Mutation of the polo-box disrupts localization and mitotic functions of the mammalian polo kinase Plk

Abstract

Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation. In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic domain, termed the polo-box. The function of the polo-box is currently undefined. The mammalian polo-like kinase Plk is a functional homologue of Saccharomyces cerevisiae Cdc5. Here, we show that Plk localizes at the spindle poles and cytokinetic neck filaments. Without impairing kinase activity, a conservative mutation in the polo-box disrupts the capacity of Plk to complement the defect associated with a cdc5-1 temperature-sensitive mutation and to localize to these subcellular structures. Our data provide evidence that the polo-box plays a critical role in Plk function, likely by directing its subcellular localization.

Figure 1

(A) Structures of Cdc5 and Plk. A hatched box denotes the kinase domain, and a closed box denotes the polo-box. Plk lacking the C-terminal domain (PlkΔC) has lost amino acid residues 356 to 604. (B) Identification of the cdc5–1 mutation site and diagram showing the polo-box mutants generated in Plk. Yeast genomic DNAs prepared from the cdc5–1 mutant (H5C1A1) and its parental wild-type strain (H4939–1b) (a gift of L. Hartwell, University of Washington) were used as templates to amplify full length cdc5–1 and CDC5 genes, respectively, by using the PCR. Restriction and complementation analyses confirmed the cloned genes (data not shown). DNA sequence analysis revealed a point mutation (conversion of Pro511 to Leu) in the cdc5–1 allele. Introduction of the P511L mutation into the wild-type Cdc5 was sufficient to abolish its capacity to complement the cdc5–1 defect (data not shown). Conserved amino acids among all of the polo family members known to date are in bold letters; arrows point to amino acids changed in the point mutations.

Figure 2

Mutations in either the ATP-binding site or the polo-box abolish the capacity of Plk to complement the cdc5–1 defect. A haploid cdc5–1 mutant strain, KKY921–2B (MATa cdc5–1 leu2 trp1 ura1) (12) was transformed with various YCplac111-GAL1-HA-PLK constructs or with YCplac111-CDC5. To examine the ability of Plk constructs to complement the cdc5–1 defect, transformants were cultured at 37°C in yeast extract/peptone + 2% galactose medium for 10 hr as described (19) and were subjected to flow cytometry analyses. Vector, YCplac111-GAL1; CDC5, YCplac111-CDC5; PlK, YCplac111-GAL1-HA-Plk; K82M, YCplac111-GAL1-HA-PlkK82M; PlkΔC, YCplac111-GAL1-HA-PlkΔC; W414F, YCplac111-GAL1-HA-PlkW414F. (A–F) A G1 population (1N arrow) and G2/M population (2N arrow) are indicated in the vector panel. Cells with a DNA content >2N are indicated by the 4N arrow (a broad cell population with the third 4N arrow). (G–L) The increase in forward scatter at the x axis reflects an increase in cell size. The broad, spread out pattern observed in the cdc5–1 mutant transformed with vector (G) results from a heterogeneous population of enlarged cells whereas wild-type cells (data not shown) and the cdc5–1 mutant complemented with Cdc5 (H) or Plk (I) expression produce a distinct bell-shaped pattern of forward scatter.

Figure 3

Mutations in the polo-box do not impair Plk kinase activity in vitro. The cdc5–1 (KKY921–2B) cells bearing various YCplac111-GAL1-HA-PLK constructs were cultured under inducing conditions for 10 hr and were harvested. The lysates were centrifuged at 15,000 × g for 30 min to clarify heavy cellular materials. From 500 μg of cellular proteins present in the S15 fraction, the wild-type and mutant forms of HA-Plk were immunoprecipitated and subjected to in vitro kinase assays. (Upper) HA-tagged Plk was immunoprecipitated with affinity-purified anti-Plk antibody, was electrophoresed, and was detected on membranes by Western analysis with anti-HA antibody. (Lower) HA-tagged Plk was immunoprecipitated with anti-Plk antibody, and in vitro kinase assays were performed as described by using casein as a substrate (19). Vector, YCplac111-GAL1; K82M, YCplac111-GAL1-HA-PlkK82M; WT, YCplac111-GAL1-HA-Plk; W414F, YCplac111-GAL1-HA-PlkW414F.

Figure 4

The requirement of the polo-box for Plk localization. To localize wild-type and mutant forms of Plk in a diploid wild-type strain, 1788, EGFP–Plk 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 were examined by confocal microscopy. PlkWT, YCplac111-GAL1-HA-EGFP-Plk; T210D, YCplac111-GAL1-HA-EGFP-PlkT210D; W414F, YCplac111-GAL1-HA-EGFP-PlkW414F; T210D/W414F, YCplac111-GAL1-HA-EGFP-PlkT210D/W414F; PlkΔC, YCplac111-GAL1-HA-EGFP-PlkΔC; Plk-N^1–49, YCplac111-GAL1-HA-EGFP-Plk-N^1–49; control, an irrelevant plasmid without EGFP. Plk-N^1–49 contains only the N-terminal 49-aa residues of Plk fused to EGFP and serves as a background EGFP signal. DIC, differential interference contrast; Plk, EGFP–Plk expression; PI, propidium iodide staining of nuclei; Plk + PI, EGFP–Plk and propidium iodide images superimposed. (Bar = 5 μm.)

Figure 5

Ectopically expressed Plk localizes at the spindle poles and bud neck filaments. EGFP–Plk fusion constructs were expressed under the control of the GAL1 promoter in a diploid wild-type strain, 1788. To enhance the signals present at the spindle poles and the cytokinetic septal structures, two tandem EGFPs were inserted into the N terminus of the Plk coding sequence. Transformants were cultured for subsequent immunostainings to examine Cdc10 and tubulin localizations. (A) Plk (green) and Cdc10 (red) localize at the neck filaments. Septin rings (red) are viewed edge on and therefore appear as lines. (B) Plk (green) localizes at the spindle poles. Spindles are visualized by microtubule staining (red). The spindles appear to emanate from the structures with which Plk associates. DIC, differential interference contrast; Plk, EGFP–Plk expression; Cdc10, Cdc10 staining; Tubulin, tubulin staining. Superimposed images are shown as Plk + Cdc10 and Plk + Tubulin. (Bar = 5 μm.)