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Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores.

Wong OK, Fang G - J. Cell Biol. (2005)

Bottom Line: Using a rephosphorylation assay in Xenopus laevis extracts, we identified the kinetochore-associated Polo-like kinase Plx1 as the kinase both necessary and sufficient for this phosphorylation.Indeed, Plx1 is the physiological 3F3/2 kinase involved in checkpoint response, as immunodepletion of Plx1 from checkpoint extracts abolished the 3F3/2 signal and blocked association of xMad2, xBubR1, xNdc80, and xNuf2 with kinetochores.Interestingly, the kinetochore localization of Plx1 is under the control of the checkpoint protein xMps1, as immunodepletion of xMps1 prevents binding of Plx1 to kinetochores.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.

ABSTRACT
Dynamic attachment of microtubules to kinetochores during mitosis generates pulling force, or tension, required for the high fidelity of chromosome separation. A lack of tension activates the spindle checkpoint and delays the anaphase onset. A key step in the tension-response pathway involves the phosphorylation of the 3F3/2 epitope by an unknown kinase on untensed kinetochores. Using a rephosphorylation assay in Xenopus laevis extracts, we identified the kinetochore-associated Polo-like kinase Plx1 as the kinase both necessary and sufficient for this phosphorylation. Indeed, Plx1 is the physiological 3F3/2 kinase involved in checkpoint response, as immunodepletion of Plx1 from checkpoint extracts abolished the 3F3/2 signal and blocked association of xMad2, xBubR1, xNdc80, and xNuf2 with kinetochores. Interestingly, the kinetochore localization of Plx1 is under the control of the checkpoint protein xMps1, as immunodepletion of xMps1 prevents binding of Plx1 to kinetochores. Thus, Plx1 couples the tension signal to cellular responses through phosphorylating the 3F3/2 epitope and targeting structural and checkpoint proteins to kinetochores.

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Plk1 is required for the generation of the 3F3/2 epitope in HeLa cells. (A, top) Schematic of double-thymidine synchronization and siRNA transfection of HeLa cells. (bottom) Western blot analysis of Plk1 and control knockdown cell lysates to determine the knockdown efficiency. p38 MAPK was used as a loading control. The volumes of HeLa cell lysates loaded were as indicated. Different amounts of cell lysates were loaded from the control knockdown sample to quantify the degree of Plk1 knockdown. (B and D–G) Plk1 or control knockdown cells were fixed at 11 h after release from the second thymindine arrest. Prometaphase cells were stained with the following antibodies: (B) Crest serum (red) and anti-Plk1 (green); (D) 3F3/2 antibody (red) and anti-Plk1 (green); (E) 3F3/2 antibody (red), Crest serum (green), and anti-Plk1 (green); (F) Plk1 (red) and Mad2 (green); (G) Plk1 (red) and BubR1 (green). In D, F, and G, arrowheads point to 3F3/2 and Plk1 signals at spindle poles. Bars, 5 μm. (C) Mean kinetochore fluorescence intensity (from randomly selected kinetochores of multiple prometaphase cells) of Plk1, 3F3/2, Mad2, and BubR1 signals from Plk1 (green) or control knockdown (red) cells. The fluorescence intensity was normalized to the corresponding values derived from control knockdown cells. Error bars represent SD. (H) HeLa cells were synchronized by double-thymidine arrest/release and transfected with siRNAs as described in A, except that transfected cells were released from the second thymidine arrest in either the presence (+ Noc) or absence (− Noc) of 100 ng/ml nocodazole. At 14 h after release from the second thymidine arrest, cells were fixed, and the mitotic index was counted (n > 150 cells for each sample).
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fig4: Plk1 is required for the generation of the 3F3/2 epitope in HeLa cells. (A, top) Schematic of double-thymidine synchronization and siRNA transfection of HeLa cells. (bottom) Western blot analysis of Plk1 and control knockdown cell lysates to determine the knockdown efficiency. p38 MAPK was used as a loading control. The volumes of HeLa cell lysates loaded were as indicated. Different amounts of cell lysates were loaded from the control knockdown sample to quantify the degree of Plk1 knockdown. (B and D–G) Plk1 or control knockdown cells were fixed at 11 h after release from the second thymindine arrest. Prometaphase cells were stained with the following antibodies: (B) Crest serum (red) and anti-Plk1 (green); (D) 3F3/2 antibody (red) and anti-Plk1 (green); (E) 3F3/2 antibody (red), Crest serum (green), and anti-Plk1 (green); (F) Plk1 (red) and Mad2 (green); (G) Plk1 (red) and BubR1 (green). In D, F, and G, arrowheads point to 3F3/2 and Plk1 signals at spindle poles. Bars, 5 μm. (C) Mean kinetochore fluorescence intensity (from randomly selected kinetochores of multiple prometaphase cells) of Plk1, 3F3/2, Mad2, and BubR1 signals from Plk1 (green) or control knockdown (red) cells. The fluorescence intensity was normalized to the corresponding values derived from control knockdown cells. Error bars represent SD. (H) HeLa cells were synchronized by double-thymidine arrest/release and transfected with siRNAs as described in A, except that transfected cells were released from the second thymidine arrest in either the presence (+ Noc) or absence (− Noc) of 100 ng/ml nocodazole. At 14 h after release from the second thymidine arrest, cells were fixed, and the mitotic index was counted (n > 150 cells for each sample).

