Limits...
Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.

Sudakin V, Chan GK, Yen TJ - J. Cell Biol. (2001)

Bottom Line: We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity.Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20.Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

View Article: PubMed Central - PubMed

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

ABSTRACT
The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

Show MeSH

Related in: MedlinePlus

Model for MCC function and regulation. MCC is present throughout the cell cycle but is incapable of inhibiting APC/C until it has undergone mitotic modifications. APC/C is normally rapidly activated in mitosis by phosphorylations and association with CDC20. These modifications may also be recognized by the MCC so that APC/C is prevented from ubiquitinating target proteins until all chromosomes are aligned. We speculate that the interaction between MCC and APC/C is not stable so APC/C is not permanently inhibited. In the presence of unattached kinetochores (•), signals initiated at kinetochores modify the APC/C or APC-bound MCC to enhance their interactions and thus prolong its inhibition. The signal from the kinetochore can either be MAD2 as proposed or a kinase cascade that is initiated at the kinetochore by hBUBR1, hBUB1, or other kinases. After chromosomes align, the signal from kinetochores decays along with the modifications. The MCC then dissociates and APC/C becomes active to drive cells out of mitosis. We believe that MCC may not inhibit APC/C during interphase because it either lacks the appropriate phosphorylations or the APC/C is bound to a different substrate specificity factor, CDH1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196190&req=5

fig8: Model for MCC function and regulation. MCC is present throughout the cell cycle but is incapable of inhibiting APC/C until it has undergone mitotic modifications. APC/C is normally rapidly activated in mitosis by phosphorylations and association with CDC20. These modifications may also be recognized by the MCC so that APC/C is prevented from ubiquitinating target proteins until all chromosomes are aligned. We speculate that the interaction between MCC and APC/C is not stable so APC/C is not permanently inhibited. In the presence of unattached kinetochores (•), signals initiated at kinetochores modify the APC/C or APC-bound MCC to enhance their interactions and thus prolong its inhibition. The signal from the kinetochore can either be MAD2 as proposed or a kinase cascade that is initiated at the kinetochore by hBUBR1, hBUB1, or other kinases. After chromosomes align, the signal from kinetochores decays along with the modifications. The MCC then dissociates and APC/C becomes active to drive cells out of mitosis. We believe that MCC may not inhibit APC/C during interphase because it either lacks the appropriate phosphorylations or the APC/C is bound to a different substrate specificity factor, CDH1.

Mentions: The finding that MCC is present during interphase and can inhibit the APC/C was unexpected because kinetochores (which are not present during interphase) are thought to generate the inhibitor of the APC/C. However, we found that only APC/C from mitotic cells was sensitive to MCC inhibition. These findings have changed our view of the checkpoint pathway in two important ways (Fig. 8) . First, our data suggest that the formation and activity of the APC/C inhibitor can be uncoupled from kinetochores. This may be an important feature considering that APC/C is known to be activated at the onset of mitosis. The existence of a preformed pool of inhibitor would rapidly block precocious ubiquitination activity by the APC/C at the onset of mitosis. It is noteworthy that only the mitotically modified form of the APC/C can be inhibited (Fig. 6, B and C; Fig. 7 A), which can explain why the preformed pool of MCC doesn't inhibit APC before mitosis. By necessity, the inhibition must be reversible so that APC/C can be activated once cells are ready to exit mitosis. We believe that in vivo, the interaction between APC/C and MCC is quite labile in the absence of unattached kinetochores. This would explain why APC/C that is purified from mitotic cells exhibits a lag before it becomes reactivated.


Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.

Sudakin V, Chan GK, Yen TJ - J. Cell Biol. (2001)

Model for MCC function and regulation. MCC is present throughout the cell cycle but is incapable of inhibiting APC/C until it has undergone mitotic modifications. APC/C is normally rapidly activated in mitosis by phosphorylations and association with CDC20. These modifications may also be recognized by the MCC so that APC/C is prevented from ubiquitinating target proteins until all chromosomes are aligned. We speculate that the interaction between MCC and APC/C is not stable so APC/C is not permanently inhibited. In the presence of unattached kinetochores (•), signals initiated at kinetochores modify the APC/C or APC-bound MCC to enhance their interactions and thus prolong its inhibition. The signal from the kinetochore can either be MAD2 as proposed or a kinase cascade that is initiated at the kinetochore by hBUBR1, hBUB1, or other kinases. After chromosomes align, the signal from kinetochores decays along with the modifications. The MCC then dissociates and APC/C becomes active to drive cells out of mitosis. We believe that MCC may not inhibit APC/C during interphase because it either lacks the appropriate phosphorylations or the APC/C is bound to a different substrate specificity factor, CDH1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196190&req=5

fig8: Model for MCC function and regulation. MCC is present throughout the cell cycle but is incapable of inhibiting APC/C until it has undergone mitotic modifications. APC/C is normally rapidly activated in mitosis by phosphorylations and association with CDC20. These modifications may also be recognized by the MCC so that APC/C is prevented from ubiquitinating target proteins until all chromosomes are aligned. We speculate that the interaction between MCC and APC/C is not stable so APC/C is not permanently inhibited. In the presence of unattached kinetochores (•), signals initiated at kinetochores modify the APC/C or APC-bound MCC to enhance their interactions and thus prolong its inhibition. The signal from the kinetochore can either be MAD2 as proposed or a kinase cascade that is initiated at the kinetochore by hBUBR1, hBUB1, or other kinases. After chromosomes align, the signal from kinetochores decays along with the modifications. The MCC then dissociates and APC/C becomes active to drive cells out of mitosis. We believe that MCC may not inhibit APC/C during interphase because it either lacks the appropriate phosphorylations or the APC/C is bound to a different substrate specificity factor, CDH1.
Mentions: The finding that MCC is present during interphase and can inhibit the APC/C was unexpected because kinetochores (which are not present during interphase) are thought to generate the inhibitor of the APC/C. However, we found that only APC/C from mitotic cells was sensitive to MCC inhibition. These findings have changed our view of the checkpoint pathway in two important ways (Fig. 8) . First, our data suggest that the formation and activity of the APC/C inhibitor can be uncoupled from kinetochores. This may be an important feature considering that APC/C is known to be activated at the onset of mitosis. The existence of a preformed pool of inhibitor would rapidly block precocious ubiquitination activity by the APC/C at the onset of mitosis. It is noteworthy that only the mitotically modified form of the APC/C can be inhibited (Fig. 6, B and C; Fig. 7 A), which can explain why the preformed pool of MCC doesn't inhibit APC before mitosis. By necessity, the inhibition must be reversible so that APC/C can be activated once cells are ready to exit mitosis. We believe that in vivo, the interaction between APC/C and MCC is quite labile in the absence of unattached kinetochores. This would explain why APC/C that is purified from mitotic cells exhibits a lag before it becomes reactivated.

Bottom Line: We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity.Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20.Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

View Article: PubMed Central - PubMed

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

ABSTRACT
The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

Show MeSH
Related in: MedlinePlus