Phosphorylation of right open reading frame 2 (Rio2) protein kinase by polo-like kinase 1 regulates mitotic progression

Abstract

Polo-like kinase 1 (Plk1) plays essential roles during multiple stages of mitosis by phosphorylating a number of substrates. Here, we report that the atypical protein kinase Rio2 is a novel substrate of Plk1 and can be phosphorylated by Plk1 at Ser-335, Ser-380, and Ser-548. Overexpression of Rio2 causes a prolonged mitotic exit whereas knockdown of Rio2 accelerates mitotic progression, suggesting that Rio2 is required for the proper mitotic progression. Overexpression of phospho-mimicking mutant Rio2 S3D but not the nonphosphorylatable mutant Rio2 S3A displays a profile similar to that of wild-type Rio2. These results indicate that the phosphorylation status of Rio2 correlates with its function in mitosis. Furthermore, time-lapse imaging data show that overexpression of Rio2 but not Rio2 S3A results in a slowed metaphase-anaphase transition. Collectively, these findings strongly indicate that the Plk1-mediated phosphorylation of Rio2 regulates metaphase-anaphase transition during mitotic progression.

FIGURE 1.

Rio2 interacts with Plk1. A, 293T cells co-transfected with FLAG-tagged Rio2 and myc-Plk1. The total cell extracts (TCE) were immunoprecipitated (IP) with FLAG antibody and immunoblotted (IB) with myc antibody. B, 293T cells transfected with pcDNA3.1, FLAG-tagged Plk1, and Plk1-KD (K82R). The cell lysates were immunoprecipitated with FLAG antibody and immunoblotted with Rio2 antibody. C, lysates from 293T cells immunoprecipitated with normal mouse serum or Plk1 antibody, respectively, followed by immunoblotting with Rio2 antibody. D, lysates from 293T cells immunoprecipitated with normal rabbit serum or Rio2 antibody and subjected to immunoblotting with Plk1 antibody. E, GST pulldown assay. Purified His-Plk1 was incubated with immobilized GST or GST-Rio2, respectively. The bound protein was detected by immunoblotting with Plk1 antibody. CBB, Coomassie Brilliant Blue.

FIGURE 2.

Rio2 interacts with the N-terminal domain of Plk1. A, schematic of Plk1 deletion mutants. B, 293T cells transfected with FLAG-tagged full-length of Plk1 or the indicated Plk1 deletion mutants. The total cell extracts (TCE) were immunoprecipitated (IP) with FLAG antibody and immunoblotted (IB) with Rio2 antibody. The total cell lysates were immunoblotted with mixed Rio2 and FLAG antibodies.

FIGURE 3.

Plk1 phosphorylates Rio2 at the sites of Ser-335, Ser-380, and Ser-548. A, purified GST or GST-Rio2 incubated with His-Plk1-TD for the in vitro kinase assay. CBB, Coomassie Brilliant Blue. B, GST-Rio2 incubated with His-Plk1-KD or His-Plk1-TD for the in vitro kinase assay. C, phosphopeptides identified by mass spectrometry. GST-Rio2 was incubated with His-Plk1-KD or His-Plk1-TD for the in vitro kinase assay in the presence of cold ATP and then subjected to mass spectrometry analysis. D, schematic figure of Rio2 deletion and point mutation mutants. E and F, GST-Rio2 mutants (E) and GST-Rio2 full-length and its Ser/Ala mutants (F) incubated with His-Plk1-TD for the in vitro kinase assay. G, 293T cells co-transfected with FLAG-tagged Plk1-TD and myc-Rio2 or myc-Rio2 S3A. The total cell extracts (TCE) were immunoprecipitated (IP) with myc antibody and followed by immunoblotting (IB) with anti-phosphoserine (pSer) and myc antibodies, respectively. H, purified GST-Rio2 wild-type or mutant Rio2 proteins (KD, S3A, or S3D) subjected to the in vitro kinase assay.

FIGURE 4.

Overexpression of Rio2 influences mitotic exit. A, tet-on inducible HeLa cells (control, Rio2, Rio2 S3A, and Rio2 S3D) were harvested at 24 h in the presence or absence of tetracycline and subjected to immunoblotting with Rio2 antibody. B and C, tet-on inducible HeLa cells (control, Rio2, Rio2 S3A, and Rio2 S3D) were synchronized at M phase in the presence of tetracycline. Then, the mitotic cells were released and harvested at the indicated times and subjected to flow cytometry analysis (B); the percentage of mitotic cells was calculated (C). D, cell lysates from the above experiment were subjected to immunoblotting with the indicated antibodies.

FIGURE 5.

Phosphorylation of Rio2 affects the metaphase-anaphase transition. A, the tet-on inducible HeLa cells (control, Rio2, and Rio2 S3A) were monitored under a microscope. The time-lapse movies were taken, and representative images at the indicated times during mitosis are shown. B, the time of metaphase-anaphase transition for tet-on inducible HeLa cells (control, Rio2, and Rio2 S3A) was assessed by video time-lapse microscopy. Results are representative of the average ± S.E. (error bars) of 100 mitotic cells from each group monitored individually. *, p < 0.01.

FIGURE 6.

Knockdown of Rio2 causes the accelerated mitotic progression. A, HeLa cells were synchronized at different stages of the cell cycle. The amounts of Rio2, Plk1, and β-actin (as a loading control) were analyzed by immunoblotting. Asy, asynchronize cells. B, HeLa cells were transfected with control siRNA or siRNA against Rio2 (siRio2-1 or siRio2-2) for 72 h. The cell lysates were harvested and immunoblotted with Rio2 and β-actin antibodies, respectively. C and D, HeLa cells were transfected with control siRNA, siRio2-1, and siRio2-2 and synchronized to M phase. Mitotic cells were released and collected at the indicated times for flow cytometry analysis (C), and the percentage of mitotic cells was calculated (D). E, cell lysates from the above experiment were subjected to immunoblotting with Rio2, cyclin B1, and β-actin antibodies. F, HeLa cells were transfected with control siRNA and siRio2-1 followed by thymidine-nocodazole or thymidine-paclitaxel (Taxol) arrest. Mitotic index was determined by FACS using Ser(P)-10-H3 antibody. G, HeLa cells were transfected with control siRNA and siRio2-1 and arrested by treatment with the indicated concentrations of nocodazole. Mitotic index was determined by FACS using Ser(P)-10-H3 antibody.