Figure 3.

Cpp1p dephosphorylates and inactivates MAP kinase in vitro. (A) Cpp1p inhibits the MBP kinase activity of Erk1. Purified active GST-Erk1 on beads was incubated with indicated concentrations of GST-Cpp1p. Following incubation, the beads were washed and assayed for MBP kinase activity. (B) Cpp1p dephosphorylates Erk1. Purified active GST-Erk1 was incubated in the absence or presence of indicated concentrations of GST-Cpp1p or GST, or with 100 μg/ml GST-Cpp1p in the presence of the phosphatase inhibitors sodium vanadate and zinc chloride. Proteins were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine antibody. (C) Cpp1p specifically dephosphorylates Erk1 on tyrosine. Erk1 was immunoprecipitated from ³²P-labeled serum-stimulated Rat 1 cells and incubated in the absence (control) or presence (Cpp1p) of soluble GST-Cpp1p. Two-dimensional phosphoamino acid analysis of the ³²P-labeled Erk1 is shown. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine.