FIGURE 7.

Okadaic acid inhibits dephosphorylation of cell surface receptors at Thr-353/354. Dephosphorylation of cell surface receptors was determined in CHO-sst2A cells in two ways. Panel A, cells were treated with 10 nm SS14 for 2 min. Panel B, cells were treated with 10 nm SS14 for 15 min in the presence of 0.45 m sucrose to block receptor internalization. In both experiments, cells were preincubated for 15 min without (●) or with (○) 10 nm okadaic acid prior to the SS14 stimulation. Following a rapid wash to remove agonist, cells were incubated for the times shown in fresh 37 °C medium without SS14 in the continued absence (●) or presence (○) of the phosphatase inhibitor. Cells were then fixed, and receptor phosphorylation at Thr-353/354 was measured with an in-cell ELISA. In two independent experiments the half-time for Thr-353/354 dephosphorylation was 7.2 ± 0.4 min after 2 min of SS14 stimulation and 7.1 ± 0.5 min following 15 min of SS14 stimulation in the presence of sucrose. Okadaic acid blocked Thr-353/354 dephosphorylation under both conditions.