Exo84 is phosphorylated by Cdk1 at M phase. (A) Exo84 is phosphorylated in a Cdk1-dependent fashion in vivo. Cells expressing myc-tagged Exo84 (chromosomally integrated) were grown to early log phase at 25°C. Exo84-myc was immunoprecipitated and probed with anti-myc and anti–phospho-CDK substrate antibody (α-pS) by Western blotting. Untagged cells were used as a negative control. Where designated, immunoprecipitated Exo84 was treated with λ-phosphatase to abolish phosphorylation (left). Exo84 was immunoprecipitated from cdk1-as cells that were treated with DMSO (mock) or the Cdk1-as inhibitor 1NM-PP1 (right). Phosphorylation of Exo84 is reduced dramatically in cdk1-as1 cells treated with 1NM-PP1. (B) Cell cycle–dependent phosphorylation of Exo84. Cells expressing Exo84-myc and Clb2-HA were synchronized at G1 with α-factor and then released and harvested at the indicated time points. The expression of Clb2-HA and the migration profiles of Exo84-myc and Cdc24 were examined by immunoblot analysis after the samples were separated on a Phos-tag PAGE gel that specifically retards the migration of phosphoproteins (see Materials and methods). (C) Exo84-myc was immunoprecipitated from asynchronous and cell cycle–arrested cells and then probed for Cdk1 phosphorylation by immunoblotting as in A. Where designated, samples were treated with λ-phosphatase (λ-Ppase). Wild-type cells were arrested in G1 phase with α-factor (α), S phase with hydroxyurea (HU), or M phase with nocodazole (Noc). Asynchronous cells are designated as “NT.” Immunoblots reveal that Exo84 is heavily phosphorylated in M phase–arrested cells. Error bars represent standard deviation (n = 3). *, P < 0.01. (D) Exo84-myc was immunoprecipitated from the wild-type and cdc20-1 mutant cells at the permissive or restrictive temperature, and then probed for Cdk1 phosphorylation by immunoblotting as in A. Error bars represent standard deviation (n = 3). *, P < 0.01. (E) Exo84 is phosphorylated by Cdk1 in vitro. GST and GST-Exo84 were purified from Escherichia coli and incubated with Cln2–Cdk1, Clb5–Cdk1, or Clb2–Cdk1 in the presence of γ-[³²P]ATP. Exo84 phosphorylation was detected by autoradiography (top). Corresponding Coomassie blue–stained gels are shown on the bottom. (F) In vitro Clb2-Cdk1 kinase assays with recombinant Exo84-A, which lacks the five Cdk1 phosphorylation sites. The phosphorylation of Exo84-A by mitotic Clb2–Cdk1 is barely detectable.