Deak J C, Cross J V, Lewis M, Qian Y, Parrott L A, Distelhorst C W, Templeton D J
Institute of Pathology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland OH 44106, USA.
Proc Natl Acad Sci U S A. 1998 May 12;95(10):5595-600. doi: 10.1073/pnas.95.10.5595.
The stress-activated protein kinase (SAPK, alternatively JNK) is activated rapidly by cell stress stimuli such as inflammatory cytokines and oxidative stress, and more slowly by the initiation of the apoptotic cell death response by events such as ligation of the Fas protein. Mitogen-activated protein kinase/Erk kinase kinase-1 (MEKK1) is an activator of SAPK, serving as a SAPK-kinase-kinase through intermediate phosphorylation of the SAPK kinase SEK1. By sequencing proteolytic cleavage products of MEKK1, we found that the proapoptotic protease caspase 3 (CPP32) cleaves MEKK1 after residue D68 both in vivo and in vitro. Cleavage of MEKK1 after D68 is blocked by viral and chemical protease inhibitors. Cleavage of MEKK1 at D68 changes the intracellular distribution of the protein from a Triton-insoluble compartment to a Triton-soluble compartment, reflected in a redistribution from a particulate to a diffuse cytoplasmic staining seen by immunofluorescence. Activation of both SAPK and MEKK1 after Fas ligation is prevented by both viral and chemical caspase 3 inhibitors, which in contrast fail to block activation of SAPK by rapidly acting cell stresses. Stress factor-induced SAPK signaling is not dependent on caspase 3 function. We propose that two mechanisms of stress signaling through MEKK1 exist. One is rapid, independent of proteases, and occurs in the particulate Triton-insoluble compartment. The other is more slowly activated and involves liberation of particulate MEKK1 by proteolytic cleavage and activation by caspase 3.
应激激活蛋白激酶（SAPK，又称JNK）可被细胞应激刺激（如炎性细胞因子和氧化应激）迅速激活，而通过诸如Fas蛋白连接等事件引发凋亡性细胞死亡反应时，其激活速度则较慢。丝裂原激活蛋白激酶/细胞外信号调节激酶激酶-1（MEKK1）是SAPK的激活剂，通过对SAPK激酶SEK1的中间磷酸化作用充当SAPK激酶激酶。通过对MEKK1的蛋白水解裂解产物进行测序，我们发现在体内和体外，促凋亡蛋白酶caspase 3（CPP32）均可在D68残基后切割MEKK1。病毒和化学蛋白酶抑制剂可阻断D68后MEKK1的切割。MEKK1在D68处的切割会使该蛋白的细胞内分布从Triton不溶性区室转变为Triton可溶性区室，这在免疫荧光观察到的从颗粒状到弥漫性细胞质染色的重新分布中得以体现。病毒和化学caspase 3抑制剂均可阻止Fas连接后SAPK和MEKK1的激活，相比之下，它们无法阻断快速作用的细胞应激对SAPK的激活。应激因子诱导的SAPK信号传导不依赖于caspase 3功能。我们提出，通过MEKK1存在两种应激信号传导机制。一种是快速的，不依赖蛋白酶，发生在颗粒状Triton不溶性区室中。另一种激活速度较慢，涉及通过蛋白水解切割释放颗粒状MEKK1并由caspase 3激活。
