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，特别是对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，我们使用了高表达ERK2的MDCK细胞，因为它们对HGF有显著的运动反应。HGF刺激细胞不仅可引起ERK1/2的快速、显著和持续的激活和核的快速堆积，而且可延长激活的ERK1/2的核滞留。在HGF刺激后30min用PD98059阻断ERK1/2的激活，可使HGF诱导的细胞运动消失。通过表达失活形式的MKP-3胞浆磷酸酶，增强激活的ERK1/2的胞浆滞留，抑制HGF诱导的细胞运动。尽管表皮生长因子刺激导致ERK1/2的快速、显著和持续的激活以及t…的快速核积聚更重要的是，它不能诱导激活的ERK1/2长时间的核滞留；EGF不能诱导细胞的运动性反应。这些结果表明，ERK1/2在细胞核中的持续活动是诱导细胞运动性反应所必需的。在细胞核中，被激活的ERK1/2被认为持续磷酸化Elk-1，导致c-fos的延长表达，最终导致基质金属蛋白酶-3/-9/-14等基因的表达，这些表达的MMPs的活性是诱导细胞运动反应所必需的。(2)我们研究了ERK激活、MMP-9表达与人类肿瘤细胞侵袭性表型的可能相关性。ERK通路在肿瘤细胞中的激活状态与肿瘤的侵袭表型密切相关，侵袭表型由细胞通过重组细胞外基质侵袭的能力决定。在检测到ERK通路的结构性激活的肿瘤细胞中，可以观察到MMP9以及MMP3、MMP14和CD44的高表达。丝裂原活化蛋白激酶/丝裂原活化蛋白激酶的特异性和强效抑制剂PD 184352阻断ERK通路，抑制MMP3、MMP9、MMP14和CD44的表达，显著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途径有望在肿瘤细胞中产生抗转移作用。(3)我们研究了Sprouty蛋白抑制RTK/ERK途径的分子机制，特别是Sprouty亚型之间的协同作用。这四种哺乳动物的Sprouty亚型通过它们的C-末端结构域相互形成同源/异源低聚物：异源寡聚不仅在过度表达外源Sprouty异构体的293T细胞中观察到，而且在成纤维细胞生长因子-2刺激的瑞士3T3细胞中也观察到。Sprouty1与Grb2特异地相互作用，而Sprouty4与SOSL相互作用。虽然任何Sprouty亚型本身都能显著抑制成纤维细胞生长因子-2诱导的ERK通路的激活，但杂寡体表现出更明显的抑制活性。Sprouty1和Sprouty4之间形成的杂寡体通过高效地抑制Grb2-Sos1复合体与FRS2的结合，对ERK的激活表现出最强的抑制作用。观察到的Sprouty亚型之间的协同作用可能代表了一种高级系统，其功能是严格调控哺乳动物细胞中RTK/ERK通路的激活状态。较少

(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