Janulis M, Silberman S, Ambegaokar A, Gutkind J S, Schultz R M
Department of Molecular and Cellular Biochemistry, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA.
J Biol Chem. 1999 Jan 8;274(2):801-13. doi: 10.1074/jbc.274.2.801.
Ras activates a multitude of downstream activities with roles in cellular proliferation, invasion and metastasis, differentiation, and programmed cell death. In this work we have evaluated the requirement of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase kinase (JNKK), and c-Jun/AP-1 activities in transformation and extracellular matrix invasion of ras oncogene expressing NIH 3T3 fibroblasts by expressing stable mutant genes that constitutively inhibit these activities. Whereas the inhibition of ERK activity reverts the transformed and invasive phenotype, the inhibition of the JNK pathway and AP-1 trans-activating activities by JNKK[K129R] and c-Jun(TAM67) had no effect on the ability of the ras oncogene-expressing cells to grow in soft agar or invade Matrigel basement membrane. Thus an elevated JNK activity and/or c-Jun/AP-1 trans-activating activity are not absolute requirements for ras transformation or invasion through basement membrane, and the dependence on AP-1 activity for transformation is cell-specific. However, inhibition of JNK kinase (JNKK) in ras-transformed cells with normally elevated JNK activity switches the protease-dependent invasive phenotype from a urokinase plasminogen activator (uPA)-dependent to a cathepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-transformed cells of low constitutive JNK activity with the JNK stimulator, anisomycin, converts the protease mRNA levels from those characteristic of a CL-dependent to a uPA-dependent phenotype. These protease phenotypes can be duplicated in untransformed NIH 3T3 cells that express platelet-derived growth factor receptors and m1 muscarinic receptors that selectively stimulate the ERK or JNK pathways, respectively. It is concluded that high ERK activity is required for both protease phenotypes, whereas the JNK pathway and c-Jun/AP-1 activity are not required for transformation but regulate a switch between uPA and CL protease phenotypes in both transformed and untransformed cells. In ras-transformed NIH 3T3 fibroblasts, the uPA- and CL-dependent protease phenotypes are redundant in their ability to invade through basement membrane.
Ras激活众多下游活性，在细胞增殖、侵袭和转移、分化以及程序性细胞死亡中发挥作用。在本研究中，我们通过表达组成性抑制这些活性的稳定突变基因，评估了细胞外信号调节蛋白激酶（ERK）、c-Jun NH2末端激酶激酶（JNKK）以及c-Jun/AP-1活性在表达ras癌基因的NIH 3T3成纤维细胞的转化和细胞外基质侵袭中的需求。虽然抑制ERK活性可逆转转化和侵袭表型，但JNKK[K129R]和c-Jun(TAM67)对JNK途径和AP-1反式激活活性的抑制对表达ras癌基因的细胞在软琼脂中生长或侵袭基质胶基底膜的能力没有影响。因此，升高的JNK活性和/或c-Jun/AP-1反式激活活性并非ras转化或通过基底膜侵袭的绝对必要条件，并且转化对AP-1活性的依赖性具有细胞特异性。然而，在JNK活性正常升高的ras转化细胞中抑制JNK激酶（JNKK），可将蛋白酶依赖性侵袭表型从尿激酶型纤溶酶原激活剂（uPA）依赖性转变为组织蛋白酶L（CL）依赖性侵袭表型。相反，用JNK刺激剂茴香霉素处理组成性JNK活性低的ras转化细胞，可使蛋白酶mRNA水平从CL依赖性特征转变为uPA依赖性表型。这些蛋白酶表型可在表达血小板衍生生长因子受体和m1毒蕈碱受体的未转化NIH 3T3细胞中重现，这两种受体分别选择性刺激ERK或JNK途径。得出的结论是，两种蛋白酶表型都需要高ERK活性，而JNK途径和c-Jun/AP-1活性在转化中并非必需，但在转化和未转化细胞中调节uPA和CL蛋白酶表型之间的转换。在ras转化的NIH 3T3成纤维细胞中，uPA和CL依赖性蛋白酶表型在通过基底膜侵袭的能力上是冗余的。
