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Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes.

Topper LM, Bastians H, Ruderman JV, Gorbsky GJ - J. Cell Biol. (2001)

Bottom Line: Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure.Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores.These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

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Proteasome inhibition does not prevent chromosome congression or CENP-E association with kinetochores but arrests cells at metaphase. (A) Live images of a Ptk1 cell incubated in MG132. The cell began chromosome condensation (0 min) after 30-min incubation in MG132. Nuclear envelope breakdown and alignment of chromosomes at the metaphase plate occurred normally. The cell arrested at metaphase as shown in the 50-min image. (B) Cell was detergent extracted and fixed after incubation in MG132 for ∼1 h. Immunolabeling with anti-kinetochore antibody (green) is followed by anti–CENP-E labeling (red) taken at the same focal plane. The yellow kinetochores represent colocalization of the two antibodies (closed arrows).
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fig6: Proteasome inhibition does not prevent chromosome congression or CENP-E association with kinetochores but arrests cells at metaphase. (A) Live images of a Ptk1 cell incubated in MG132. The cell began chromosome condensation (0 min) after 30-min incubation in MG132. Nuclear envelope breakdown and alignment of chromosomes at the metaphase plate occurred normally. The cell arrested at metaphase as shown in the 50-min image. (B) Cell was detergent extracted and fixed after incubation in MG132 for ∼1 h. Immunolabeling with anti-kinetochore antibody (green) is followed by anti–CENP-E labeling (red) taken at the same focal plane. The yellow kinetochores represent colocalization of the two antibodies (closed arrows).

Mentions: The injection of Cdc34 protein might affect the degradation of proteins during prometaphase, either causing an unscheduled degradation or inhibiting a required degradation. Most previous studies with rodent and HeLa cells have shown that proteasome inhibition blocks anaphase onset but does not affect chromosome alignment at the metaphase plate (Sherwood et al., 1993; Wojcik et al., 1996). Similarly, we found that treatment of Ptk1 and LLC-Pk cells with proteasome inhibitors MG132 or β-lactone–induced metaphase arrest without impairment of chromosome movement in prometaphase (Fig. 6 A).


Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes.

Topper LM, Bastians H, Ruderman JV, Gorbsky GJ - J. Cell Biol. (2001)

Proteasome inhibition does not prevent chromosome congression or CENP-E association with kinetochores but arrests cells at metaphase. (A) Live images of a Ptk1 cell incubated in MG132. The cell began chromosome condensation (0 min) after 30-min incubation in MG132. Nuclear envelope breakdown and alignment of chromosomes at the metaphase plate occurred normally. The cell arrested at metaphase as shown in the 50-min image. (B) Cell was detergent extracted and fixed after incubation in MG132 for ∼1 h. Immunolabeling with anti-kinetochore antibody (green) is followed by anti–CENP-E labeling (red) taken at the same focal plane. The yellow kinetochores represent colocalization of the two antibodies (closed arrows).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196447&req=5

fig6: Proteasome inhibition does not prevent chromosome congression or CENP-E association with kinetochores but arrests cells at metaphase. (A) Live images of a Ptk1 cell incubated in MG132. The cell began chromosome condensation (0 min) after 30-min incubation in MG132. Nuclear envelope breakdown and alignment of chromosomes at the metaphase plate occurred normally. The cell arrested at metaphase as shown in the 50-min image. (B) Cell was detergent extracted and fixed after incubation in MG132 for ∼1 h. Immunolabeling with anti-kinetochore antibody (green) is followed by anti–CENP-E labeling (red) taken at the same focal plane. The yellow kinetochores represent colocalization of the two antibodies (closed arrows).
Mentions: The injection of Cdc34 protein might affect the degradation of proteins during prometaphase, either causing an unscheduled degradation or inhibiting a required degradation. Most previous studies with rodent and HeLa cells have shown that proteasome inhibition blocks anaphase onset but does not affect chromosome alignment at the metaphase plate (Sherwood et al., 1993; Wojcik et al., 1996). Similarly, we found that treatment of Ptk1 and LLC-Pk cells with proteasome inhibitors MG132 or β-lactone–induced metaphase arrest without impairment of chromosome movement in prometaphase (Fig. 6 A).

Bottom Line: Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure.Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores.These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

Show MeSH
Related in: MedlinePlus