Seifert Anne, Allan Lindsey A, Clarke Paul R
Biomedical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, UK.
FEBS J. 2008 Dec;275(24):6268-80. doi: 10.1111/j.1742-4658.2008.06751.x. Epub 2008 Nov 7.
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.
DYRK1A是双特异性酪氨酸磷酸化调节蛋白激酶家族的成员，与唐氏综合征有关。在此，我们确定半胱天冬酶9（一种内在凋亡途径的关键成分）是DYRK1A的底物。通过短干扰RNA从人细胞中耗尽DYRK1A可抑制半胱天冬酶9在抑制位点Thr125的基础磷酸化。Harmine也可阻断Thr125的DYRK1A依赖性磷酸化，证实了这种β-咔啉生物碱在细胞中作为DYRK1A有效抑制剂的用途。我们表明，Harmine不仅抑制成熟DYRK1A的蛋白质丝氨酸/苏氨酸激酶活性，还抑制其在翻译过程中酪氨酸的自磷酸化，表明Harmine可阻止活性酶的形成。当在细胞中共表达时，DYRK1A与半胱天冬酶9相互作用，强烈诱导Thr125磷酸化并抑制半胱天冬酶9的自动加工。DYRK1A对半胱天冬酶9的磷酸化涉及共定位到细胞核。这些结果表明，DYRK1A通过对该蛋白酶的基础抑制性磷酸化来设定半胱天冬酶9激活的阈值。通过对半胱天冬酶9的抑制性磷酸化来调节细胞凋亡可能在DYRK1A在发育和发病机制中的功能中起作用。
