Pombo C M, Bonventre J V, Avruch J, Woodgett J R, Kyriakis J M, Force T
Cardiac Unit, Massachusetts General Hospital 02129.
J Biol Chem. 1994 Oct 21;269(42):26546-51.
The signal transduction pathways that mediate activation of trans acting factors controlling an organ's response to ischemia are unknown. The stress-activated protein kinases (SAPKs), a subfamily of the extracellular signal-regulated kinases (ERKs), phosphorylate c-Jun within the amino-terminal transactivation domain and are activated in response to a variety of cellular stresses. We determined whether SAPKs are activated in response to ischemia, an extreme, albeit common, pathophysiologic stress. Rats underwent 40 min of renal ischemia followed by reperfusion for 0, 5, 20, or 90 min. SAPKs were immunoprecipitated from kidney lysates and kinase activity assayed with recombinant GST-c-Jun(1-135), containing the amino-terminal transactivation domain of c-Jun as substrate. SAPKs were not activated by ischemia alone, but reperfusion for as little as 5 min was associated with a 4.6-fold increase in kinase activity. Kinase activity was increased 7.6-fold at 20 min following reperfusion and remained elevated at 90 min of reperfusion (4.9-fold). In contrast, activity of the related ERK-1 and -2 was increased only 1.3-fold and only at the 5-min reperfusion time point. When SAPKs were immunodepleted from kidney extracts prior to incubation of the extracts with agarose-coupled GST-c-Jun(1-135), it was found that SAPKs accounted for the majority of the amino-terminal c-Jun kinase activity of kidney at 5 min following reperfusion. In Madin-Darby canine kidney epithelial cells, ATP repletion, following ATP depletion induced by chemical anoxia, was associated with a 9-15-fold activation of SAPKs with a similar time course of activation to that seen in the kidney after ischemia and reperfusion. In conclusion, the SAPKs are markedly activated very early after reperfusion of ischemic kidney and following ATP repletion of anoxic cells in culture. We propose that this activation of SAPKs may trigger part of the kidney's early genetic response to ischemia, possibly by enhancing trans acting activity of c-Jun.
介导控制器官对缺血反应的反式作用因子激活的信号转导途径尚不清楚。应激激活蛋白激酶（SAPKs）是细胞外信号调节激酶（ERKs）的一个亚家族，可在氨基末端反式激活域内磷酸化c-Jun，并在对多种细胞应激的反应中被激活。我们确定了SAPKs是否会在缺血（一种极端但常见的病理生理应激）反应中被激活。对大鼠进行40分钟的肾脏缺血，然后再灌注0、5、20或90分钟。从肾脏裂解物中免疫沉淀SAPKs，并用含有c-Jun氨基末端反式激活域的重组GST-c-Jun(1-135)作为底物测定激酶活性。单独的缺血不会激活SAPKs，但再灌注仅5分钟就会使激酶活性增加4.6倍。再灌注20分钟时激酶活性增加7.6倍，在再灌注90分钟时仍保持升高（4.9倍）。相比之下，相关的ERK-1和-2的活性仅在再灌注5分钟时增加了1.3倍。当在将肾脏提取物与琼脂糖偶联的GST-c-Jun(1-135)孵育之前从肾脏提取物中免疫去除SAPKs时，发现再灌注5分钟后，SAPKs占肾脏氨基末端c-Jun激酶活性的大部分。在Madin-Darby犬肾上皮细胞中，化学性缺氧诱导的ATP耗竭后的ATP补充与SAPKs 9-15倍的激活相关，其激活的时间进程与缺血再灌注后肾脏中观察到的相似。总之，缺血肾脏再灌注后以及培养的缺氧细胞ATP补充后，SAPKs会在很早的时候就被显著激活。我们提出，SAPKs的这种激活可能会触发肾脏对缺血的早期基因反应的一部分，可能是通过增强c-Jun的反式作用活性来实现的。
