Role of MAP Kinase Cascade in the Regulation of Diverse Cellular Functions

(1)我们研究了肝细胞生长因子(HGF)诱导细胞运动反应的信号通路，特别关注细胞核内细胞外信号调节激酶(ERK)活性的可能要求。为了进行分析，我们利用过度表达 ERK2 的 MDCK 细胞，因为它们对 HGF 具有显着的运动反应。 HGF 对细胞的刺激不仅诱导 ERK1/2 快速、显着和持续的激活及其快速核积累，而且还诱导激活的 ERK1/2 的长期核保留。 HGF 刺激后 30 分钟，通过 PD98059 处理细胞来中断 ERK1/2 激活，导致 HGF 诱导的细胞运动消失。通过表达无活性形式的 MKP-3 细​​胞质磷酸酶，强制活化的 ERK1/2 保留在细胞质中，抑制 HGF 诱导的细胞运动。虽然 EGF 对细胞的刺激会诱导 ERK1/2 快速、显着和持续的激活以及细胞核的快速积累，但它不会诱导激活的 ERK1/2 的长期核保留；EGF 无法诱导细胞运动反应。这些结果表明，细胞核中 ERK1/2 的持续活性是诱导细胞运动反应所必需的。在细胞核中，激活的ERK1/2被认为持续磷酸化Elk-1，导致c-fos表达延长，最终导致基质金属蛋白酶(mmp)-3/-9/-14等多个基因的表达；这些表达的MMP的活性是诱导细胞运动反应所必需的。(2)我们研究了人肿瘤细胞中ERK激活、MMP-9表达和侵袭表型之间可能的相关性。肿瘤细胞中ERK通路的激活状态与侵袭表型密切相关，侵袭表型由细胞通过重建的细胞外基质侵袭的能力决定。在检测到 ERK 通路组成型激活的肿瘤细胞中，观察到 MMP-9 以及 MMP-3、MMP-14 和 CD44 表达升高。丝裂原激活蛋白(MAP)激酶/ERK激酶(MEK)特异强效抑制剂PD 184352通过阻断ERK通路，抑制MMP-3、MMP-9、MMP-14和CD44的表达，显着抑制肿瘤细胞的侵袭性。这些结果表明，除了抗增殖作用外，特异性阻断 ERK 通路有望在肿瘤细胞中产生抗转移作用。(3)我们研究了 Sprouty 蛋白对 RTK/ERK 通路产生抑制作用的分子机制，特别关注 Sprouty 亚型之间的合作。四种哺乳动物 Sprouty 亚型通过其 C 端结构域彼此形成同源/异源寡聚体：异源寡聚化不仅在过表达外源 Sprouty 亚型的 293T 细胞中观察到，而且在用成纤维细胞生长因子 (FGF)-2 刺激的 Swiss 3T3 细胞中也观察到。 Sprouty1 专门与 Grb2 相互作用，而 Sprouty4 与 Sosl 相互作用。尽管任何 Sprouty 异构体本身都会显着抑制 FGF-2 诱导的 ERK 通路激活，但异源寡聚物表现出更明显的抑制活性。 Sprouty1 和 Sprouty4 之间形成的异源寡聚物通过其高效抑制 Grb2-Sos1 复合物与 FRS2 结合的能力，对 ERK 激活表现出最有效的抑制作用。在 Sprouty 同种型之间观察到的协同相互作用可能代表了一个先进的系统，该系统的功能是严格调节哺乳动物细胞中 RTK/ERK 通路的激活状态。较少的

(1)We have examined the signaling pathway of hepatocyte growth factor(HGF) to induce the cell motility response, with special focus on a possible requirement of the extracellular signal-regulated kinase(ERK) activity in the nucleus. For the analysis, we utilize MDCK cells over-expressing ERK2 because of their prominent motility response to HGF. HGF stimulation of the cells induces not only a rapid, marked and sustained activation of ERK1/2 and a rapid nuclear accumulation of them but also a prolonged nuclear retention of the activated ERK1/2. Interruption of the ERK1/2 activation by PD98059-treatment of the cells 30 min after HGF-stimulation results in the abolishment of HGF-induced cell motility. Enforced cytoplasmic retention of the activated ERK1/2 by expressing an inactive form of MKP-3 cytoplasmic phosphatase inhibits the HGF-induced cell motility. Although EGF stimulation of the cells induces a rapid, marked and sustained activation of ERK1/2 and a rapid nuclear accumulation of t … More hem, it does not induce the prolonged nuclear retention of the activated ERK1/2;EGF fails to induce the cell motility response. These results suggest that sustained activity of ERK1/2 in the nucleus is required for the induction of cell motility response. In the nucleus, the activated ERK1/2 are suggested to continuously phosphorylate Elk-1 leading to the prolonged expression of c-fos, which finally results in the expression of several genes such as matrix metalloproteinase (mmp)-3/-9/-14;the activities of such expressed MMPs is required for the induction of cell motility response.(2)We have examined a possible correlation between ERK activation, MMP-9 expression and invasive phenotype in human tumor cells. Activation state of the ERK pathway in tumor cells was well correlated with the invasive phenotype, which was determined by the ability of cells to invade through reconstituted extracellular matrix. Elevated expression of MMP-9 as well as of MMP-3,MMP-14 and CD44 was observed in tumor cells in which constitutive activation of the ERK pathway is detected. Blockade of the ERK pathway by treatment with PD 184352,a specific and powerful inhibitor of mitogen-activated protein(MAP) kinase/ERK kinase(MEK), suppressed the expression of MMP-3,MMP-9,MMP-14 and CD44,and inhibited markedly the invasiveness of tumor cells. These results imply that, in addition to anti-proliferative effects, specific blockade of the ERK pathway is expected to result in anti-metastatic effects in tumorcells.(3)We have examined the molecular mechanisms by which Sprouty proteins elicit their inhibitory effects on the RTK/ERK pathway, with special focus on the co-operation among Sprouty isoforms. The four mammalian Sprouty isoforms form homo-/hetero-oligomers with each other via their C-terminal domains : hetero-oligomerization is observed not only in 293T cells that overexpress exogenous Sprouty isoforms but also in Swiss 3T3 cells stimulated with fibroblast growth factor(FGF)-2. Sprouty1 specifically interacts with Grb2,whereas Sprouty4 interacts with Sosl. Although any of the Sprouty isoforms by itself inhibits the FGF-2-induced activation of the ERK pathway significantly, hetero-oligomers show a more pronounced inhibitory activity. The hetero-oligomer formed between Sprouty1 and Sprouty4 exhibits the most potent inhibitory effect on ERK activation via its highly effective ability to suppress the association of Grb2-Sos1 complex with FRS2. The cooperative interactions observed among Sprouty isoforms could represent an advanced system which functions to strictly regulate the activation state of the RTK/ERK pathway in mammalian cells. Less