A calmodulin-regulated protein kinase linked to neuron survival is a substrate for the calmodulin-regulated death-associated protein kinase

Andrew M. Schumacher, James P. Schavocky, Anastasia V. Velentza, Salida Mirzoeva, D. Martin Watterson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Death-associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase and a drug-discovery target for neurodegenerative diseases. However, a protein substrate relevant to neuronal death had not been described. We identified human brain CaM-regulated protein kinase kinase (CaMKK), an enzyme key to neuronal survival, as the first relevant substrate protein by using a focused proteomics-and informatics-based approach that can be generalized to protein kinase open reading frames identified in genome projects without prior knowledge of biochemical context. First, DAPK-interacting proteins were detected in yeast two-hybrid screens and in immunoprecipitates of brain extracts. Second, potential phosphorylation site sequences in yeast two-hybrid hits were identified on the basis of our previous results from positional-scanning synthetic-peptide substrate libraries and molecular modeling. Third, reconstitution assays using purified components demonstrated that DAPK phosphorylates CaMKK with a stoichiometry of nearly 1 mol of phosphate per mole of CaMKK and a Km value of 3 μM. Fourth, S511 was identified as the phosphorylation site by peptide mapping using mass spectrometry, site-directed mutagenesis, and Western blot analysis with a site-directed antisera targeting the phosphorylated sequence. Fifth, a potential mechanism of action was identified on the basis of the location of S511 near the CaM recognition domain of CaMKK and demonstrated by attenuation of CaM-stimulated CaMKK autophosphorylation after DAPK phosphorylation. The results raise the possibility of a CaM-regulated protein kinase cascade as a key mechanism in acute neurodegeneration amenable to therapeutic targeting.

Original languageEnglish (US)
Pages (from-to)8116-8124
Number of pages9
JournalBiochemistry
Volume43
Issue number25
DOIs
StatePublished - Jun 29 2004

ASJC Scopus subject areas

  • Biochemistry

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