FIGURE 5:

Mutating Tyr280/Tyr-261 in Erk1/2 causes loss-of-function effects on their biological properties in mammalian cells. (A, B) pCEFL vectors carrying cDNAs encoding the indicated Erk1 (A) or Erk2 (B) molecules, a control empty vector, or vectors encoding Ha-Ras(V12) or Mek(EE) were introduced into HEK293 cells. At 48 h posttransfection, cells were exposed to 50 ng/ml EGF for 10 min and harvested. Protein lysates prepared from these cells were analyzed by Western blot analysis, using antibodies that specifically recognize phospho-Erk or the relevant tag (His-Erk1 or HA-Erk2). (C) NIH3T3 cells transfected with the indicated vectors were allowed to grow in the presence of G-418 to high density and reach confluence. Continuation of proliferation and appearance of foci was monitored. Cells were fixed and stained with crystal violet 4 wk after transfection. We estimate that the following number of foci appear when 1 μg of plasmid is introduced into the cells: ∼500 foci with a plasmid carrying Ha-ras(V12), ∼60 with a plasmid carrying ERK1(R84S), ∼10 with a plasmid carrying MEK(EE), and 0 with plasmids carrying ERK1(R84S + Y280C) or ERK1(R84S + Y280A).