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Cdc20 directs proteasome-mediated degradation of the tumor suppressor SMAR1 in higher grades of cancer through the anaphase promoting complex.

ABSTRACT: The Tumor suppressor SMAR1 (scaffold matrix attachment region binding protein 1) has a crucial role in maintaining genomic stability, cell cycle progression and apoptosis.Our previous finding showed that it is highly suppressed in higher grade of cancer. However, the underlying mechanism of this suppression was not well understood. In this study, we show that SMAR1 expression levels are controlled at the proteasomal level by five RING finger E3 ubiquitin ligases including, Cdc20, a substrate receptor of ubiquitin ligase APC/C complex. We found that Cdc20 binds and promotes proteasomal degradation of SMAR1 in a D-box motif dependent manner. Further, our results demonstrated that Cdc20 promotes proteasomal degradation of SMAR1 through K48-linked specific polyubiquitylation, and that short hairpin RNA mediated inactivation of Cdc20 leads to significant stabilization of SMAR1. These findings suggest that Cdc20 is responsible for maintaining the cellular levels of SMAR1. However, since Cdc20 fails to target SMAR1 upon exposure to genotoxic stresses, SMAR1 helps to maintain genomic stability under these conditions through its DNA damage repair activity. Interestingly, Cdc20-mediated degradation of SMAR1 promotes cell migration and invasion.The reciprocal relationship of the duo is evident in breast cancer cell lines as well as in patient samples, suggesting that Cdc20 functions as an important negative regulator of SMAR1 in higher grades of cancer. Our study reveals for the first time, the molecular mechanism associated with lower levels of expression of the important tumor suppressor SMAR1 in higher grades of breast cancer.

SUBMITTER: Paul D

PROVIDER: S-EPMC5520925 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The G2/M regulator histone demethylase PHF8 is targeted for degradation by the anaphase-promoting complex containing CDC20.

Project description:Monomethylated histone H4 lysine 20 (H4K20me1) is tightly regulated during the cell cycle. The H4K20me1 demethylase PHF8 transcriptionally regulates many cell cycle genes and is therefore predicted to play key roles in the cell cycle. Here, we show that PHF8 protein levels are the highest during G2 phase and mitosis, and we found PHF8 protein stability to be regulated by the ubiquitin-proteasome system. Purification of the PHF8 complex led to the identification of many subunits of the anaphase-promoting complex (APC) associated with PHF8. We showed that PHF8 interacts with the CDC20-containing APC (APC(cdc20)) primarily during mitosis. In addition, we defined a novel, KEN- and D-box-independent, LXPKXLF motif on PHF8 that is required for binding to CDC20. Through various in vivo and in vitro assays, we demonstrate that mutations of the LXPKXLF motif abrogate polyubiquitylation of PHF8 by the APC. APC substrates are typically cell cycle regulators, and consistent with this, the loss of PHF8 leads to prolonged G2 phase and defective mitosis. Furthermore, we provide evidence that PHF8 plays an important role in transcriptional activation of key G2/M genes during G2 phase. Taken together, these findings suggest that PHF8 is regulated by APC(cdc20) and plays an important role in the G2/M transition.

| S-EPMC3811896 | biostudies-other

Cdc20 is required for the post-anaphase, KEN-dependent degradation of centromere protein F.