Mentions: To address whether the relationship between Plx1 and the 3F3/2 epitope is conserved during evolution, we tested the role of Plk1 in phosphorylation of the 3F3/2 epitope in HeLa cells. We transfected small interfering RNAs (siRNAs) into synchronized HeLa cells to achieve the maximal knockdown and analyzed the immediate phenotype during the first mitosis after transfection (Fig. 4 A). Western blot analysis indicated that the level of Plk1 was efficiently reduced through RNAi by 95% (Fig. 4 A). The reduction of Plk1 is also drastic on kinetochores at the cellular level. Whereas 100% of prometaphase cells (n = 56) in the control-transfected sample had strong Plk1 signals on kinetochores, 89.9% of prometaphase cells (n = 148) in the Plk1 knockdown sample had no detectable Plk1 signal on kinetochores (Fig. 4 B). The remaining 10.1% of cells had weak but visible Plk1 signals on kinetochores. To quantify the level of Plk1 knockdown on individual kinetochores, cells were costained with Crest serum and Plk1. Crest serum recognizes structural components at inner centromeres not affected by Plk1 knockdown (Fig. 4 B) and was used here as an internal reference to normalize the Plk1 fluorescence intensity. Upon knockdown of Plk1, the normalized fluorescence intensity of Plk1 signals at prometaphase kinetochores was reduced to ∼7.3% of that of control cells (Fig. 4, B and C). We next examined the level of the kinetochore 3F3/2 epitope in prometaphase cells in which Plk1 was not detectable on kinetochores. Prometaphase cells in control knockdown cells contained strong 3F3/2 signals at kinetochores (Fig. 4, D and E). In agreement with our results from extract experiments, the 3F3/2 signals were absent in Plk1 knockdown cells (Fig. 4, C and D). To exclude the possibility that Plk1 is generally required for the assembly of mitotic kinetochores, but not specifically involved in the phosphorylation of the 3F3/2 epitope, we triple stained the Plk1 knockdown cells with 3F3/2, Plk1, and Crest serum. Cells with efficient knockdown of Plk1 were identified by the absence of Plk1 signals at the spindle poles. In these cells, the 3F3/2 epitope was absent, but the Crest signals persisted, indicating that knockdown of Plk1 does not affect the integrity of the inner centromere structure (Fig. 4 E). Thus, Plk1 is also required for the formation of the 3F3/2 phosphoepitope in mammalian cells, suggesting a common mechanism for tension sensing/response from X. laevis to human.


Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores.

