The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism

J Biol Chem. 2001 Dec 14;276(50):47460-7. doi: 10.1074/jbc.M105133200. Epub 2001 Sep 28.


Death-associated protein kinase is a calcium/calmodulin serine/threonine kinase, which positively mediates programmed cell death in a variety of systems. Here we addressed its mode of regulation and identified a mechanism that restrains its apoptotic function in growing cells and enables its activation during cell death. It involves autophosphorylation of Ser(308) within the calmodulin (CaM)-regulatory domain, which occurs at basal state, in the absence of Ca(2+)/CaM, and is inversely correlated with substrate phosphorylation. This type of phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C(6)-ceramide. The substitution of Ser(308) to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca(2+)/CaM-independent substrate phosphorylation as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. Conversely, mutation to aspartic acid reduces the binding of the protein to CaM and abrogates almost completely the death-promoting function of the protein. These results are consistent with a molecular model in which phosphorylation on Ser(308) stabilizes a locked conformation of the CaM-regulatory domain within the catalytic cleft and simultaneously also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device, which keeps death-associated protein kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Apoptosis Regulatory Proteins
  • Apoptosis*
  • Aspartic Acid / genetics
  • Binding Sites
  • Blotting, Western
  • Calcium / metabolism
  • Calcium-Calmodulin-Dependent Protein Kinases / chemistry*
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Calmodulin / metabolism
  • Catalysis
  • Catalytic Domain
  • Cell Death
  • Cell Line
  • Ceramides / chemistry
  • Ceramides / metabolism
  • Death-Associated Protein Kinases
  • Electrophoresis, Polyacrylamide Gel
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Luminescent Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Phosphorylation
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Serine / chemistry
  • Serine / metabolism
  • Transfection


  • Apoptosis Regulatory Proteins
  • Calmodulin
  • Ceramides
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Aspartic Acid
  • Serine
  • Death-Associated Protein Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Calcium