Project description:Progression through mitosis and cytokinesis requires the sequential proteolysis of several cell-cycle regulators. This proteolysis is mediated by the ubiquitin-proteasome system, with the E3 ligase being the anaphase-promoting complex, also known as the cyclosome (APC/C). The APC/C is regulated by two activators, namely Cdc20 and Cdh1. The current view is that prior to anaphase, the APC/C is activated by Cdc20, but that following anaphase, APC/C switches to Cdh1-dependent activation. However, here we present an analysis of the kinetochore protein Cenp-F that is inconsistent with this notion. Although it has long been appreciated that Cenp-F is degraded sometime during or after mitosis, exactly when and how has not been clear. Here we show that degradation of Cenp-F initiates about six minutes after anaphase, and that this is dependent on a C-terminal KEN-box. Although these two observations are consistent with Cenp-F being a substrate of Cdh1-activated APC/C, Cenp-F is degraded normally in Cdh1-null cells. By contrast, RNAi-mediated repression of APC/C subunits or Cdc20 does inhibit Cenp-F degradation. These findings therefore suggest that the APC/C does not simply 'switch' upon anaphase onset; rather, our observations indicate that Cdc20 also contributes to post-anaphase activation of the APC/C. We also show that the post-anaphase, KEN-box-dependent degradation of Cenp-F requires it to be farnesylated, a post-translational modification usually linked to membrane association. Because so many of the behaviours of Cenp-F are farnesylation-dependent, we suggest that this modification plays a more global role in Cenp-F function.

| S-EPMC2816182 | biostudies-literature

HTLV-I Tax directly binds the Cdc20-associated anaphase-promoting complex and activates it ahead of schedule.

Project description:Expression of the human T lymphotropic virus type I (HTLV-I) transactivator/oncoprotein, Tax, leads to faulty mitosis as reflected by chromosome aneuploidy, cytokinesis failure, and formation of micro- and multinucleated cells. Here we show that HTLV-I-transformed T cells progress through S/G(2)/M phases of the cell cycle with a delay. This delay is correlated with a decrease in the levels of cyclin A, cyclin B1, and securin. In tax-expressing cells, the Cdc20-associated anaphase promoting complex (APC(Cdc20)), an E3 ubiquitin ligase that controls metaphase to anaphase transition, becomes active before cellular entry into mitosis as evidenced by premature cyclin B1 polyubiquitination and degradation during S/G(2). Consistent with the notion that Tax activates APC(Cdc20) directly, Tax is found to coimmunoprecipitate with Cdc20 and Cdc27/APC3. The APC(Cdc20) activity prematurely activated by Tax remains sensitive to spindle checkpoint inhibition. Unscheduled activation of APC(Cdc20) by Tax provides an explanation for the mitotic abnormalities in HTLV-I-infected cells and is likely to play an important role in the development of adult T cell leukemia.

| S-EPMC544051 | biostudies-literature

Substrate degradation by the anaphase promoting complex occurs during mitotic slippage.

Project description:Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.

| S-EPMC3163903 | biostudies-literature

Regulated degradation of spindle assembly factors by the anaphase-promoting complex.

Project description:The ubiquitin ligase anaphase-promoting complex (APC/C) is essential for cell division in all eukaryotes. Loss of APC/C activity arrests cells at metaphase and results in severe aberrations of the mitotic spindle, but how the APC/C regulates spindle formation is not understood. Here, we report that the APC/C promotes the ubiquitination and degradation of four proteins required for Ran-dependent spindle assembly: Bard1, Hmmr, HURP, and NuSAP. Among these substrates, HURP and NuSAP can be degraded during spindle formation when the spindle checkpoint is active. Their degradation requires additional layers of regulation, and both SAFs are only degraded after being released from their inhibitor importin beta by Ran(GTP). Our findings reveal a tightly regulated mechanism by which the APC/C and the GTPase Ran control the abundance of active spindle assembly factors to achieve the accurate formation of the mitotic spindle.

| S-EPMC2883244 | biostudies-literature

Degradation of p21Cip1 through anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20) ubiquitin ligase complex-mediated ubiquitylation is inhibited by cyclin-dependent kinase 2 in cardiomyocytes.

Project description:Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.

| S-EPMC3243522 | biostudies-literature

Deregulation of Rb-E2F1 axis causes chromosomal instability by engaging the transactivation function of Cdc20-anaphase-promoting complex/cyclosome.

