Boudreau Robert T M, Conrad David M, Hoskin David W
Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, 5850 University Ave., Halifax, Nova Scotia, Canada B3H 1X5.
Cell Signal. 2007 Jan;19(1):139-51. doi: 10.1016/j.cellsig.2006.05.030. Epub 2006 Jun 7.
Serine/threonine phosphatase regulation of phosphorylation-mediated intracellular signaling controls a number of important processes in mammalian cells. In this study, we show that constitutively active protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase, is essential for T leukemia cell survival. Jurkat and CCRF-CEM T leukemia cells treated with the PP2A-selective inhibitor okadaic acid (OA) showed a dose- and time-dependent induction of apoptosis, as indicated by loss of mitochondrial transmembrane potential (delta psi(m)), cleavage-induced activation of caspase-3, -8, and -9, and DNA fragmentation. In addition, caspase-8 or caspase-9 inhibition with z-IETD-fmk or z-LEHD-fmk, respectively, largely prevented OA-induced apoptosis. Although OA treatment did not affect constitutive Bcl-2 expression, overexpression of Bcl-2 prevented both OA-induced DNA fragmentation and dissipation of delta psi(m). Furthermore, inhibition of caspase-3, -8, or -9 partially protected against OA-induced loss of delta psi(m). In addition, caspase-9 and caspase-3 inhibition largely prevented procaspase-3 and procaspase-8 cleavage, respectively, while caspase-8 inhibition partially interfered with procaspase-9 cleavage in OA-treated T leukemia cells. Thus, PP2A inhibition triggered the intrinsic pathway of apoptosis, which was enhanced by a mitochondrial feedback amplification loop. PP2A has also been implicated in the regulation of p38 mitogen-activated protein kinase (MAPK). Co-immunoprecipitation analysis revealed a physical association between the catalytic subunit of PP2A and p38 MAPK in T leukemia cells. Moreover, OA treatment caused p38 MAPK to be phosphorylated in a dose- and time-dependent fashion, indicating that PP2A prevented p38 MAPK activation. Although p38 MAPK activation usually promotes apoptosis, pharmacologic inhibition of p38 MAPK exacerbated OA-induced DNA fragmentation and loss of delta psi(m) in T leukemia cells, suggesting that, in this instance, the p38 MAPK signaling pathway promoted cell survival. Collectively, these findings indicate that PP2A and p38 MAPK have coordinate effects on signaling pathways that regulate the survival of T leukemia cells.
丝氨酸/苏氨酸磷酸酶对磷酸化介导的细胞内信号传导的调节控制着哺乳动物细胞中的许多重要过程。在本研究中，我们表明，作为一种丝氨酸/苏氨酸磷酸酶的组成型活性蛋白磷酸酶2A（PP2A）对于T白血病细胞的存活至关重要。用PP2A选择性抑制剂冈田酸（OA）处理的Jurkat和CCRF-CEM T白血病细胞显示出剂量和时间依赖性的凋亡诱导，这通过线粒体跨膜电位（Δψm）的丧失、裂解诱导的半胱天冬酶-3、-8和-9的激活以及DNA片段化来表明。此外，分别用z-IETD-fmk或z-LEHD-fmk抑制半胱天冬酶-8或半胱天冬酶-9在很大程度上防止了OA诱导的凋亡。尽管OA处理不影响组成型Bcl-2表达，但Bcl-2的过表达防止了OA诱导的DNA片段化和Δψm的消散。此外，抑制半胱天冬酶-3、-8或-9部分保护细胞免受OA诱导的Δψm丧失。另外，抑制半胱天冬酶-9和半胱天冬酶-3分别在很大程度上防止了前体半胱天冬酶-3和前体半胱天冬酶-8的裂解，而抑制半胱天冬酶-8在OA处理的T白血病细胞中部分干扰了前体半胱天冬酶-9的裂解。因此，PP2A抑制触发了凋亡的内在途径，该途径通过线粒体反馈放大环得到增强。PP2A还参与了p38丝裂原活化蛋白激酶（MAPK）的调节。免疫共沉淀分析揭示了T白血病细胞中PP2A催化亚基与p38 MAPK之间的物理关联。此外，OA处理导致p38 MAPK以剂量和时间依赖性方式磷酸化，表明PP2A阻止了p38 MAPK的激活。尽管p38 MAPK激活通常促进凋亡，但药理学抑制p38 MAPK加剧了OA诱导的T白血病细胞中的DNA片段化和Δψm丧失，这表明在这种情况下，p38 MAPK信号通路促进了细胞存活。总的来说，这些发现表明PP2A和p38 MAPK对调节T白血病细胞存活的信号通路具有协同作用。
