A Calmodulin-Regulated Protein Kinase Linked to Neuron Survival Is a Substrate for the Calmodulin-Regulated Death-Associated Protein Kinase

• Published in: Biochemistry (Easton) Vol. 43; no. 25; pp. 8116 - 8124
• Main Authors: Schumacher, Andrew M, Schavocky, James P, Velentza, Anastasia V, Mirzoeva, Salida, Watterson, D. Martin
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 29.06.2004
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Animals; Apoptosis Regulatory Proteins; Binding Sites; Brain - cytology; Brain - enzymology; Brain - metabolism; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Calcium-Calmodulin-Dependent Protein Kinases - genetics; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Calcium-Calmodulin-Dependent Protein Kinases - physiology; Calmodulin - metabolism; Cell Survival - physiology; Death-Associated Protein Kinases; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Neurons - cytology; Neurons - enzymology; Neurons - metabolism; Phosphorylation; Precipitin Tests; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Rats; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; Two-Hybrid System Techniques; Yeasts - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi049589v

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 K m 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.