Schwertfeger K L, Hunter S, Heasley L E, Levresse V, Leon R P, DeGregori J, Anderson S M
Department of Pathology, University of Colorado Health Sciences Center, Denver 80262, USA.
Mol Endocrinol. 2000 Oct;14(10):1592-602. doi: 10.1210/mend.14.10.0536.
In recent years the mitogen-activated protein (MAP) kinase family has expanded to include both c-jun N-terminal kinases (JNKs), and the p38/HOG1 family in addition to the extracellular regulated kinase (ERK) family. These kinases are activated by a variety of growth factors, as well as extra- and intracellular insults such as osmotic stress, UV light, and chemotherapeutic agents. Stimulation of the PRL-dependent Nb2 cell line with PRL results in the rapid activation of JNK as determined by the glutathione-S-transferase (GST)-jun kinase assay. Activation was maximal 30 min after stimulation with 50 nM rat PRL (rPRL) and decreased after that time. Dose response studies indicated that concentrations as low as 10 nM rPRL resulted in maximal activation. The interleukin-3 (IL-3)-dependent myeloid progenitor cell line 32Dcl3 was transfected with the long, Nb2, and short forms of the rat PRL receptor (rPRLR), as well as the long form of the human PRLR (hPRLR). The long and Nb2 forms of the PRLR were able to stimulate activation of JNK; however, the short form of the rPRLR was not. This corresponds with the inability of the short form of the rPRLR to stimulate proliferation of 32Dcl3 cells. Activation of JNK in 32Dcl3 cells expressing the long form of the hPRLR was maximal at 30 min after stimulation with 100 nM ovine PRL (oPRL) and declined after that time. Dose response studies indicated that activation of JNK was maximal after 30 min at a concentration of 10 nM, and the amount of activated JNK declined at the highest concentration of oPRL, 100 nM. Immunoblot analysis with an antibody that recognizes the activated (phosphorylated) forms of JNK1 and JNK2 indicated that both JNK1 and JNK2 isoforms were activated in 32D/hPRLR cells stimulated with oPRL. A recombinant human adenovirus expressing a kinase-inactive mutant of JNK1 (APF mutant) was used to determine the biological effect of blocking JNK activity in Nb2 cells. Expression of the JNK1-APF mutant inhibited cellular proliferation and induced DNA fragmentation typical of cells undergoing apoptosis. These data suggest that activation of JNKs may be important in mitogenic signaling and/or suppression of apoptosis in Nb2 cells.
近年来，丝裂原活化蛋白（MAP）激酶家族不断扩展，除细胞外调节激酶（ERK）家族外，还包括c-jun氨基末端激酶（JNKs）和p38/HOG1家族。这些激酶可被多种生长因子以及细胞外和细胞内刺激激活，如渗透压应激、紫外线和化疗药物。用催乳素（PRL）刺激PRL依赖的Nb2细胞系，通过谷胱甘肽-S-转移酶（GST）-jun激酶测定法可确定JNK迅速被激活。用50 nM大鼠PRL（rPRL）刺激后30分钟激活达到最大值，之后下降。剂量反应研究表明，低至10 nM的rPRL浓度即可导致最大激活。将白细胞介素-3（IL-3）依赖的髓系祖细胞系32Dcl3转染大鼠PRL受体（rPRLR）的长、Nb2和短形式，以及人PRLR（hPRLR）的长形式。PRLR的长形式和Nb2形式能够刺激JNK激活；然而，rPRLR的短形式则不能。这与rPRLR短形式无法刺激32Dcl3细胞增殖相一致。在用100 nM绵羊PRL（oPRL）刺激后30分钟，表达hPRLR长形式的32Dcl3细胞中JNK激活达到最大值，之后下降。剂量反应研究表明，在10 nM浓度下30分钟后JNK激活达到最大值，在最高浓度100 nM的oPRL时，激活的JNK量下降。用识别JNK1和JNK2激活（磷酸化）形式的抗体进行免疫印迹分析表明，在oPRL刺激的32D/hPRLR细胞中，JNK1和JNK2亚型均被激活。使用表达JNK1激酶失活突变体（APF突变体）的重组人腺病毒来确定阻断Nb2细胞中JNK活性的生物学效应。JNK1-APF突变体的表达抑制细胞增殖并诱导典型的凋亡细胞DNA片段化。这些数据表明，JNKs的激活在Nb2细胞的有丝分裂信号传导和/或凋亡抑制中可能很重要。
