The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism.
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.
Shohat
G
G
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Spivak-Kroizman
T
T
Cohen
O
O
Bialik
S
S
Shani
G
G
Berrisi
H
H
Eisenstein
M
M
Kimchi
A
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D016923:Cell Death; D002460:Cell Line; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D051017:Apoptosis Regulatory Proteins; D002147:Calmodulin; D002518:Ceramides; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D012694:Serine; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium