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

The 3F3/2 epitope in X. laevis is a phosphospecific kinetochore antigen. (A) Asynchronous XTC cells in mitosis (M) and in interphase (I) were stained with the 3F3/2 antibody and with an anti–xCenp-A antibody. The majority of the xCenp-A dots in M cells contained the 3F3/2 signals, although a few lacked the 3F3/2 staining, likely because of the dephosphorylation that occurred during sample processing. (B and C) XTC cells arrested with taxol or released from taxol arrest were stained for the 3F3/2 antigen and for xCenp-A or xBub1. Insets show a magnified image of the boxed areas. (D) Nuclei purified from checkpoint extracts (top) were dephosphorylated with λ-phosphatase (middle) and then rephosphorylated with ATP (bottom). (A–D) Red, 3F3/2; green, xCenp-A or xBub1. Arrowheads and arrow point to the 3F3/2 signals on centrosomes and at the midbody, respectively. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171348&req=5

fig1: The 3F3/2 epitope in X. laevis is a phosphospecific kinetochore antigen. (A) Asynchronous XTC cells in mitosis (M) and in interphase (I) were stained with the 3F3/2 antibody and with an anti–xCenp-A antibody. The majority of the xCenp-A dots in M cells contained the 3F3/2 signals, although a few lacked the 3F3/2 staining, likely because of the dephosphorylation that occurred during sample processing. (B and C) XTC cells arrested with taxol or released from taxol arrest were stained for the 3F3/2 antigen and for xCenp-A or xBub1. Insets show a magnified image of the boxed areas. (D) Nuclei purified from checkpoint extracts (top) were dephosphorylated with λ-phosphatase (middle) and then rephosphorylated with ATP (bottom). (A–D) Red, 3F3/2; green, xCenp-A or xBub1. Arrowheads and arrow point to the 3F3/2 signals on centrosomes and at the midbody, respectively. Bars, 5 μm.

Mentions: To investigate the biochemistry of the 3F3/2 pathway, we analyzed the 3F3/2 epitope in X. laevis cells and extracts. Asynchronous X. laevis tissue culture (XTC) cells were costained with a 3F3/2 antibody and an antibody against xCenp-A, which is a structural component of kinetochores. The 3F3/2 epitope was present only in mitotic cells, but not in interphase cells (Fig. 1 A). In addition to centrosomal staining, the 3F3/2 epitope colocalized with xCenp-A in mitotic cells, indicating that the 3F3/2 antibody recognizes a mitotic kinetochore epitope (Fig. 1 A). To determine the sensitivity of the 3F3/2 epitope to tension across sister kinetochores, XTC cells were treated with taxol. In early prometaphase cells treated with taxol, strong 3F3/2 signals colocalized with xCenp-A and xBub1, which is a kinetochore-associated checkpoint protein (Fig. 1, B and C). As cells were released from taxol arrest, both the 3F3/2 epitope and xBub1 were diminished from kinetochores, whereas xCenp-A remained at kinetochores (Fig. 1, B and C). Thus, in X. laevis cells, the 3F3/2 antibody recognizes unattached kinetochores that are not under tension, a finding that is consistent with previous reports in mammalian cells (Gorbsky and Ricketts, 1993; Nicklas et al., 1995).


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

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

The 3F3/2 epitope in X. laevis is a phosphospecific kinetochore antigen. (A) Asynchronous XTC cells in mitosis (M) and in interphase (I) were stained with the 3F3/2 antibody and with an anti–xCenp-A antibody. The majority of the xCenp-A dots in M cells contained the 3F3/2 signals, although a few lacked the 3F3/2 staining, likely because of the dephosphorylation that occurred during sample processing. (B and C) XTC cells arrested with taxol or released from taxol arrest were stained for the 3F3/2 antigen and for xCenp-A or xBub1. Insets show a magnified image of the boxed areas. (D) Nuclei purified from checkpoint extracts (top) were dephosphorylated with λ-phosphatase (middle) and then rephosphorylated with ATP (bottom). (A–D) Red, 3F3/2; green, xCenp-A or xBub1. Arrowheads and arrow point to the 3F3/2 signals on centrosomes and at the midbody, respectively. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: The 3F3/2 epitope in X. laevis is a phosphospecific kinetochore antigen. (A) Asynchronous XTC cells in mitosis (M) and in interphase (I) were stained with the 3F3/2 antibody and with an anti–xCenp-A antibody. The majority of the xCenp-A dots in M cells contained the 3F3/2 signals, although a few lacked the 3F3/2 staining, likely because of the dephosphorylation that occurred during sample processing. (B and C) XTC cells arrested with taxol or released from taxol arrest were stained for the 3F3/2 antigen and for xCenp-A or xBub1. Insets show a magnified image of the boxed areas. (D) Nuclei purified from checkpoint extracts (top) were dephosphorylated with λ-phosphatase (middle) and then rephosphorylated with ATP (bottom). (A–D) Red, 3F3/2; green, xCenp-A or xBub1. Arrowheads and arrow point to the 3F3/2 signals on centrosomes and at the midbody, respectively. Bars, 5 μm.
Mentions: To investigate the biochemistry of the 3F3/2 pathway, we analyzed the 3F3/2 epitope in X. laevis cells and extracts. Asynchronous X. laevis tissue culture (XTC) cells were costained with a 3F3/2 antibody and an antibody against xCenp-A, which is a structural component of kinetochores. The 3F3/2 epitope was present only in mitotic cells, but not in interphase cells (Fig. 1 A). In addition to centrosomal staining, the 3F3/2 epitope colocalized with xCenp-A in mitotic cells, indicating that the 3F3/2 antibody recognizes a mitotic kinetochore epitope (Fig. 1 A). To determine the sensitivity of the 3F3/2 epitope to tension across sister kinetochores, XTC cells were treated with taxol. In early prometaphase cells treated with taxol, strong 3F3/2 signals colocalized with xCenp-A and xBub1, which is a kinetochore-associated checkpoint protein (Fig. 1, B and C). As cells were released from taxol arrest, both the 3F3/2 epitope and xBub1 were diminished from kinetochores, whereas xCenp-A remained at kinetochores (Fig. 1, B and C). Thus, in X. laevis cells, the 3F3/2 antibody recognizes unattached kinetochores that are not under tension, a finding that is consistent with previous reports in mammalian cells (Gorbsky and Ricketts, 1993; Nicklas et al., 1995).

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