DYRK1A phosphorylates caspase 9 at an inhibitory site and is potently inhibited in human cells by harmine.
DYRK1A is a member of the dual-specificity tyrosine-phosphorylation-regulated protein kinase family and is implicated in Down's syndrome. Here, we identify the cysteine aspartyl protease caspase 9, a critical component of the intrinsic apoptotic pathway, as a substrate of DYRK1A. Depletion of DYRK1A from human cells by short interfering RNA inhibits the basal phosphorylation of caspase 9 at an inhibitory site, Thr125. DYRK1A-dependent phosphorylation of Thr125 is also blocked by harmine, confirming the use of this beta-carboline alkaloid as a potent inhibitor of DYRK1A in cells. We show that harmine not only inhibits the protein-serine/threonine kinase activity of mature DYRK1A, but also its autophosphorylation on tyrosine during translation, indicating that harmine prevents formation of the active enzyme. When co-expressed in cells, DYRK1A interacts with caspase 9, strongly induces Thr125 phosphorylation and inhibits caspase 9 auto-processing. Phosphorylation of caspase 9 by DYRK1A involves co-localization to the nucleus. These results indicate that DYRK1A sets a threshold for the activation of caspase 9 through basal inhibitory phosphorylation of this protease. Regulation of apoptosis through inhibitory phosphorylation of caspase 9 may play a role in the function of DYRK1A during development and in pathogenesis.
Seifert
Anne
A
Biomedical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, UK.
Allan
Lindsey A
LA
Clarke
Paul R
PR
D017209:Apoptosis; D001665:Binding Sites; D053453:Caspase 9 / Q000378:metabolism*; D061945:Caspase Inhibitors; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D017124:Conserved Sequence; D006247:Harmine / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D013912:Threonine / Q000378:metabolism; D014162:Transfection; D000097603:Dyrk Kinases
D053453:Caspase 9; D061945:Caspase Inhibitors; D006247:Harmine; D010769:Phosphothreonine; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D034741:RNA, Small Interfering; D013912:Threonine; D000097603:Dyrk Kinases