Wong OK, Fang G - J. Cell Biol. (2005)

Plk1 is required for the generation of the 3F3/2 epitope in HeLa cells. (A, top) Schematic of double-thymidine synchronization and siRNA transfection of HeLa cells. (bottom) Western blot analysis of Plk1 and control knockdown cell lysates to determine the knockdown efficiency. p38 MAPK was used as a loading control. The volumes of HeLa cell lysates loaded were as indicated. Different amounts of cell lysates were loaded from the control knockdown sample to quantify the degree of Plk1 knockdown. (B and D–G) Plk1 or control knockdown cells were fixed at 11 h after release from the second thymindine arrest. Prometaphase cells were stained with the following antibodies: (B) Crest serum (red) and anti-Plk1 (green); (D) 3F3/2 antibody (red) and anti-Plk1 (green); (E) 3F3/2 antibody (red), Crest serum (green), and anti-Plk1 (green); (F) Plk1 (red) and Mad2 (green); (G) Plk1 (red) and BubR1 (green). In D, F, and G, arrowheads point to 3F3/2 and Plk1 signals at spindle poles. Bars, 5 μm. (C) Mean kinetochore fluorescence intensity (from randomly selected kinetochores of multiple prometaphase cells) of Plk1, 3F3/2, Mad2, and BubR1 signals from Plk1 (green) or control knockdown (red) cells. The fluorescence intensity was normalized to the corresponding values derived from control knockdown cells. Error bars represent SD. (H) HeLa cells were synchronized by double-thymidine arrest/release and transfected with siRNAs as described in A, except that transfected cells were released from the second thymidine arrest in either the presence (+ Noc) or absence (− Noc) of 100 ng/ml nocodazole. At 14 h after release from the second thymidine arrest, cells were fixed, and the mitotic index was counted (n > 150 cells for each sample).
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fig4: Plk1 is required for the generation of the 3F3/2 epitope in HeLa cells. (A, top) Schematic of double-thymidine synchronization and siRNA transfection of HeLa cells. (bottom) Western blot analysis of Plk1 and control knockdown cell lysates to determine the knockdown efficiency. p38 MAPK was used as a loading control. The volumes of HeLa cell lysates loaded were as indicated. Different amounts of cell lysates were loaded from the control knockdown sample to quantify the degree of Plk1 knockdown. (B and D–G) Plk1 or control knockdown cells were fixed at 11 h after release from the second thymindine arrest. Prometaphase cells were stained with the following antibodies: (B) Crest serum (red) and anti-Plk1 (green); (D) 3F3/2 antibody (red) and anti-Plk1 (green); (E) 3F3/2 antibody (red), Crest serum (green), and anti-Plk1 (green); (F) Plk1 (red) and Mad2 (green); (G) Plk1 (red) and BubR1 (green). In D, F, and G, arrowheads point to 3F3/2 and Plk1 signals at spindle poles. Bars, 5 μm. (C) Mean kinetochore fluorescence intensity (from randomly selected kinetochores of multiple prometaphase cells) of Plk1, 3F3/2, Mad2, and BubR1 signals from Plk1 (green) or control knockdown (red) cells. The fluorescence intensity was normalized to the corresponding values derived from control knockdown cells. Error bars represent SD. (H) HeLa cells were synchronized by double-thymidine arrest/release and transfected with siRNAs as described in A, except that transfected cells were released from the second thymidine arrest in either the presence (+ Noc) or absence (− Noc) of 100 ng/ml nocodazole. At 14 h after release from the second thymidine arrest, cells were fixed, and the mitotic index was counted (n > 150 cells for each sample).
Mentions: To address whether the relationship between Plx1 and the 3F3/2 epitope is conserved during evolution, we tested the role of Plk1 in phosphorylation of the 3F3/2 epitope in HeLa cells. We transfected small interfering RNAs (siRNAs) into synchronized HeLa cells to achieve the maximal knockdown and analyzed the immediate phenotype during the first mitosis after transfection (Fig. 4 A). Western blot analysis indicated that the level of Plk1 was efficiently reduced through RNAi by 95% (Fig. 4 A). The reduction of Plk1 is also drastic on kinetochores at the cellular level. Whereas 100% of prometaphase cells (n = 56) in the control-transfected sample had strong Plk1 signals on kinetochores, 89.9% of prometaphase cells (n = 148) in the Plk1 knockdown sample had no detectable Plk1 signal on kinetochores (Fig. 4 B). The remaining 10.1% of cells had weak but visible Plk1 signals on kinetochores. To quantify the level of Plk1 knockdown on individual kinetochores, cells were costained with Crest serum and Plk1. Crest serum recognizes structural components at inner centromeres not affected by Plk1 knockdown (Fig. 4 B) and was used here as an internal reference to normalize the Plk1 fluorescence intensity. Upon knockdown of Plk1, the normalized fluorescence intensity of Plk1 signals at prometaphase kinetochores was reduced to ∼7.3% of that of control cells (Fig. 4, B and C). We next examined the level of the kinetochore 3F3/2 epitope in prometaphase cells in which Plk1 was not detectable on kinetochores. Prometaphase cells in control knockdown cells contained strong 3F3/2 signals at kinetochores (Fig. 4, D and E). In agreement with our results from extract experiments, the 3F3/2 signals were absent in Plk1 knockdown cells (Fig. 4, C and D). To exclude the possibility that Plk1 is generally required for the assembly of mitotic kinetochores, but not specifically involved in the phosphorylation of the 3F3/2 epitope, we triple stained the Plk1 knockdown cells with 3F3/2, Plk1, and Crest serum. Cells with efficient knockdown of Plk1 were identified by the absence of Plk1 signals at the spindle poles. In these cells, the 3F3/2 epitope was absent, but the Crest signals persisted, indicating that knockdown of Plk1 does not affect the integrity of the inner centromere structure (Fig. 4 E). Thus, Plk1 is also required for the formation of the 3F3/2 phosphoepitope in mammalian cells, suggesting a common mechanism for tension sensing/response from X. laevis to human.

