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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 and Plx1 are required for the assembly of the outer kinetochore structure. (A) Nuclei were purified from mock-, xMps1-, or xMps1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 5. Purified nuclei were stained for xNdc80 or xNuf2. (B) Nuclei were purified from mock-, Plx1-, or Plx1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 3. Purified nuclei were stained for xNdc80 and xNuf2. ID, immunodepletion; AB, add-back. Bars, 5 μm.
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fig6: xMps1 and Plx1 are required for the assembly of the outer kinetochore structure. (A) Nuclei were purified from mock-, xMps1-, or xMps1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 5. Purified nuclei were stained for xNdc80 or xNuf2. (B) Nuclei were purified from mock-, Plx1-, or Plx1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 3. Purified nuclei were stained for xNdc80 and xNuf2. ID, immunodepletion; AB, add-back. Bars, 5 μm.

Mentions: To determine whether xMps1 and Plx1 control the outer kinetochore structure, nuclei from xMps1- or Plx1-depleted extracts were stained with xNdc80 and xNuf2. xNdc80 and xNuf2 form a complex required for microtubule attachment to kinetochores (DeLuca et al., 2002). Depletion of xMps1 completely removed xNdc80 and xNuf2 from the kinetochores, and their kinetochore localization can only be rescued by the addition of wild-type xMps1 but not xMps1-KD (Fig. 6 A). Similarly, Plx1 is also required for the kinetochore localization of xNdc80 and xNuf2 (Fig. 6 B). Interestingly, addition of either wild type or Plx1-KD was sufficient to rescue the kinetochore localization of xNdc80 and xNuf2, indicating a kinase-independent function of Plx1 at kinetochores, although we cannot exclude the possibility that the residual endogenous Plx1, even with >99.5% depletion, acted together with Plx1-KD to mediate the rescue. Together, our data suggest that both xMps1 and Plx1 control the assembly of the outer kinetochore structure.


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

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

xMps1 and Plx1 are required for the assembly of the outer kinetochore structure. (A) Nuclei were purified from mock-, xMps1-, or xMps1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 5. Purified nuclei were stained for xNdc80 or xNuf2. (B) Nuclei were purified from mock-, Plx1-, or Plx1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 3. Purified nuclei were stained for xNdc80 and xNuf2. ID, immunodepletion; AB, add-back. Bars, 5 μm.
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Related In: Results  -  Collection

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fig6: xMps1 and Plx1 are required for the assembly of the outer kinetochore structure. (A) Nuclei were purified from mock-, xMps1-, or xMps1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 5. Purified nuclei were stained for xNdc80 or xNuf2. (B) Nuclei were purified from mock-, Plx1-, or Plx1-depleted extracts with the add-back of the indicated proteins, as described in Fig. 3. Purified nuclei were stained for xNdc80 and xNuf2. ID, immunodepletion; AB, add-back. Bars, 5 μm.
Mentions: To determine whether xMps1 and Plx1 control the outer kinetochore structure, nuclei from xMps1- or Plx1-depleted extracts were stained with xNdc80 and xNuf2. xNdc80 and xNuf2 form a complex required for microtubule attachment to kinetochores (DeLuca et al., 2002). Depletion of xMps1 completely removed xNdc80 and xNuf2 from the kinetochores, and their kinetochore localization can only be rescued by the addition of wild-type xMps1 but not xMps1-KD (Fig. 6 A). Similarly, Plx1 is also required for the kinetochore localization of xNdc80 and xNuf2 (Fig. 6 B). Interestingly, addition of either wild type or Plx1-KD was sufficient to rescue the kinetochore localization of xNdc80 and xNuf2, indicating a kinase-independent function of Plx1 at kinetochores, although we cannot exclude the possibility that the residual endogenous Plx1, even with >99.5% depletion, acted together with Plx1-KD to mediate the rescue. Together, our data suggest that both xMps1 and Plx1 control the assembly of the outer kinetochore structure.

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