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Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC.

Chan GK, Jablonski SA, Sudakin V, Hittle JC, Yen TJ - J. Cell Biol. (1999)

Bottom Line: Cell. 89:727-735).Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells.The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E-dependent activities at the kinetochore and regulating cyclosome/APC activity.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

ABSTRACT
Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1. hBUB1 and hBUBR1 bind to kinetochores where they are postulated to be components of the mitotic checkpoint that monitors kinetochore activities to determine if chromosomes have achieved alignment at the spindle equator (Jablonski, S.A., G.K.T. Chan, C.A. Cooke, W.C. Earnshaw, and T.J. Yen. 1998. Chromosoma. 107:386-396). In support of this, hBUB1 and the homologous mouse BUB1 have been shown to be important for the mitotic checkpoint (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Willson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300-303; Taylor, S.S., and F. McKeon. 1997. Cell. 89:727-735). We now demonstrate that hBUBR1 is also an essential component of the mitotic checkpoint. hBUBR1 is required by cells that are exposed to microtubule inhibitors to arrest in mitosis. Additionally, hBUBR1 is essential for normal mitotic progression as it prevents cells from prematurely entering anaphase. We establish that one of hBUBR1's checkpoint functions is to monitor kinetochore activities that depend on the kinetochore motor CENP-E. hBUBR1 is expressed throughout the cell cycle, but its kinase activity is detected after cells have entered mitosis. hBUBR1 kinase activity was rapidly stimulated when the spindle was disrupted in mitotic cells. Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells. The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E-dependent activities at the kinetochore and regulating cyclosome/APC activity.

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hBUBR1 expression, phosphorylation, and kinase activity during the cell cycle. (A) K562 and Hela cells were separated according to their position in the cell cycle by centrifugal elutriation. hBUBR1 was immunoprecipitated from equal amounts of protein from each fraction, and then tested for autokinase activity (top) or probed with hBUBR1 antibodies to compare its cell cycle expression profile (middle and bottom). (B) Comparison of autokinase activity (top) and levels of hBUBR1 (middle) and hBUB3 found in hBUBR1 immunoprecipitates from asynchronous interphase cells (A) or cells blocked in mitosis by nocodazole (N). (C) hBUBR1 is hyperphosphorylated in mitosis. hBUBR1 immunoprecipitated from mitotically blocked Hela cells were incubated in the presence (lane 1) or absence (lane 2) of lambda protein phosphatase (λPP). (lanes 3–8) Comparison of hyperphosphorylation status of hBUBR1 in normal mitotic Hela cells (lane 3, M), nocodazole-blocked mitotic cells (lane 4, N), or cells released from the nocodazole block for 1, 2, 3, and 4 h (lanes 5–8, respectively). Cells were determined to have reentered G1 by phase-contrast microscopy.
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Figure 5: hBUBR1 expression, phosphorylation, and kinase activity during the cell cycle. (A) K562 and Hela cells were separated according to their position in the cell cycle by centrifugal elutriation. hBUBR1 was immunoprecipitated from equal amounts of protein from each fraction, and then tested for autokinase activity (top) or probed with hBUBR1 antibodies to compare its cell cycle expression profile (middle and bottom). (B) Comparison of autokinase activity (top) and levels of hBUBR1 (middle) and hBUB3 found in hBUBR1 immunoprecipitates from asynchronous interphase cells (A) or cells blocked in mitosis by nocodazole (N). (C) hBUBR1 is hyperphosphorylated in mitosis. hBUBR1 immunoprecipitated from mitotically blocked Hela cells were incubated in the presence (lane 1) or absence (lane 2) of lambda protein phosphatase (λPP). (lanes 3–8) Comparison of hyperphosphorylation status of hBUBR1 in normal mitotic Hela cells (lane 3, M), nocodazole-blocked mitotic cells (lane 4, N), or cells released from the nocodazole block for 1, 2, 3, and 4 h (lanes 5–8, respectively). Cells were determined to have reentered G1 by phase-contrast microscopy.

Mentions: Having established that hBUBR1 is an essential component of the mitotic checkpoint, we wanted to investigate its mechanism of action by characterizing its expression and kinase activity as a function of the cell cycle. hBUBR1 was immunoprecipitated from K562 erythroleukemic cells and Hela cells that were separated into different phases of the cell cycles by centrifugal elutriation. Portions of the immunocomplexes were used to determine steady-state levels of protein and kinase activity. We assayed the immunoprecipitated hBUBR1 for autokinase activity as a recent study showed that a transfected hBUBR1 exhibited autokinase activity (Taylor et al. 1998). However, hBUBR1 isolated from K562 and Hela (data not shown) exhibited no detectable autokinase activity (Fig. 5 A, top) or kinase activities towards exogenous substrates (data not shown) even though it was expressed throughout the cell cycle of both cell types (Fig. 5 A, middle and bottom). hBUBR1 levels remained fairly constant throughout the cell cycle of K562 cells, whereas hBUBR1 levels in Hela cells fluctuated with the cell cycle. hBUBR1 level was lowest in G1 and steadily increased as cells progressed towards mitosis.


Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC.

