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 mit ogenic stimulation. The molecular events downstream of Mek and Erk nec essary to promote cell cycle entry are largely undefined. In order to study signals emanating from Mek independent of upstream proteins capa ble of activating multiple sig naling pathways, we fused the hormone-b inding domain of the estrogen receptor (ER) to the C terminus of const itutively activated Mek1 phosphorylation site mutants. Although 4-OH-t amoxifen stimulation of NIH-3T3 cells expressing constitutively activa ted Mek-ER resulted in only a small increase in specific activity of t he 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 fusi on protein. Induction of constitutively activated Mek-ER in NIH-3T3 ce lls resulted in accelerated S phase entry, proliferation in low serum, morphological transformation, and anchorage independent growth, Endog enous Erk1 and Erk2 were phosphorylated with kinetics similar to the e levation of Mek-ER activity. However, elevated Mek-ER activity attenua ted subsequent stimulation of Erk1 and Erk2 by serum. 4-OH-tamoxifen s timulation of Mek-ER-expressing fibroblasts also resulted in up-regula tion of cyclin D1 expression and down-regulation of p27(Kip1) expressi on, establishing a direct link between Mek1 and the cell cycle machine ry.