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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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xMps1 controls the kinetochore localization of Plx1. (A) CSF extracts were either mock depleted or depleted of xMps1 and then added back with the indicated proteins. Nuclei were purified from depleted checkpoint extracts and stained for xMps1, Plx1, and xCenp-A. ID, immunodepletion; AB, add-back. Bar, 5 μm. (B) Depletion efficiency was determined by Western blot analysis of equal volumes of mock- (lane 1) or xMps1-depleted (lane 2) extracts as well as xMps1-depleted extracts with the add-back of xMps1 (lane 3) or xMps1-KD (lane 4).
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fig5: xMps1 controls the kinetochore localization of Plx1. (A) CSF extracts were either mock depleted or depleted of xMps1 and then added back with the indicated proteins. Nuclei were purified from depleted checkpoint extracts and stained for xMps1, Plx1, and xCenp-A. ID, immunodepletion; AB, add-back. Bar, 5 μm. (B) Depletion efficiency was determined by Western blot analysis of equal volumes of mock- (lane 1) or xMps1-depleted (lane 2) extracts as well as xMps1-depleted extracts with the add-back of xMps1 (lane 3) or xMps1-KD (lane 4).

Mentions: We next asked whether Plx1 itself is also under the control of upstream checkpoint components, such as xMps1. xMps1 was depleted from CSF extracts before the assembly of the checkpoint extracts, and the kinetochore localization of Plx1 was examined in the depleted checkpoint extracts. Interestingly, depletion of xMps1 completely abolished the Plx1 signal at kinetochores without affecting the level of the Plx1 protein in extracts (Fig. 5, A and B). Addition of in vitro–translated wild-type xMps1, but not the xMps1 kinase-dead mutant (xMps1-KD), rescued the kinetochore localization of Plx1 (Fig. 5 A). In all cases, xCenp-A localization was not affected, indicating that depletion of xMps1 did not affect the structural integrity of inner centromeres. We conclude that the checkpoint kinase xMps1 is required to recruit Plx1 to kinetochores.


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

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

xMps1 controls the kinetochore localization of Plx1. (A) CSF extracts were either mock depleted or depleted of xMps1 and then added back with the indicated proteins. Nuclei were purified from depleted checkpoint extracts and stained for xMps1, Plx1, and xCenp-A. ID, immunodepletion; AB, add-back. Bar, 5 μm. (B) Depletion efficiency was determined by Western blot analysis of equal volumes of mock- (lane 1) or xMps1-depleted (lane 2) extracts as well as xMps1-depleted extracts with the add-back of xMps1 (lane 3) or xMps1-KD (lane 4).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171348&req=5

fig5: xMps1 controls the kinetochore localization of Plx1. (A) CSF extracts were either mock depleted or depleted of xMps1 and then added back with the indicated proteins. Nuclei were purified from depleted checkpoint extracts and stained for xMps1, Plx1, and xCenp-A. ID, immunodepletion; AB, add-back. Bar, 5 μm. (B) Depletion efficiency was determined by Western blot analysis of equal volumes of mock- (lane 1) or xMps1-depleted (lane 2) extracts as well as xMps1-depleted extracts with the add-back of xMps1 (lane 3) or xMps1-KD (lane 4).
Mentions: We next asked whether Plx1 itself is also under the control of upstream checkpoint components, such as xMps1. xMps1 was depleted from CSF extracts before the assembly of the checkpoint extracts, and the kinetochore localization of Plx1 was examined in the depleted checkpoint extracts. Interestingly, depletion of xMps1 completely abolished the Plx1 signal at kinetochores without affecting the level of the Plx1 protein in extracts (Fig. 5, A and B). Addition of in vitro–translated wild-type xMps1, but not the xMps1 kinase-dead mutant (xMps1-KD), rescued the kinetochore localization of Plx1 (Fig. 5 A). In all cases, xCenp-A localization was not affected, indicating that depletion of xMps1 did not affect the structural integrity of inner centromeres. We conclude that the checkpoint kinase xMps1 is required to recruit Plx1 to kinetochores.

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