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，特别关注了细胞核中细胞外信号调节激酶（ERK）活性的可能要求。为了进行分析，我们使用了过度表达ERK2的MDCK细胞，因为它们对HGF有显著的运动反应。HGF刺激细胞不仅诱导ERK1/2的快速、显著和持续的激活和它们的快速核积累，而且激活的ERK1/2的核保留时间延长。在hgf刺激后30分钟用pd98059处理细胞，阻断ERK1/2的激活，导致hgf诱导的细胞运动消失。通过表达一种无活性的MKP-3细胞质磷酸酶，使活化的ERK1/2在细胞质中保留，从而抑制hgf诱导的细胞运动。虽然EGF刺激细胞诱导ERK1/2的快速、显著和持续的激活和t的快速核积累，但它不会诱导活化的ERK1/2的长时间核保留；EGF不能诱导细胞运动反应。这些结果表明，ERK1/2在细胞核内的持续活性是诱导细胞运动反应所必需的。在细胞核中，活化的ERK1/2可能持续磷酸化Elk-1，导致c-fos的表达延长，最终导致基质金属蛋白酶(mmp)-3/-9/-14等基因的表达；这种表达的MMPs的活性是诱导细胞运动反应所必需的。(2)我们研究了人类肿瘤细胞中ERK活化、MMP-9表达与侵袭性表型之间可能存在的相关性。肿瘤细胞中ERK通路的激活状态与侵袭表型密切相关，这是由细胞通过重建的细胞外基质侵袭的能力决定的。在检测到ERK通路组成性激活的肿瘤细胞中，观察到MMP-9以及MMP-3、MMP-14和CD44的表达升高。PD 184352是一种特异性强效的丝裂原活化蛋白（MAP）激酶/ERK激酶（MEK）抑制剂，通过阻断ERK通路，抑制MMP-3、MMP-9、MMP-14和CD44的表达，显著抑制肿瘤细胞的侵袭。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通路的抑制作用的分子机制，特别关注了Sprouty亚型之间的合作。四种哺乳动物的Sprouty亚型通过其c端结构域相互形成同源/异聚物：异聚现象不仅在过表达外源Sprouty亚型的293T细胞中观察到，而且在受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。尽管任何一种Sprouty亚型本身都能显著抑制fgf -2诱导的ERK通路的激活，但异聚物寡聚物表现出更明显的抑制活性。Sprouty1和Sprouty4之间形成的异聚物通过有效抑制Grb2-Sos1复合物与FRS2的结合，对ERK活化表现出最有效的抑制作用。Sprouty亚型之间的合作相互作用可能代表了哺乳动物细胞中严格调节RTK/ERK通路激活状态的高级系统。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