Essential function of the polo box of Cdc5 in subcellular localization and induction of cytokinetic structures

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 C-terminal domain, termed the polo box. Here we show that the budding-yeast polo kinase Cdc5, when fused to green fluorescent protein and expressed under its endogenous promoter, localizes at spindle poles and the mother bud neck. Overexpression of Cdc5 can induce a class of cells with abnormally elongated buds in a polo box- and kinase activity-dependent manner. In addition to localizing at the spindle poles and cytokinetic neck filaments, Cdc5 induces and localizes to additional septin ring structures within the elongated buds. Without impairing kinase activity, conservative mutations in the polo box abolish the ability of Cdc5 to functionally complement the defect associated with a cdc5-1 temperature-sensitive mutation, to localize to the spindle poles and cytokinetic neck filaments, and to induce elongated cells with ectopic septin ring structures. Consistent with the polo box-dependent subcellular localization, the C-terminal domain of Cdc5, but not its polo box mutant, is sufficient for subcellular localization, and its overexpression appears to inhibit cytokinesis. These data provide evidence that the polo box is required to direct Cdc5 to specific subcellular locations and induce or organize cytokinetic structures.

FIG. 1

(A) Structures of the polo box in Cdc5 and Plk. Conserved amino acids in the polo box among all the members of the polo subfamily are shown in bold type. Arrows indicate amino acids changed in the point mutations in Cdc5 and Plk. (B) Either the W517F/V518A/L530A (FAA) mutations in the polo box or the N209A (NA) mutation that inactivates Cdc5 kinase activity abolishes the capacity of Cdc5 to functionally complement the cdc5-1 defect. A haploid cdc5-1 mutant strain, KKY921-2B (MATa cdc5-1 leu2 trp1 ura1) (22), was transformed with various YCplac111-Cdc5 constructs that express Cdc5 under its endogenous promoter. Transformants were selected on synthetic minimal media lacking leucine, streaked onto YEP–2% glucose, and incubated for 3 days at the indicated temperatures. Plasmids transformed are as follows (clockwise from top left): Vector, YCplac111; NA, YCplac111-cdc5N209A; WT, YCplac111-Cdc5; and FAA, YCplac111-cdc5W517F/V518A/L530A.

FIG. 2

GFP tagging of endogenous Cdc5 reveals localization of Cdc5 at the mother bud neck. Endogenous Cdc5 was C-terminally tagged with five copies of GFP proteins at the CDC5 locus of a wild-type 1783 (MATa EG123) strain (see Materials and Methods). Microscopic examination revealed that in addition to one or two fluorescent dots, one or occasionally two fluorescent bands (arrows) were present at the mother bud neck in exponentially growing cells (data not shown). The dot signals are likely to be spindle poles as reported previously (46). To enhance the signals, cells were treated with nocodazole at 15 μg/ml for 5 h and examined by confocal microscopy. DIC, differential interference contrast; Cdc5, Cdc5-GFP. Bar, 5 μm.

FIG. 3

Localization of ectopically expressed wild-type and mutant forms of Cdc5 in a diploid wild-type strain, 1788. To localize Cdc5, EGFP-Cdc5 fusion constructs were generated and expressed under the control of the GAL1 promoter for 4 h. Transformants expressing EGFP fusion constructs were stained with propidium iodide to visualize chromosomal DNA and examined by confocal microscopy. Arrows indicate rings at mother bud necks. Note that EGFP-Cdc5 forms a ring structure at the mother bud neck (NA panel). Vector, YCplac22-GAL1; NA, YCplac22-GAL1-HA-EGFP-cdc5N209A; WT, YCplac22-GAL1-HA-EGFP-Cdc5; FAA, YCplac22-GAL1-HA-EGFP-cdc5W517F/V518A/L530A. DIC, differential interference contrast; Cdc5, EGFP-Cdc5 expression; PI, propidium iodide staining of nuclei; Cdc5 + PI, EGFP-Cdc5 and propidium iodide images superimposed. Bar, 5 μm.

FIG. 4

Ectopically expressed Cdc5 localizes at the spindle poles and bud neck filaments. The EGFP-Cdc5 fusion constructs (YCplac22-GAL1-HA-EGFP-Cdc5) were expressed under the control of the GAL1 promoter in a diploid wild-type strain, 1788, for 4 h. Transformants were subjected to subsequent immunostainings to examine Cdc10 and tubulin localization. (A) EGFP-Cdc5 (green) and Cdc10 (red) localize at the neck filaments. Septin rings (red) are viewed edge-on and therefore appear as lines. Occasionally, rings of septin structures were also observed. (B) EGFP-Cdc5 (green) localizes at the spindle poles. Spindles are visualized by microtubule staining (red). The spindles appear to emanate from the structures with which Cdc5 associates. Not all the dotted EGFP-Cdc5 signals were associated with spindle poles (see the text). DIC, differential interference contrast; Cdc5, EGFP-Cdc5 expression; Cdc10, anti-Cdc10 staining; Tubulin, antitubulin staining. Superimposed images are shown as Cdc5+Cdc10 and Cdc5+Tubulin. Bar, 5 μm.

