Limits...
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.

Show MeSH

Related in: MedlinePlus

Plx1 is both necessary and sufficient to phosphorylate the 3F3/2 epitope at kinetochores. (A) Nuclei purified from checkpoint extracts carrying the 3F3/2 epitope (left) were dephosphorylated, treated with NEM, and rephosphorylated with either ATP (middle) or ATP plus CSF extract (right). Nuclei were then stained for the 3F3/2 epitope and xBub1. (B) Immunodepletion of kinetochore-associated kinases from CSF extracts. CSF extracts were depleted of xMps1 (lane 1), xBub1 (lane 4), xBubR1 (lane 8), and Plx1 (lane 11). 1 μl of depleted extracts and mock-depleted extracts (lanes 2, 5, 9, and 12) and 0.05 μl of input extracts (lanes 3, 6, 7, and 10) were analyzed by Western blotting to determine the depletion efficiency. xMad1 and xMad2 were shown here to demonstrate the specificity of the immunodepletion. (C and D) Rephosphorylation of 3F3/2 by extracts depleted of kinetochore-associated kinases. Nuclei were prepared as in A and rephosphorylated with either ATP (first column in C) or ATP plus depleted extracts prepared in B. ID, immunodepletion. (E) Mean kinetochore fluorescence intensity of xBub1 (green) and 3F3/2 (red) signals from samples rephosphorylated with xBubR1- or Plx1-depleted extracts. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. (F) Nuclei from checkpoint extracts were dephosphorylated and rephosphorylated with ATP or with ATP plus recombinant His6-Plx1. (A, C, D, and F) Red, 3F3/2; green, xBub1. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171348&req=5

fig2: Plx1 is both necessary and sufficient to phosphorylate the 3F3/2 epitope at kinetochores. (A) Nuclei purified from checkpoint extracts carrying the 3F3/2 epitope (left) were dephosphorylated, treated with NEM, and rephosphorylated with either ATP (middle) or ATP plus CSF extract (right). Nuclei were then stained for the 3F3/2 epitope and xBub1. (B) Immunodepletion of kinetochore-associated kinases from CSF extracts. CSF extracts were depleted of xMps1 (lane 1), xBub1 (lane 4), xBubR1 (lane 8), and Plx1 (lane 11). 1 μl of depleted extracts and mock-depleted extracts (lanes 2, 5, 9, and 12) and 0.05 μl of input extracts (lanes 3, 6, 7, and 10) were analyzed by Western blotting to determine the depletion efficiency. xMad1 and xMad2 were shown here to demonstrate the specificity of the immunodepletion. (C and D) Rephosphorylation of 3F3/2 by extracts depleted of kinetochore-associated kinases. Nuclei were prepared as in A and rephosphorylated with either ATP (first column in C) or ATP plus depleted extracts prepared in B. ID, immunodepletion. (E) Mean kinetochore fluorescence intensity of xBub1 (green) and 3F3/2 (red) signals from samples rephosphorylated with xBubR1- or Plx1-depleted extracts. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. (F) Nuclei from checkpoint extracts were dephosphorylated and rephosphorylated with ATP or with ATP plus recombinant His6-Plx1. (A, C, D, and F) Red, 3F3/2; green, xBub1. Bars, 5 μm.

Mentions: We used a candidate approach to identify the 3F3/2 kinase. Six kinetochore-associated mitotic kinases—xMps1, xBub1, xBubR1, xAurora B, Plx1, and Cdc2/cyclin B (for reviews see Lens and Medema, 2003; Taylor et al., 2004)—were tested. To assay for the 3F3/2 kinase activity, we modified a rephosphorylation assay developed in mammalian cells (Nicklas et al., 1995). Nuclei purified from X. laevis checkpoint extracts were treated first with λ-phosphatase to remove the phosphoepitope and then with N-ethyl maleimide (NEM), a sulfhydryl alkylating agent that covalently modifies cysteine residues, to inactivate kinetochore-associated kinases. The resulting nuclei were used as substrates in an in vitro kinase assay. Addition of ATP alone did not recover the 3F3/2 signals, whereas ATP plus X. laevis meiotic metaphase extracts (cytostatic factor [CSF] extracts) regenerated the 3F3/2 epitope (Fig. 2 A), indicating that CSF extracts contain the 3F3/2 kinase.


