Death-associated protein kinase phosphorylates mammalian ribosomal protein S6 and reduces protein synthesis.
Death-associated protein kinase (DAPK) is a pro-apoptotic, calcium/calmodulin-regulated protein kinase that is a drug discovery target for neurodegenerative disorders. Despite the potential profound physiological role of DAPK in neuronal function and pathophysiology, the endogenous substrate(s) of this kinase and the mechanisms via which DAPK elicits its biological action remain largely unknown. We report here that the mammalian 40S ribosomal protein S6 is a DAPK substrate. Results from immunoprecipitation experiments are consistent with endogenous DAPK being associated with endogenous S6 in rat brain. When S6 is a component of the 40S ribosomal subunit complex, DAPK selectively phosphorylates it at serine 235, one of the five sites in S6 that are phosphorylated by the S6 kinase family of proteins. The amino acid sequence flanking serine 235 matches the established pattern for DAPK peptide and protein substrates. Kinetic analyses using purified 40S subunits revealed a K(m) value of 9 microM, consistent with S6 being a potential physiological substrate of DAPK. This enzyme-substrate relationship has functional significance. DAPK suppresses translation in rabbit reticulocyte lysate, and treatment of neuroblastoma cells with a stimulator of DAPK reduces protein synthesis. In both cases, suppression of translation correlates with increased phosphorylation of S6 at serine 235. These results demonstrate that DAPK is a S6 kinase and provide evidence for a novel role of DAPK in the regulation of translation.
Schumacher
Andrew M
AM
Center for Drug Discovery and Chemical Biology, Northwestern University, 303 East Chicago Avenue, W-896, Chicago, Illinois 60611, USA.
Velentza
Anastasia V
AV
Watterson
D Martin
DM
Dresios
John
J
D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D064550:Death-Associated Protein Kinases; D004997:Ethinyl Estradiol / Q000378:metabolism; D019290:Megestrol Acetate / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects*; D051381:Rats; D038601:Ribosomal Protein S6 / Q000378:metabolism*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D038601:Ribosomal Protein S6; D004997:Ethinyl Estradiol; C026340:Oraconal; D064550:Death-Associated Protein Kinases; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019290:Megestrol Acetate