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.