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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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Plx1 is the physiological 3F3/2 kinase. (A) Extracts were mock depleted (lane 6) or depleted of Plx1 (lanes 3–5). Plx1 (lane 4) or Plx1-KD (lane 5) was translated in CSF extracts that had been depleted of endogenous Plx1 and then added to the Plx1-depleted extracts. Lanes 1 and 2 show input extracts. The volumes of extracts loaded were as indicated. Different amounts of input extracts were loaded to quantify the degree of depletion and add-back. (B and D–F) 3F3/2, Plx1, xCenp-A, xMad2, and xBubR1 staining of nuclei purified from checkpoint extracts that had undergone immunodepletion (ID) and add-back (AB) of the indicated proteins. Red, 3F3/2; green, Plx1, xCenp-A, xMad2, and xBubR1. (C) Mean kinetochore fluorescence intensity (from 15 randomly selected kinetochores) of xBubR1 (green) and 3F3/2 (red) signals from samples that were depleted of Plx1 and then added back with the indicated proteins. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. Bars, 5 μm.
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fig3: Plx1 is the physiological 3F3/2 kinase. (A) Extracts were mock depleted (lane 6) or depleted of Plx1 (lanes 3–5). Plx1 (lane 4) or Plx1-KD (lane 5) was translated in CSF extracts that had been depleted of endogenous Plx1 and then added to the Plx1-depleted extracts. Lanes 1 and 2 show input extracts. The volumes of extracts loaded were as indicated. Different amounts of input extracts were loaded to quantify the degree of depletion and add-back. (B and D–F) 3F3/2, Plx1, xCenp-A, xMad2, and xBubR1 staining of nuclei purified from checkpoint extracts that had undergone immunodepletion (ID) and add-back (AB) of the indicated proteins. Red, 3F3/2; green, Plx1, xCenp-A, xMad2, and xBubR1. (C) Mean kinetochore fluorescence intensity (from 15 randomly selected kinetochores) of xBubR1 (green) and 3F3/2 (red) signals from samples that were depleted of Plx1 and then added back with the indicated proteins. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. Bars, 5 μm.

Mentions: If Plx1 is indeed the 3F3/2 kinase, immunodepletion of Plx1 should abolish the 3F3/2 epitope on kinetochores in spindle checkpoint extracts. To test this prediction, we immunodepleted Plx1 from CSF extracts to >99.5%, and then assembled the checkpoint extracts by adding sperm nuclei and nocodazole (Fig. 3 A). Immunodepletion of Plx1 abolished its kinetochore staining (Fig. 3 B), indicating that Plx1 is indeed a kinetochore protein in X. laevis extracts. Upon depletion of Plx1, the 3F3/2 epitope was also absent from kinetochores, whereas the localization of xCenp-A was not affected (Fig. 3 B). To rescue the Plx1-depletion phenotype, we translated recombinant Plx1, using in vitro–transcribed Plx1 mRNA, in X. laevis CSF extracts that had been depleted of endogenous Plx1 to >99.5%. Addition of translated Plx1 to the depleted extracts at only ∼5% of the endogenous Plx1 level efficiently restored both the Plx1 and 3F3/2 signals at kinetochores. In contrast, addition of an in vitro–translated Plx1 kinase-dead mutant (Plx1-KD) failed to restore the 3F3/2 signals, despite the fact that Plx1-KD was efficiently targeted to kinetochores (Fig. 3 B). A quantitative analysis of 3F3/2 kinetochore fluorescence intensity in these samples further supported the requirement of the Plx1 kinase activity for phosphorylating the 3F3/2 epitope (Fig. 3 C). Together, our data (Figs. 2 and 3) indicate that Plx1 is the physiological kinase for the 3F3/2 epitope in checkpoint extracts. We found that Plx1 has an extremely high affinity for kinetochores, as immunodepletion of Plx1 by 99% affected neither its localization at kinetochores nor the phosphorylation of the 3F3/2 epitope in checkpoint extracts (not depicted). This high affinity of Plx1 to kinetochores also explains why the addition of only 5% of wild-type Plx1 to the depleted extracts is sufficient to rescue the 3F3/2 signal. Therefore, all the depletion experiments in this paper were performed with a depletion efficiency of >99.5% for Plx1.


