Figure 5.

Cdc2 inhibits the DNA-binding activity of bacterially produced Ste11. (A) Purified GST-Ste11 (10–100 ng) was either directly assayed by EMSA for DNA-binding activity or was first incubated with either fission yeast Cdc2 immune complexes or with 2.5 ng (50 U) of recombinant human Cdc2–Cyclin B₁ complex (New England Biolabs) in the presence of ATP. (B) Ten nanograms of GST-Ste11 and GST-Ste11^T82A were either directly assayed by EMSA for DNA-binding activity or were first incubated with ATP and Cdc2 immunoprecipitated from fission yeast cells. (C) Twenty-five nanograms of GST-Ste11 and GST-Ste11^T82A were either directly assayed by EMSA for DNA-binding activity or were first incubated with 0.5 ng of human Cdc2–Cyclin B₁ complex in the presence or the absence of ATP or AMP-PNP. In addition, lanes 9 + 11 and lanes 10 + 12 contained anti-Cyclin B₁ and anti-Ste11 antibodies, respectively. (D) In a similar assay, the effect of adding Protein Phosphatase 1 (Pp1) to Cdc2–Cyclin B₁-phosphorylated GST-Ste11 was examined (cf. lanes 4 and 6). In addition the DNA-binding activity of Cdc2–Cyclin B₁ was assayed in the absence of GST-Ste11 (lanes 7 + 8). (E) In vitro kinase assay. Protein kinase activities of recombinant human Cdc2–Cyclin B₁ were measured using histone H1 (H1), wild-type Ste11_1–113 (Ste11), and Ste11^T82A_1–113 (Ste11^T82A) as substrates. Kinase assays were carried out for 15 min at 30°C, and the samples were separated by SDS-PAGE, followed by autoradiography and Coomassie staining.