FIG. 5

Multiple GFP-Spc42 dot signals induced by overexpression of Cdc5 colocalize with Cdc5. A GFP-Spc42 strain transformed with either the YCplac-ADE2-GAL1-HA-GST construct or YCplac-ADE2-GAL1-HA-GST-Cdc5N209AΔDB were cultured under induction conditions for 12 h, harvested, and subjected to immunostaining with anti-HA antibody. Expression of kinase-inactive cdc5N209AΔDB resulted in slightly more cells with multiple GFP-Spc42 dots than did use of the wild type; therefore, cdc5N209AΔDB was used for this experiment. (A) GFP-Spc42 dot signals in cells transformed with vector. One or two fluorescent dot signals are evident. (B) Overexpression of Cdc5 results in induction of and colocalization with multiple GFP-Spc42-containing structures. When Cdc5 was overexpressed for 12 h, an average of four or five fluorescent dot signals were observed in approximately 25% of the population. Multiple GFP-Spc42 (green) dots observed with Cdc5 overexpression largely colocalize with multiple Cdc5 (red) signals. Immunostaining was carried out by the method of Rout and Kilmartin (42) with modifications (see Materials and Methods). HA-GST-cdc5N209AΔDB was localized by using affinity-purified monoclonal anti-HA antibody at 2 μg/ml and TRITC-conjugated goat anti-mouse IgG at a 1:800 dilution. Due to the methanol-acetone fixation, GFP-Spc42 signal intensity was diminished. It is noteworthy that not all the GFP-Spc42 and Cdc5 signals are colocalized. This is probably due to the perturbed spindle pole body organization or fragmented spindle pole bodies induced by Cdc5 overexpression (see the text). Overexpression of cdc5N209AΔDB severely reduced cell growth and resulted in an increase in cell size. DIC, differential interference contrast; Spc42, GFP-Spc42 signal; vector, YCplac-ADE2-GAL1-HA-GST construct; Cdc5, YCplac-ADE2-GAL1-HA-GST-cdc5N209AΔDB. Superimposed images are shown as Spc42+vector and Spc42+Cdc5. Bar, 5 μm.

FIG. 6

Induction of additional septal structures by ectopic expression of Cdc5 in a diploid wild-type strain, 1788. Cells transformed with pKK507 (YCplac22-GAL1-Cdc5) induced cells with abnormally elongated buds. Transformants were cultured in YEP–2% galactose for 12 h prior to fixation. Subsequent immunostainings with anti-Cdc10 antibody revealed the presence of multiple septal structures within the abnormally elongated buds. Localization of Cdc10 is shown in two examples of elongated cells. DIC, differential interference contrast; Cdc10, anti-Cdc10 staining; PI, propidium iodide staining of nuclei; Cdc10+PI, superimposed images of anti-Cdc10 staining and propidium iodide staining. Bar, 5 μm.

FIG. 7

Induction of additional septal structures and actin polarization by expression of EGFP-cdc5ΔDB in a diploid wild-type strain, 1788. To enhance the signals present at the additional septal structures, the N-terminal 66 amino acid residues containing the two putative destruction boxes of Cdc5 (46) were deleted. Transformants were cultured for 12 h under inducing conditions and subjected to subsequent stainings to examine tubulin, Cdc10, and actin localization. Both normal (NA; cdc5N209AΔDB transformants) and elongated (WT; wild-type cdc5ΔDB transformants) cells are shown. (A) Cdc5 (green) localizes at the spindle poles. Spindles are visualized by microtubule staining (red). In the elongated cells, the signals at the native neck filaments were much weaker than those at the ectopic sites within the elongated buds. (B) Cdc5 (green) and Cdc10 (red) colocalize at the neck filaments and additional septal structures. (C) Cdc5 (green) can induce actin accumulation (red) at the additional septal structures. DIC, differential interference contrast; Cdc5, EGFP-cdc5ΔDB expression; Tubulin, antitubulin staining; Cdc10, anti-Cdc10 staining; Actin, rhodamine-conjugated phalloidin staining. Superimposed images are shown as Cdc5+Tubulin, Cdc5+Cdc10, and Cdc5+Actin. Bar, 5 μm.

