Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome

Abstract

Early mitotic inhibitor 1 (Emi1) inhibits the activity of the anaphase promoting complex/cyclosome (APC/C), which is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. Levels of Emi1 oscillate in the cell cycle: it accumulates in the S phase and is rapidly degraded in prometaphase. The degradation of Emi1 in early mitosis is necessary for the activation of APC/C in late mitosis. Previous studies have shown that Emi1 is targeted for degradation in mitosis by a Skp1-Cullin1 F-box protein (SCF) ubiquitin ligase complex that contains the F-box protein beta-TrCP. As with other substrates of SCF(beta-TrCP), the phosphorylation of Emi1 on a DSGxxS sequence is required for this process. However, the protein kinase(s) involved has not been identified. We find that Polo-like kinase 1 (Plk1), a protein kinase that accumulates in mitosis, markedly stimulates the ligation of Emi1 to ubiquitin by purified SCF(beta-TrCP). Cdk1-cyclin B, another major mitotic protein kinase, has no influence on this process by itself but stimulates the action of Plk1 at low, physiological concentrations. Plk1 phosphorylates serine residues in the DSGxxS sequence of Emi1, as suggested by the reduced phosphorylation of a derivative in which the two serines were mutated to nonphosphorylatable amino acids. Transfection with an small interfering RNA duplex directed against Plk1 caused the accumulation of Emi1 in mitotically arrested HeLa cells. It is suggested that phosphorylation of Emi1 by Plk1 is involved in its degradation in mitosis.

Fig. 1.

Plk1 is required for the ligation of Emi1 to ubiquitin (Ub) by SCF^β-TrCP. (A) The ubiquitylation of Emi1 is stimulated by β-TrCP in extracts from M-phase cells but not from S-phase cells. The ligation of ubiquitin to wild-type (lanes 1–5) or S145N/S149N mutant (lanes 6 and 7) is shown. Emi1 was determined as described in Methods in the presence of 30 μg of protein extract from HeLa cells arrested in the S phase (lanes 2 and 3) or M phase (lanes 4–7). Where indicated, 0.5 μlof β-TrCP-Skp1 was added. (B) Plk1 stimulates the ubiquitylation of Emi1 by purified SCF^β-TrCP. Ligation of wild-type or S145/S149N mutant Emi1 to ubiquitin was determined as described in Methods in the presence of the indicated concentrations of Plk1. (C) Requirement for enzymatic activity of Plk1. The ubiquitylation of Emi1 by SCF^β-TrCP was determined in the presence of a 160 nM wild-type or catalytically inactive (N172A) mutant of Plk1, as indicated. (D) Requirement for different components of SCF^β-TrCP. Where indicated, Cul1-Roc1 or β-TrCP-Skp1 was added at the amounts specified in Methods. Plk1 was added at 160 nM, and the ligation of Emi1 to ubiquitin was assayed.

Fig. 2.

Plk1 and Cdk1-cyclin B synergistically stimulate the ubiquitylation of Emi1 by SCF^β-TrCP. (A) Synergistic effect of the two protein kinases on the ligation of Emi1 to ubiquitin (Ub). The ubiquitylation of Emi1 by SCF^β-TrCP was determined as described in Methods in the presence of the indicated concentrations of Plk1. Where indicated, 500 units of Cdk1-cyclin B were added. (B) Influence of concentrations of Plk1 on the synergistic action of Cdk1-cyclin B. Plk1 was supplemented at the indicated concentrations in the absence or presence of 500 units of Cdk1-cyclin B. The ligation of Emi1 to ubiquitin was assayed as described in Methods in the presence of 1 mM okadaic acid and was quantified by PhosphorImager analysis. Background values of Emi1 ubiquitylation in the absence of Plk1 (≈10% of Emi1 ubiquitylated) were subtracted. (C) Phosphorylation of Emi1 and not of some other component of the ubiquitylation system is required for Emi1–ubiquitin ligation. In a two-stage experiment, the first incubation contained Tris buffer, MgCl₂, DTT, ovalbumin, an ATP-regenerating system in concentrations similar to those described in Methods, and okadaic acid at 1 μM. Where indicated, 0.1 μlof ³⁵S-Emi1, 500 units of Cdk1-cyclin B, and 20 nM Plk1 were added. Where indicated, SCF^β-TrCP was added in a mixture that also included 0.1 μM E1, 10 μM UbcH5, 100 μM methylated ubiquitin, and 0.5 μM ubiquitin aldehyde. After incubation at 30°C for 30 min, 10 μM staurosporine was added to stop kinase action. Subsequently, the indicated components were added, and Emi1–ubiquitin ligation was determined after a second incubation (30°C, 30 min).

Fig. 3.

Phosphorylation of DSGxxS sequence of Emi1 by Plk1. (A) Emi1–ubiquitin (Ub) ligation is competed by the wild-type 131–160 fragment but not by the mutant fragment. Ubiquitylation of ³⁵S-Emi1 by SCF^β-TrCP was determined in the presence of the indicated concentrations of wild-type or S145/S149N mutant GST-131-160 fragment of Emi1. (B) Phosphorylation of wild-type and mutant GST-Emi1 131–160 fragments by Plk1. The Plk1-dependent incorporation of ³²P-phosphate from [γ-³²P]ATP into wild-type or S145/S149N mutant GST-Emi1 131–160 fragments was determined as described in Methods. Results were expressed in arbitrary units.

Fig. 4.

Influence of depletion of Plk1 on levels of Emi1 in mitotically arrested HeLa cells. (A) Effects of transfection with siRNA duplex directed against Plk1 on levels of Plk1, cyclin B, and Emi1 proteins. HeLa cells were either mock-treated or transfected with siRNA directed against human Plk1, as described in Methods. Where indicated, nocodazole (Noc, 0.5 μg/ml) was added 16 h before collection of cells. Cells were collected 24 h after transfection, and samples were immunoblotted with antibodies directed against Plk1 (Top), cyclin B (Middle), and C-terminal peptide of Emi1 (Bottom). (B) Specificity of anti-Emi1 antibody. Extracts from asynchronous (As) or nocodazole-arrested cells and 0.1 ng of recombinant Emi1 (Rec) were separated on 10% SDS/polyacrylamide gels, transferred to nitrocellulose, and blotted with an antibody directed against a C-terminal peptide of Emi1 (1 μg/ml) in the absence (lanes 1–3) or presence (lanes 4–6) of C-terminal peptide (0.1 μg/ml). Asterisks denote crossreacting proteins; the number on the right indicates the position of molecular mass marker protein (kDa).