Project description:The E2F family of transcription factors regulates genes involved in various aspects of the cell cycle. Beyond the well-documented role in G1/S transition, mitotic regulation by E2F has also been reported. Proper mitotic progression is monitored by the spindle assembly checkpoint (SAC). The SAC ensures bipolar separation of chromosomes and thus prevents aneuploidy. There are limited reports on the regulation of the SAC by E2F. Our previous work identified the SAC protein Cdc20 as a novel transcriptional regulator of the mitotic ubiquitin carrier protein UbcH10. However, none of the Cdc20 transcription complex proteins have any known DNA binding domain. Here we show that an E2F1-DP1 heterodimer is involved in recruitment of the Cdc20 transcription complex to the UBCH10 promoter and in transactivation of the gene. We further show that inactivation of Rb can facilitate this transactivation process. Moreover, this E2F1-mediated regulation of UbcH10 influences mitotic progression. Deregulation of this pathway results in premature anaphase, chromosomal abnormalities, and aneuploidy. We conclude that excess E2F1 due to Rb inactivation recruits the complex of Cdc20 and the anaphase-promoting complex/cyclosome (Cdc20-APC/C) to deregulate the expression of UBCH10, leading to chromosomal instability in cancer cells.

| S-EPMC4272426 | biostudies-literature

Proteasome-dependent disruption of the E3 ubiquitin ligase anaphase-promoting complex by HCMV protein pUL21a.

Project description:The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109-110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity.

| S-EPMC3390409 | biostudies-other

Anaphase promoting complex-dependent degradation of transcriptional repressors Nrm1 and Yhp1 in Saccharomyces cerevisiae.

Project description:The anaphase-promoting complex/cyclosome (APC/C) is an essential ubiquitin ligase that targets cell cycle proteins for proteasome-mediated degradation in mitosis and G1. The APC regulates a number of cell cycle processes, including spindle assembly, mitotic exit, and cytokinesis, but the full range of its functions is still unknown. To better understand cellular pathways controlled by the APC, we performed a proteomic screen to identify additional APC substrates. We analyzed cell cycle-regulated proteins whose expression peaked during the period when other APC substrates were expressed. Subsequent analysis identified several proteins, including the transcriptional repressors Nrm1 and Yhp1, as authentic APC substrates. We found that APC(Cdh1) targeted Nrm1 and Yhp1 for degradation in early G1 through Destruction-box motifs and that the degradation of these repressors coincided with transcriptional activation of MBF and Mcm1 target genes, respectively. In addition, Nrm1 was stabilized by phosphorylation, most likely by the budding yeast cyclin-dependent protein kinase, Cdc28. We found that expression of stabilized forms of Nrm1 and Yhp1 resulted in reduced cell fitness, due at least in part to incomplete activation of G1-specific genes. Therefore, in addition to its known functions, APC-mediated targeting of Nrm1 and Yhp1 coordinates transcription of multiple genes in G1 with other cell cycle events.

| S-EPMC3128521 | biostudies-literature

The anaphase-promoting complex regulates the degradation of the inner nuclear membrane protein Mps3.

Project description:The nucleus is enclosed by the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). While the ONM is continuous with the endoplasmic reticulum (ER), the INM is independent and separates the nucleoplasm from the ER lumen. Turnover of ER proteins has been well characterized by the ER-associated protein degradation (ERAD) pathway, but very little is known about turnover of resident INM proteins. Here we show that the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase, regulates the degradation of Mps3, a conserved integral protein of the INM. Turnover of Mps3 requires the ubiquitin-conjugating enzyme Ubc7, but was independent of the known ERAD ubiquitin ligases Doa10 and Hrd1 as well as the recently discovered Asi1-Asi3 complex. Using a genetic approach, we have found that Cdh1, a coactivator of APC/C, modulates Mps3 stability. APC/C controls Mps3 degradation through Mps3's N terminus, which resides in the nucleoplasm and possesses two putative APC/C-dependent destruction motifs. Accumulation of Mps3 at the INM impairs nuclear morphological changes and cell division. Our findings therefore reveal an unexpected mechanism of APC/C-mediated protein degradation at the INM that coordinates nuclear morphogenesis and cell cycle progression.

| S-EPMC6400550 | biostudies-literature

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