using high-density protein

Cathrin Schnack1, Bastian Hengerer, Frank Gillardon

  • 1Boehringer Ingelheim Pharma GmbH & Co. KG, CNS Research, Biberach an der Riss, Germany.

Proteomics
|May 21, 2008
PubMed

Insights

Cyclin-dependent kinase 5 (Cdk5) phosphorylates protein phosphatase 1, regulatory subunit 14A (PPP1R14A), revealing a new role in CNS development. This study also assessed Cdk5 inhibitor selectivity, aiding neuroprotection research.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • Cyclin-dependent kinase 5 (Cdk5) is crucial for central nervous system (CNS) development and implicated in neurodegeneration.
  • Over 20 substrates of Cdk5 have been identified, but their cellular functions remain largely unknown.
  • High-throughput protein microarrays offer a method for parallel identification of kinase substrates.

Purpose of the Study:

  • To identify novel substrates of Cdk5/p25 using protein microarray technology.
  • To confirm the phosphorylation of identified substrates.
  • To characterize the selectivity of the Cdk5 inhibitor indolinone A.

Main Methods:

  • Utilized Protoarray protein microarrays to screen for Cdk5 substrates.
  • Employed secondary assays to validate phosphorylation events.
  • Applied Functional Kinome Arrays to assess inhibitor selectivity.

Main Results:

  • Identified protein phosphatase 1, regulatory subunit 14A (PPP1R14A) as a novel substrate of Cdk5/p25.
  • Confirmed PPP1R14A phosphorylation by Cdk5/p25.
  • Validated the selectivity of indolinone A, identifying two additional targeted kinases.

Conclusions:

  • The identification of PPP1R14A as a Cdk5 substrate may elucidate Cdk5's role in synaptic signaling.
  • Understanding indolinone A's targets deepens insights into its neuroprotective mechanisms.
  • Protein array techniques require careful interpretation due to potential false negatives from protein conformation issues.

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