FIG. 8

(A) The FAA mutations do not influence the level of Cdc5 expression. The wild-type 1788 cells bearing various YCplac22-GAL1-HA-EGFP-Cdc5 constructs were cultured under inducing conditions for 12 h and harvested. The same amount (400 μg each) of total cellular lysates prepared from various transformants was loaded onto each lane. After the proteins were transferred onto a polyvinylidene difluoride membrane, proteins interacting with the anti-GFP antibody were detected by immunoblotting. Vector, YCplac22-GAL1; NA, YCplac22-GAL1-HA-EGFP-cdc5N209AΔDB; WT, YCplac22-GAL1-HA-EGFP-cdc5ΔDB; FAA, YCplac22-GAL1-HA-EGFP-cdc5W517F/V518A/L530AΔDB. (B) The FAA mutations in the polo box do not impair Cdc5 kinase activity in vitro. Total cellular lysates prepared as described above were subjected to further centrifugation at 15,000 × g for 30 min to clarify heavy cellular materials. Equal amounts (1 mg each) of the resulting supernatants (S15) were diluted to 1 ml with TBSN buffer supplemented with protease inhibitors and then incubated with anti-HA antibodies. Protein A-Sepharose 4B was added, and the mixture was incubated for an additional 1 h to precipitate the antibodies. Immune complex kinase assays were carried out with casein as the substrate. Cs, casein. (Top) Reaction mixtures were electrophoresed, and the proteins were transferred onto a polyvinylidene difluoride membrane and exposed to detect kinase activities. (Bottom) The same blot was subjected to immunoblotting with anti-EGFP antibody to determine the amount of HA-EGFP-Cdc5 protein present in each immunoprecipitate. Lane designations are as defined for panel A.

FIG. 9

Inhibition of cytokinesis by expression of the C-terminal domain of Cdc5 is abolished by the introduction of the FAA mutations in the polo box. (A) Introduction of the FAA mutations into the polo box abolish localization of HA-EGFP-cdc5 · C-term. Wild-type 1788 cells bearing either YCplac22-GAL1-HA-EGFP-cdc5 · C-term or YCplac22-GAL1-HA-EGFP-cdc5 · C-termFAA were cultured in YEP–2% galactose medium for 12 h and then examined under the confocal microscope. HA-EGFP-cdc5 · C-term localizes as dot signals in the cytoplasm and bands at the cytokinetic neck filaments. Examples of either normal cells (top) or chains of connected cells (middle) are shown. The FAA mutant yielded only dispersed signals (bottom). (Left) Differential interference contrast; (right) HA-EGFP-cdc5 · C-term expression. cdc5 · C-term, HA-EGFP-cdc5 · C-term; cdc5 · C-termFAA, HA-EGFP-cdc5 · C-termFAA. Bar, 5 μm. (B) The FAA mutations do not influence the expression level of HA-EGFP-cdc5 · C-term. Wild-type 1788 cells bearing various constructs were cultured under inducing conditions for 12 h and harvested. The same amount (400 μg each) of total cellular lysates prepared from various transformants was loaded onto each lane. After the proteins were transferred onto a polyvinylidene difluoride membrane, proteins interacting with an anti-GFP antibody were detected by immunoblotting. cdc5 · C-term denotes the HA-GFP-cdc5 · C-term proteins expressed. Vector, YCplac22-GAL1; WT, YCplac22-GAL1-HA-EGFP-cdc5 · C-term; FAA, YCplac22-GAL1-HA-EGFP-cdc5 · C-termFAA.

FIG. 10

Expression of HA-EGFP-cdc5 · C-term, but not its FAA mutant, results in the induction of chains of connected cells. Wild-type 1788 cells bearing YCplac22-GAL1-HA-EGFP-cdc5 · C-term was cultured in YEP–2% galactose medium for 12 h and then examined under the confocal microscope. (A) Localization of HA-EGFP-cdc5 · C-term at the multiple cytokinetic neck filaments between the connected cells. To visualize clear morphology of the connected cells, live cells were examined under the microscope without fixation. (B) Nuclei were present in most of the cell bodies present in the connected cells. To visualize nuclei in these cells, cells were fixed and then treated with propidium iodide. (C) The presence of septin ring structures between the cell bodies of connected cells. Septin rings were observed by staining cells with an anti-Cdc10 antibody. (D) Accumulation of actin was often evident between the cell bodies. DIC, differential interference contrast; cdc5 · C-term, HA-EGFP-cdc5 · C-term; PI, propidium iodide staining; Cdc10, anti-Cdc10 staining; Actin, rhodamine-conjugated phalloidin staining. Superimposed images are shown as cdc5 · C-term + PI, cdc5 · C-term + Cdc10, and cdc5 · C-term + Actin. Bar, 5 μm.