A calmodulin-regulated protein kinase linked to neuron survival is a substrate for the calmodulin-regulated death-associated protein kinase.
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 microM. 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.
Schumacher
Andrew M
AM
Drug Discovery Program, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.
Schavocky
James P
JP
Velentza
Anastasia V
AV
Mirzoeva
Salida
S
Watterson
D Martin
DM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D001921:Brain / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D064550:Death-Associated Protein Kinases; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; D002147:Calmodulin; D011994:Recombinant Proteins; D064550:Death-Associated Protein Kinases; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D017871:Calcium-Calmodulin-Dependent Protein Kinases