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

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

Plx1 is both necessary and sufficient to phosphorylate the 3F3/2 epitope at kinetochores. (A) Nuclei purified from checkpoint extracts carrying the 3F3/2 epitope (left) were dephosphorylated, treated with NEM, and rephosphorylated with either ATP (middle) or ATP plus CSF extract (right). Nuclei were then stained for the 3F3/2 epitope and xBub1. (B) Immunodepletion of kinetochore-associated kinases from CSF extracts. CSF extracts were depleted of xMps1 (lane 1), xBub1 (lane 4), xBubR1 (lane 8), and Plx1 (lane 11). 1 μl of depleted extracts and mock-depleted extracts (lanes 2, 5, 9, and 12) and 0.05 μl of input extracts (lanes 3, 6, 7, and 10) were analyzed by Western blotting to determine the depletion efficiency. xMad1 and xMad2 were shown here to demonstrate the specificity of the immunodepletion. (C and D) Rephosphorylation of 3F3/2 by extracts depleted of kinetochore-associated kinases. Nuclei were prepared as in A and rephosphorylated with either ATP (first column in C) or ATP plus depleted extracts prepared in B. ID, immunodepletion. (E) Mean kinetochore fluorescence intensity of xBub1 (green) and 3F3/2 (red) signals from samples rephosphorylated with xBubR1- or Plx1-depleted extracts. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. (F) Nuclei from checkpoint extracts were dephosphorylated and rephosphorylated with ATP or with ATP plus recombinant His6-Plx1. (A, C, D, and F) Red, 3F3/2; green, xBub1. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171348&req=5

fig2: Plx1 is both necessary and sufficient to phosphorylate the 3F3/2 epitope at kinetochores. (A) Nuclei purified from checkpoint extracts carrying the 3F3/2 epitope (left) were dephosphorylated, treated with NEM, and rephosphorylated with either ATP (middle) or ATP plus CSF extract (right). Nuclei were then stained for the 3F3/2 epitope and xBub1. (B) Immunodepletion of kinetochore-associated kinases from CSF extracts. CSF extracts were depleted of xMps1 (lane 1), xBub1 (lane 4), xBubR1 (lane 8), and Plx1 (lane 11). 1 μl of depleted extracts and mock-depleted extracts (lanes 2, 5, 9, and 12) and 0.05 μl of input extracts (lanes 3, 6, 7, and 10) were analyzed by Western blotting to determine the depletion efficiency. xMad1 and xMad2 were shown here to demonstrate the specificity of the immunodepletion. (C and D) Rephosphorylation of 3F3/2 by extracts depleted of kinetochore-associated kinases. Nuclei were prepared as in A and rephosphorylated with either ATP (first column in C) or ATP plus depleted extracts prepared in B. ID, immunodepletion. (E) Mean kinetochore fluorescence intensity of xBub1 (green) and 3F3/2 (red) signals from samples rephosphorylated with xBubR1- or Plx1-depleted extracts. The fluorescence intensity was normalized to the corresponding values derived from mock-depleted extracts. Error bars represent SD. (F) Nuclei from checkpoint extracts were dephosphorylated and rephosphorylated with ATP or with ATP plus recombinant His6-Plx1. (A, C, D, and F) Red, 3F3/2; green, xBub1. Bars, 5 μm.
Mentions: We used a candidate approach to identify the 3F3/2 kinase. Six kinetochore-associated mitotic kinases—xMps1, xBub1, xBubR1, xAurora B, Plx1, and Cdc2/cyclin B (for reviews see Lens and Medema, 2003; Taylor et al., 2004)—were tested. To assay for the 3F3/2 kinase activity, we modified a rephosphorylation assay developed in mammalian cells (Nicklas et al., 1995). Nuclei purified from X. laevis checkpoint extracts were treated first with λ-phosphatase to remove the phosphoepitope and then with N-ethyl maleimide (NEM), a sulfhydryl alkylating agent that covalently modifies cysteine residues, to inactivate kinetochore-associated kinases. The resulting nuclei were used as substrates in an in vitro kinase assay. Addition of ATP alone did not recover the 3F3/2 signals, whereas ATP plus X. laevis meiotic metaphase extracts (cytostatic factor [CSF] extracts) regenerated the 3F3/2 epitope (Fig. 2 A), indicating that CSF extracts contain the 3F3/2 kinase.

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