Chan GK, Jablonski SA, Sudakin V, Hittle JC, Yen TJ - J. Cell Biol. (1999)

hBUBR1 expression, phosphorylation, and kinase activity during the cell cycle. (A) K562 and Hela cells were separated according to their position in the cell cycle by centrifugal elutriation. hBUBR1 was immunoprecipitated from equal amounts of protein from each fraction, and then tested for autokinase activity (top) or probed with hBUBR1 antibodies to compare its cell cycle expression profile (middle and bottom). (B) Comparison of autokinase activity (top) and levels of hBUBR1 (middle) and hBUB3 found in hBUBR1 immunoprecipitates from asynchronous interphase cells (A) or cells blocked in mitosis by nocodazole (N). (C) hBUBR1 is hyperphosphorylated in mitosis. hBUBR1 immunoprecipitated from mitotically blocked Hela cells were incubated in the presence (lane 1) or absence (lane 2) of lambda protein phosphatase (λPP). (lanes 3–8) Comparison of hyperphosphorylation status of hBUBR1 in normal mitotic Hela cells (lane 3, M), nocodazole-blocked mitotic cells (lane 4, N), or cells released from the nocodazole block for 1, 2, 3, and 4 h (lanes 5–8, respectively). Cells were determined to have reentered G1 by phase-contrast microscopy.
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Related In: Results  -  Collection

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Figure 5: hBUBR1 expression, phosphorylation, and kinase activity during the cell cycle. (A) K562 and Hela cells were separated according to their position in the cell cycle by centrifugal elutriation. hBUBR1 was immunoprecipitated from equal amounts of protein from each fraction, and then tested for autokinase activity (top) or probed with hBUBR1 antibodies to compare its cell cycle expression profile (middle and bottom). (B) Comparison of autokinase activity (top) and levels of hBUBR1 (middle) and hBUB3 found in hBUBR1 immunoprecipitates from asynchronous interphase cells (A) or cells blocked in mitosis by nocodazole (N). (C) hBUBR1 is hyperphosphorylated in mitosis. hBUBR1 immunoprecipitated from mitotically blocked Hela cells were incubated in the presence (lane 1) or absence (lane 2) of lambda protein phosphatase (λPP). (lanes 3–8) Comparison of hyperphosphorylation status of hBUBR1 in normal mitotic Hela cells (lane 3, M), nocodazole-blocked mitotic cells (lane 4, N), or cells released from the nocodazole block for 1, 2, 3, and 4 h (lanes 5–8, respectively). Cells were determined to have reentered G1 by phase-contrast microscopy.
Mentions: Having established that hBUBR1 is an essential component of the mitotic checkpoint, we wanted to investigate its mechanism of action by characterizing its expression and kinase activity as a function of the cell cycle. hBUBR1 was immunoprecipitated from K562 erythroleukemic cells and Hela cells that were separated into different phases of the cell cycles by centrifugal elutriation. Portions of the immunocomplexes were used to determine steady-state levels of protein and kinase activity. We assayed the immunoprecipitated hBUBR1 for autokinase activity as a recent study showed that a transfected hBUBR1 exhibited autokinase activity (Taylor et al. 1998). However, hBUBR1 isolated from K562 and Hela (data not shown) exhibited no detectable autokinase activity (Fig. 5 A, top) or kinase activities towards exogenous substrates (data not shown) even though it was expressed throughout the cell cycle of both cell types (Fig. 5 A, middle and bottom). hBUBR1 levels remained fairly constant throughout the cell cycle of K562 cells, whereas hBUBR1 levels in Hela cells fluctuated with the cell cycle. hBUBR1 level was lowest in G1 and steadily increased as cells progressed towards mitosis.

Bottom Line: Cell. 89:727-735).Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells.The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E-dependent activities at the kinetochore and regulating cyclosome/APC activity.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

ABSTRACT
Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1. hBUB1 and hBUBR1 bind to kinetochores where they are postulated to be components of the mitotic checkpoint that monitors kinetochore activities to determine if chromosomes have achieved alignment at the spindle equator (Jablonski, S.A., G.K.T. Chan, C.A. Cooke, W.C. Earnshaw, and T.J. Yen. 1998. Chromosoma. 107:386-396). In support of this, hBUB1 and the homologous mouse BUB1 have been shown to be important for the mitotic checkpoint (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Willson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300-303; Taylor, S.S., and F. McKeon. 1997. Cell. 89:727-735). We now demonstrate that hBUBR1 is also an essential component of the mitotic checkpoint. hBUBR1 is required by cells that are exposed to microtubule inhibitors to arrest in mitosis. Additionally, hBUBR1 is essential for normal mitotic progression as it prevents cells from prematurely entering anaphase. We establish that one of hBUBR1's checkpoint functions is to monitor kinetochore activities that depend on the kinetochore motor CENP-E. hBUBR1 is expressed throughout the cell cycle, but its kinase activity is detected after cells have entered mitosis. hBUBR1 kinase activity was rapidly stimulated when the spindle was disrupted in mitotic cells. Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells. The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E-dependent activities at the kinetochore and regulating cyclosome/APC activity.

Show MeSH
Related in: MedlinePlus