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

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(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