An analysis of Mek1 signaling in cell proliferation and transformation.
The Mek1 dual specificity protein kinase phosphorylates and activates the mitogen-activated protein kinases Erk1 and Erk2 in response to mitogenic stimulation. The molecular events downstream of Mek and Erk necessary to promote cell cycle entry are largely undefined. In order to study signals emanating from Mek independent of upstream proteins capable of activating multiple signaling pathways, we fused the hormone-binding domain of the estrogen receptor (ER) to the C terminus of constitutively activated Mek1 phosphorylation site mutants. Although 4-OH-tamoxifen stimulation of NIH-3T3 cells expressing constitutively activated Mek-ER resulted in only a small increase in specific activity of the fusion protein, a 5-10 fold increase in total cellular Mek activity was observed over a period of 1-2 days due to an accumulation of fusion protein. Induction of constitutively activated Mek-ER in NIH-3T3 cells resulted in accelerated S phase entry, proliferation in low serum, morphological transformation, and anchorage independent growth. Endogenous Erk1 and Erk2 were phosphorylated with kinetics similar to the elevation of Mek-ER activity. However, elevated Mek-ER activity attenuated subsequent stimulation of Erk1 and Erk2 by serum. 4-OH-tamoxifen stimulation of Mek-ER-expressing fibroblasts also resulted in up-regulation of cyclin D1 expression and down-regulation of p27(Kip1) expression, establishing a direct link between Mek1 and the cell cycle machinery.
Greulich
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Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA. heidi@biosun.harvard.edu
Erikson
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RL
D016475:3T3 Cells; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D005347:Fibroblasts / Q000166:cytology; D048369:MAP Kinase Kinase 1; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction
D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases