Figure 7. The strength and duration of ERK activation is determined by MEK and PP2A-B56γ1 activities.

(A) The activity of PP2A-B56γ1 phosphatase measured by immune complex fluorometric phosphatase assay using 4-methylumbelliferyl phosphate as a substrate. The closed squares indicate the measured phosphatase activity. Two areas demarcated by dotted lines (“transient phosphorylation” and “IER3”) were deduced from the effect of two distinct factors, i.e., the rapid inactivation of phosphatase activity (probably due to phosphorylation of the C subunit of PP2A) and the later PP2A inactivation reaction by IER3. C subunits of PP2A in the immune complex were detected by western blotting using anti-C subunit antibodies. Since faint blot at 3 min shows non-specific binding to Sepharose beads, densities of non-immune controls (C) and of 3 min are being compared. (B) The kinetics of ERK and MEK activities in Calu1 cells were examined by western blotting. The plot was constructed using data from 3 (0 to 15 min) or 9 (0, 15 to 60 min) repeated experiments.