The polo-box-dependent induction of ectopic septal structures by a mammalian polo kinase, plk, in Saccharomyces cerevisiae

Abstract

Members of the polo subfamily of protein kinases play pivotal roles in cell-cycle control and proliferation. In addition to a high degree of sequence similarity in the kinase domain, polo kinases contain a strikingly conserved motif termed "polo-box" in the noncatalytic C-terminal domain. We have previously shown that the mammalian polo-like kinase Plk is a functional homolog of Saccharomyces cerevisiae Cdc5. Here, we show that, in a polo-box- and kinase activity-dependent manner, ectopic expression of Plk in budding yeast can induce a class of cells with abnormally elongated buds. In addition to localization at spindle poles and cytokinetic neck filaments, Plk induces and localizes to ectopic septin ring structures within the elongated buds. In contrast, mutations in the polo-box abolish both localization to, and induction of, septal structures. Consistent with the polo-box-dependent subcellular localization, the C-terminal domain of Plk, but not its polo-box mutant, is sufficient for subcellular localization. Our data suggest that Plk may contribute a signal to initiate or promote cytokinetic event(s) and that an intact polo-box is required for regulation of these cellular processes.

Figure 1

Coexpression of wild-type or mutant forms of Plk with GST-Cdc10 in the diploid wild-type S. cerevisiae strain 1788 (isogenic diploid of EG123). (A) Wild-type and mutant forms of Plk were expressed either alone or with GST-Cdc10. Total cellular protein (200 μg of each) was analyzed for HA-Plk expression with anti-Plk Ab (Upper), or for GST-Cdc10 expression with anti-GST Ab (Lower). (B) Total cell extracts were prepared and clarified by centrifugation at 15,000 × g for 30 min. An equal amount (500 μg) of the resulting supernatants (S15) was subjected to immune complex kinase assays with an anti-Plk Ab. Levels of immunoprecipitated HA-Plk were detected with an anti-HA Ab (Upper). HA-Plk kinase activity was measured, using casein as the in vitro substrate (Lower). K82M, YCplac111-GAL1-HA-PlkK82M; WT, YCplac111-GAL1-HA-Plk; W414F, YCplac111-GAL1-HA-PlkW414F; T210D, YCplac111-GAL1-HA-PlkT210D; T210D/W414F, YCplac111-GAL1-HA-PlkT210D/W414F; GST-Cdc10, YCplac33-GAL1-GST-CDC10.

Figure 2

Induction of additional septal structures and actin polarization by expression of an activated form of Plk, PlkT210D, in a diploid wild-type strain, 1788. To visualize localization of Plk at additional septal structures, YCplac111-GAL1-HA-EGFP-PlkT210D and YCplac33-GAL1-Cdc10 were cotransformed to increase the population of cells with abnormally elongated buds (see Table 1). Transformants were cultured for subsequent stainings to examine Cdc10, tubulin, and actin localization. (A) Plk (green) and Cdc10 (red) colocalize at the neck filaments and additional septal structures. Septin rings (red) are viewed edge on and therefore appear as lines. (B) Plk (green) localizes at the spindle poles. Spindles were visualized by microtubule staining (red). It is apparent that Plk is present at both ends of spindle structures. (C) Plk (green) induces actin accumulation (red) at the additional septal structures. DIC, differential interference contrast; Plk, EGFP-Plk expression; Cdc10, immunostaining with an anti-Cdc10 Ab. Superimposed images are shown as Plk + Cdc10, Plk + Tubulin, and Plk + Actin. (Bar, 5 μm.)

Figure 3

The C-terminal domain of Plk (PlkΔN) is sufficient to localize at spindle poles and cytokinetic neck filaments. The W414F/L427A (FA) mutations in the polo-box abolish localization of the PlkΔN at these sites. To localize wild-type and mutant forms of the C-terminal domain of Plk in a diploid wild-type strain, 1788, EGFP-PlkΔN and EGFP-PlkΔN/FA fusion constructs were generated and expressed under the control of the GAL1 promoter. Transformants expressing EGFP fusion constructs were stained with propidium iodide to visualize chromosomal DNA and examined by confocal microscopy. PlkΔN, YCplac111-GAL1-HA-EGFP-PlkΔN; PlkΔN/FA, YCplac111-GAL1-HA-EGFP-PlkΔN/W414F/ L427A; Control, YCplac111-GAL1-HA-EGFP. DIC, differential interference contrast; PlkΔN + PI, EGFP-PlkΔN and propidium iodide images superimposed. (Bar, 5 μm.)

Figure 4

Localization of the C-terminal domain of Plk at spindle poles and neck filaments. For localization of PlkΔN, transformants were cultured under the induction conditions for 2 hr and prepared for subsequent immunostaining to examine Cdc10 and tubulin. (A) EGFP-PlkΔN localizes at the spindle poles. Spindles were visualized by microtubule staining (red). (B) Plk (green) colocalizes with Cdc10 (red) at the neck filaments. Occasionally EGFP-PlkΔN ring structure was apparent (Right). DIC, differential interference contrast; PlkΔN, EGFP-PlkΔN expression. Superimposed images are shown as PlkΔN + Tubulin and PlkΔN + Cdc10. (Bar, 5 μm.)

Figure 5

The FA mutations in the polo-box do not significantly influence the expression level of PlkΔN. The wild-type 1788 cells bearing various YCplac22-GAL1-HA-EGFP-PlkΔN constructs were cultured under inducing conditions for 2 hr and then harvested. An equal amount (200 μg each) of total cellular lysates prepared from various transformants was loaded onto each lane. After the proteins were transferred onto a poly(vinylidene difluoride) membrane, proteins interacting with the anti-GFP Ab were detected by immunoblotting. EGFP, YCplac22-GAL1-HA-EGFP; EGFP-PlkΔN, YCplac22-GAL1-HA-EGFP-PlkΔN; EGFP-PlkΔN/FA, YCplac22-GAL1-HA-EGFP-PlkΔN/W414F/L427A. Although the expression level of the FA mutant induced for 10 hr becomes greater than that of wild type induced for 2 hr, it failed to yield distinctly localized signals (see text).