Figure 4. Kinase cdc2p (Cdk1) and its antagonist phosphatase clp1p (Cdc14) regulate the phosphorylation states of klp9p and ase1p.

(A) Time-lapsed images of cells expressing either cdc13p-GFP (cyclin B, binding partner of cdc2p) or clp1p-GFP during mitosis. Prior to anaphase B, cdc13p-GFP localizes to the spindle. At anaphase B onset (yellow arrow head), cdc13p-GFP delocalizes from the spindle (yellow dashed oval). In contrast, prior to anaphase B, clp1p-GFP localizes to the spindle poles and nucleolus. At anaphase B onset (yellow arrow head), clp1p-GFP localizes to the spindle and subsequently the spindle midzone (yellow dashed oval). Bar, 5 μm.

(B) Klp9p and ase1p are phosphorylated by cdc2p in vitro. Recombinant His-klp9p and His-klp9p^{phosphoinhibit} (His-klp9p^S>A) and His-ase1p and His-ase1p^{phosphoinhibit} (His-ase1p^S>A) were incubated with cdc2p.

(C) Klp9p and ase1p are dephosphorylated by clp1p in vitro. Recombinant GST-klp9p and GST-ase1p was first phosphorylated by cdc2p. The products were next incubated with recombinant MBP-clp1p. (Asterisk * corresponds to MBP-clp1p)

(D) Ase1-phosphoinhibit version ase1p^S>A cannot enter the nucleus during closed mitosis. Shown are cells expressing mCherry-atb2 (tubulin) and GFP-tagged ase1^wt, ase1^{phosphoinhibit} (ase1^S>A), or ase1^phosphomimic (ase1^S>D). At mitosis ase1^wt and ase1^S>D localize to the spindle midzone. In contrast, ase1^S>A is not strongly visible at the spindle midzone (yellow dashed circle). Bar, 5 μm.

(E) Dual GFP- and NLS-tagged versions of ase1^wt, ase1^{phosphoinhibit} (ase1^S>A), or ase1^phosphomimic (ase1^S>D). Prominent is the strong signal of ase1^phosphomimic (ase1^S>D), which now can enter the nucleus. Arrow heads mark the spindle poles. Bar, 5 μm.