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

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 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.

Death-Associated Protein Kinase Phosphorylates Mammalian Ribosomal Protein S6 and Reduces Protein Synthesis

“…Both proteins were found in immunoprecipitates from rat brain and kinetic analyses show that S6 is a DAPK substrate. Further, the amino acid sequence surrounding the site of phosphorylation in S6 by DAPK matches the pattern revealed by positional scanning peptide library screening for DAPK substrate preferences (21) and by the proteomics-based mapping of DAPK phosphorylation sites in protein substrates (8). Second, DAPK phosphorylates S6 in the context of the 40S ribosomal subunit at position Ser235, but does not phosphorylate S6 at any of the other four sites phosphorylated by previously discovered S6Ks.…”

“…Although these findings support the notion that calcium/calmodulin-regulated pathways affect the molecular mechanisms that influence synaptic plasticity (47,48), clearly, much remains to be done as a follow up to the novel finding reported here that DAPK is capable of regulating protein synthesis with a possible role in neuronal function. Regardless, the key findings reported here and previously (2,5,8) provide an emerging view of DAPK as a kinase potentially capable of selective modulation of neuronal homeostasis in rapid response to diverse stimuli, with apoptosis as one possible outcome that can be attenuated by targeted therapeutics in disease-relevant events.…”

DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death

“…It is possible that the previously identified DAPk substrate syntaxin-1A, a component of the v-SNARE complex, may be involved. 35 Notably, the CaM-dependent kinase kinase b (CaMKKb), another substrate of DAPk, 36 has been implicated in mediating autophagy induced by ionomycin and inducers of ER stress. 29 However, phosphorylation of CaMKKb by DAPk on Ser511 attenuates the former's CaM-dependent autophosphorylation, a step necessary for its activation, suggesting that DAPk actually inhibits CaMKK function.…”

[P2–202]: Dapk1 Signaling Pathways in Stroke: From Mechanisms to Therapies

“…It has been demonstrated that DAPK1 phosphorylates diverse substrates via its kinase domain. These substrates include the MLC [19], beclin-1 [37,38], zipper-interacting protein kinase (ZIPK) [39,40], calmodulin-regulated protein kinase kinase (CaMKK) [41], and syntaxin-1A [42]. The requirement of DAPK catalytic activity for its proposed cell functions and the validation of protein kinases as therapeutic targets in human disease make the identification of substrates of DAPK1 in ischemia extremely important.…”