Enslen H, Tokumitsu H, Stork P J, Davis R J, Soderling T R
Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10803-8. doi: 10.1073/pnas.93.20.10803.
Membrane depolarization of NG108 cells gives rapid (< 5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK). To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively). In the absence of depolarization, CaM-KKc transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were co-transfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38). The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays. We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases. Mutation of the phosphorylation site (Thr196) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade. These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription.
NG108细胞的膜去极化可快速（<5分钟）激活钙/钙调蛋白依赖性蛋白激酶IV（CaM-KIV）以及c-Jun氨基末端激酶（JNK）。为了研究丝裂原活化蛋白激酶（ERK、JNK和p38）的钙依赖性激活是否可能由CaM激酶级联介导，我们用CaM激酶激酶和/或CaM-KIV（分别为CaM-KKc和CaM-KIVc）的组成型活性突变体转染了缺乏CaM-KIV的PC12细胞。在没有去极化的情况下，CaM-KKc转染对荧光素酶报告基因的Elk依赖性转录没有影响，而单独的CaM-KIVc或与CaM-KKc联合使用分别产生7至10倍和60至80倍的刺激，这被丝裂原活化蛋白（MAP）激酶磷酸酶共转染所阻断。当MAP激酶的表位标签构建体与CaM-KKc加CaM-KIVc共转染时，免疫沉淀的MAP激酶被激活2倍（ERK-2）和7至10倍（JNK-1和p38）。使用特异性的c-Jun或ATF2依赖性转录测定法进一步研究了JNK和p38途径。我们发现，CaM-KIVc使c-Jun/ATF2依赖性转录增强7至10倍，CaM-KKc加CaM-KIVc使其增强20至30倍。在Jun依赖性转录的情况下，这种效应不是由于活化的CaM-KIV直接磷酸化c-Jun，因为转录被显性负性JNK和两种MAP激酶磷酸酶所阻断。CaM-KIV中介导其被CaM-KIV激酶激活作用的磷酸化位点（Thr196）发生突变，可阻止CaM激酶级联对Elk-1、c-Jun和ATF2的激活。这些结果建立了一种新的钙依赖性机制来调节MAP激酶途径及随后的转录。