Bottom Line: Using a rephosphorylation assay in Xenopus laevis extracts, we identified the kinetochore-associated Polo-like kinase Plx1 as the kinase both necessary and sufficient for this phosphorylation.Indeed, Plx1 is the physiological 3F3/2 kinase involved in checkpoint response, as immunodepletion of Plx1 from checkpoint extracts abolished the 3F3/2 signal and blocked association of xMad2, xBubR1, xNdc80, and xNuf2 with kinetochores.Interestingly, the kinetochore localization of Plx1 is under the control of the checkpoint protein xMps1, as immunodepletion of xMps1 prevents binding of Plx1 to kinetochores.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.

ABSTRACT
Dynamic attachment of microtubules to kinetochores during mitosis generates pulling force, or tension, required for the high fidelity of chromosome separation. A lack of tension activates the spindle checkpoint and delays the anaphase onset. A key step in the tension-response pathway involves the phosphorylation of the 3F3/2 epitope by an unknown kinase on untensed kinetochores. Using a rephosphorylation assay in Xenopus laevis extracts, we identified the kinetochore-associated Polo-like kinase Plx1 as the kinase both necessary and sufficient for this phosphorylation. Indeed, Plx1 is the physiological 3F3/2 kinase involved in checkpoint response, as immunodepletion of Plx1 from checkpoint extracts abolished the 3F3/2 signal and blocked association of xMad2, xBubR1, xNdc80, and xNuf2 with kinetochores. Interestingly, the kinetochore localization of Plx1 is under the control of the checkpoint protein xMps1, as immunodepletion of xMps1 prevents binding of Plx1 to kinetochores. Thus, Plx1 couples the tension signal to cellular responses through phosphorylating the 3F3/2 epitope and targeting structural and checkpoint proteins to kinetochores.

Show MeSH
Related in: MedlinePlus