Meier R, Rouse J, Cuenda A, Nebreda A R, Cohen P
MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Scotland.
Eur J Biochem. 1996 Mar 15;236(3):796-805. doi: 10.1111/j.1432-1033.1996.00796.x.
The identities of the upstream activators of the mitogen-activated protein (MAP) kinase homologues termed stress-activated-protein (SAP) kinase-1 (also known as JNK or SAPK) and SAP kinase-2 (also known as p38, RK and CSBP) were investigated in rat PC12 cells and human KB cells after exposure to cellular stresses and cytokines. In PC12 cells, the same two upstream activators, SAP kinase kinase-1 (SAPKK-1) and SAPKK-2 were activated after exposure to osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor anisomycin, and more weakly in response to sodium arsenite. SAPKK-1 was capable of activating both SAP kinase-1 and SAP kinase-2 and was similar, if not identical, to the previously described MAP kinase kinase homologue MKK4, as judged by immunological criteria and by its ability to be activated by MEK kinase in vitro. In contrast, SAPKK-2 activated SAP kinase-2, but not SAP kinase-1 in vitro. In KB cells, five distinct upstream activators of SAP kinase-1 and SAP kinase-2 were induced, namely SAPKK-1, SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, whose appearance depended on the nature of the stimulus. SAPKK-3, which was strongly induced by every stimulus tested (osmotic shock, ultraviolet irradiation, anisomycin or IL-1), accounted for about 95% of the SAP kinase-2 activator activity in these cells, did not activate SAP kinase-1 and eluted from Mono S at a lower salt concentration than SAPKK-2. SAPKK-4 and SAPKK-5 were also eluted from Mono S at higher NaC1 concentrations than SAPKK-3 and these enzymes activated SAP kinase-1 but not SAP kinase-2. SAPKK-4 was the only SAP kinase-1 activator induced by interleukin-1 or ultraviolet irradiation, while two SAP kinase-1 activators, SAPKK-1 and SAPKK-5, were induced by osmotic shock or anisomycin. SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, were not activated by MEK kinase in vitro, were separable from the major activator(s) of p42 MAP kinase, and were not recognised by anti-MKK4 antibodies. At least two of these enzymes are likely to be novel MAP kinase kinase homologues. Our results demonstrate unexpected complexity in the upstream regulation of stress and cytokine-stimulated kinase cascades and indicate that the selection of the appropriate SAPKK varies with both the stimulus and the cell type.
在大鼠PC12细胞和人KB细胞中，在暴露于细胞应激和细胞因子后，对被称为应激激活蛋白（SAP）激酶-1（也称为JNK或SAPK）和SAP激酶-2（也称为p38、RK和CSBP）的丝裂原活化蛋白（MAP）激酶同系物的上游激活剂的身份进行了研究。在PC12细胞中，在暴露于渗透压休克、紫外线照射或蛋白质合成抑制剂茴香霉素后，相同的两种上游激活剂，即SAP激酶激酶-1（SAPKK-1）和SAPKK-2被激活，而对亚砷酸钠的反应较弱。SAPKK-1能够激活SAP激酶-1和SAP激酶-2，并且从免疫学标准及其在体外被MEK激酶激活的能力判断，它与先前描述的MAP激酶激酶同系物MKK4相似（如果不是相同的话）。相反，SAPKK-2在体外激活SAP激酶-2，但不激活SAP激酶-1。在KB细胞中，诱导出了五种不同的SAP激酶-1和SAP激酶-2的上游激活剂，即SAPKK-1、SAPKK-2、SAPKK-3、SAPKK-4和SAPKK-5，它们的出现取决于刺激的性质。SAPKK-3在每种测试刺激（渗透压休克、紫外线照射、茴香霉素或IL-1）下都被强烈诱导，在这些细胞中占SAP激酶-2激活剂活性的约95%，不激活SAP激酶-1，并且在比SAPKK-2更低的盐浓度下从Mono S柱上洗脱。SAPKK-4和SAPKK-5也在比SAPKK-3更高的NaCl浓度下从Mono S柱上洗脱，并且这些酶激活SAP激酶-1但不激活SAP激酶-2。SAPKK-4是白细胞介素-1或紫外线照射诱导的唯一的SAP激酶-1激活剂，而两种SAP激酶-1激活剂，即SAPKK-1和SAPKK-5，是由渗透压休克或茴香霉素诱导的。SAPKK-2、SAPKK-3、SAPKK-4和SAPKK-5在体外不被MEK激酶激活，可与p42 MAP激酶的主要激活剂分离，并且不被抗MKK4抗体识别。这些酶中至少有两种可能是新的MAP激酶激酶同系物。我们的结果证明了应激和细胞因子刺激的激酶级联反应上游调节中意想不到的复杂性，并表明合适的SAPKK的选择随刺激和细胞类型而变化。
