Figure 1

Effects of MEK1 and MEK2 mutations on their intrinsic kinase activity. (A) Intrinsic kinase activity of WT-MEK1/2 and their Q56/60 P and Y130/134 C mutants. Cos7 cells were transfected with the indicated GFP constructs, and 24 hours later the cells were serum starved for 14 hours after which the cells were harvested in Buffer H, sonicated (2 × 7 sec) and centrifuged, and the extracts were then subjected anion exchange mini-colons. Then the amount of protein was determined using anti pMEK and gMEK Abs, and the amount of phosphorylated MEK was equalized by adjusting the small amounts of phosphorylated MEK intensity (final amounts are shown in A bottom panels). This was done in order to follow changes in the intrinsic activity without the influence of the activatory phosphorylations. The partially purified MEKs were then used for in-vitro kinase assay using GST-ERK2 as a substrate (30 °C indicated times). The reaction was performed for 0, 5 and 10 min, after which the reaction was terminated with SB and boiling, and the sample were subjected to western blotting with anti-pERK and gERK Abs (A top panels). (B) Quantification of the results in (A). The catalytic activity of MEK was determined as the intensity of the ERK bands per min. The bar-graphs are averages and standard errors of 3 distinct experiments. *P < 0.01 according to student’s t-test. (C) In vitro ERK phosphorylation by IPed MEKs and their mutants. Cos7 cells were transfected with the indicated GFP constructs for 24 hours, serum starved for 14 hours and then the cells were harvested in Buffer H, sonicated (2 × 7 sec) and centrifuged. The MEKs and the mutants were IPed with anti GFP Ab, washed extensively, and then subjected to in vitro kinase assay using GST-ERK2 as a substrate (30 °C, indicated times). The reaction was terminated and the sample were subjected to western blotting with anti-pERK gERK, pMEK and gMEK Abs.