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

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

Plx1 is the physiological 3F3/2 kinase. (A) Extracts were mock depleted (lane 6) or depleted of Plx1 (lanes 3–5). Plx1 (lane 4) or Plx1-KD (lane 5) was translated in CSF extracts that had been depleted of endogenous Plx1 and then added to the Plx1-depleted extracts. Lanes 1 and 2 show input extracts. The volumes of extracts loaded were as indicated. Different amounts of input extracts were loaded to quantify the degree of depletion and add-back. (B and D–F) 3F3/2, Plx1, xCenp-A, xMad2, and xBubR1 staining of nuclei purified from checkpoint extracts that had undergone immunodepletion (ID) and add-back (AB) of the indicated proteins. Red, 3F3/2; green, Plx1, xCenp-A, xMad2, and xBubR1. (C) Mean kinetochore fluorescence intensity (from 15 randomly selected kinetochores) of xBubR1 (green) and 3F3/2 (red) signals from samples that were depleted of Plx1 and then added back with the indicated proteins. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. Bars, 5 μm.
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fig3: Plx1 is the physiological 3F3/2 kinase. (A) Extracts were mock depleted (lane 6) or depleted of Plx1 (lanes 3–5). Plx1 (lane 4) or Plx1-KD (lane 5) was translated in CSF extracts that had been depleted of endogenous Plx1 and then added to the Plx1-depleted extracts. Lanes 1 and 2 show input extracts. The volumes of extracts loaded were as indicated. Different amounts of input extracts were loaded to quantify the degree of depletion and add-back. (B and D–F) 3F3/2, Plx1, xCenp-A, xMad2, and xBubR1 staining of nuclei purified from checkpoint extracts that had undergone immunodepletion (ID) and add-back (AB) of the indicated proteins. Red, 3F3/2; green, Plx1, xCenp-A, xMad2, and xBubR1. (C) Mean kinetochore fluorescence intensity (from 15 randomly selected kinetochores) of xBubR1 (green) and 3F3/2 (red) signals from samples that were depleted of Plx1 and then added back with the indicated proteins. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. Bars, 5 μm.
Mentions: If Plx1 is indeed the 3F3/2 kinase, immunodepletion of Plx1 should abolish the 3F3/2 epitope on kinetochores in spindle checkpoint extracts. To test this prediction, we immunodepleted Plx1 from CSF extracts to >99.5%, and then assembled the checkpoint extracts by adding sperm nuclei and nocodazole (Fig. 3 A). Immunodepletion of Plx1 abolished its kinetochore staining (Fig. 3 B), indicating that Plx1 is indeed a kinetochore protein in X. laevis extracts. Upon depletion of Plx1, the 3F3/2 epitope was also absent from kinetochores, whereas the localization of xCenp-A was not affected (Fig. 3 B). To rescue the Plx1-depletion phenotype, we translated recombinant Plx1, using in vitro–transcribed Plx1 mRNA, in X. laevis CSF extracts that had been depleted of endogenous Plx1 to >99.5%. Addition of translated Plx1 to the depleted extracts at only ∼5% of the endogenous Plx1 level efficiently restored both the Plx1 and 3F3/2 signals at kinetochores. In contrast, addition of an in vitro–translated Plx1 kinase-dead mutant (Plx1-KD) failed to restore the 3F3/2 signals, despite the fact that Plx1-KD was efficiently targeted to kinetochores (Fig. 3 B). A quantitative analysis of 3F3/2 kinetochore fluorescence intensity in these samples further supported the requirement of the Plx1 kinase activity for phosphorylating the 3F3/2 epitope (Fig. 3 C). Together, our data (Figs. 2 and 3) indicate that Plx1 is the physiological kinase for the 3F3/2 epitope in checkpoint extracts. We found that Plx1 has an extremely high affinity for kinetochores, as immunodepletion of Plx1 by 99% affected neither its localization at kinetochores nor the phosphorylation of the 3F3/2 epitope in checkpoint extracts (not depicted). This high affinity of Plx1 to kinetochores also explains why the addition of only 5% of wild-type Plx1 to the depleted extracts is sufficient to rescue the 3F3/2 signal. Therefore, all the depletion experiments in this paper were performed with a depletion efficiency of >99.5% for Plx1.

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