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Cdh1-anaphase-promoting complex targets Skp2 for destruction in transforming growth factor beta-induced growth inhibition.

ABSTRACT: As a subunit of a ubiquitin ligase, Skp2 is implicated in facilitating cell cycle progression via degradation of various protein targets. We report here that Skp2 is rapidly degraded following cellular stimulation by the cytokine transforming growth factor beta (TGF-beta) and that this degradation stabilizes the cell cycle arrest protein p27. The Skp2 degradation is mediated by Cdh1-anaphase-promoting complex (APC), as shown by depletion of Cdh1 with small interfering RNA, and by reconstitution of ubiquitylation reactions in a purified system. Blockage of Skp2 degradation greatly reduces TGF-beta-induced cell cycle arrest, as does expression of a nondegradable Skp2 mutant. Furthermore, we demonstrate that TGF-beta-induced Skp2 degradation is mediated by the Smad cascade. The degradation of Skp2 stabilizes p27, thereby ensuring TGF-beta-induced cell cycle arrest. These results identify a novel mechanism for tumor suppression by TGF-beta and explain why dysfunction of APC in the TGF-beta pathway in responsive cells is associated with cancer.

SUBMITTER: Liu W

PROVIDER: S-EPMC1899917 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.

Project description:Anaphase promoting complex (APC)-Cdh1 targets multiple mitotic proteins for degradation upon exit from mitosis into G1; inhibitory phosphorylation of Cdh1 by cyclin-dependent kinase (CDK) and Polo kinase has been proposed to prevent the premature degradation of substrates in the ensuing cell cycle. Here, we demonstrate essentiality of CDK phosphorylation of Cdh1 in Saccharomyces cerevisiae by exact endogenous gene replacement of CDH1 with CDK-unphosphorylatable CDH1-m11; in contrast, neither Cdh1 polo kinase sites nor polo interaction motifs are required. CDH1-m11 cells arrest in the first cycle with replicated DNA and sustained polarized growth; most cells have monopolar spindles. Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation. In contrast, expression of undegradable mitotic cyclin results in both SPB separation and the restoration of isotropic growth. A minority of CDH1-m11 cells arrest with short bipolar spindles that fail to progress to anaphase; this can be accounted for by a failure to accumulate Cdc20 and consequent failure to cleave cohesin. Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

| S-EPMC2836972 | biostudies-literature

TGF-? activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth.

Project description:We previously reported that aberrant TGF-?/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27(kip1)(p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation, as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-? signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-? increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-?-mediated inhibition of proliferation. Protein synthesis was not required for TGF-?-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-?-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-? signaling.

| S-EPMC4889277 | biostudies-literature

Regulation of the anaphase-promoting complex-separase cascade by transforming growth factor-beta modulates mitotic progression in bone marrow stromal cells.

Project description:Alteration of the tumor microenvironment by aberrant stromal cells influences many aspects of cell biology, including differentiation of stem cells and tumor metastasis. The role of transforming growth factor (TGF)-beta signaling in stromal cells of the tissue microenvironment is critical to both pathways. We examined murine marrow stromal cells with deletion of Smad3 and found that they have an altered cell cycle profile, with a higher fraction of cells in G2/M phase. Deletion of Smad3 significantly abrogates TGF-beta signaling and suppresses phosphorylation of CDC27-anaphase-promoting complex (APC) during mitosis, thereby resulting in elevated cyclin-dependent kinase (CDK)1 activity via increased levels of cyclin B. Enhanced CDK1 activity due to deregulation of APC leads in turn to hyperphosphorylation of separase, impeding chromatid separation. A residue Ser1126Ala mutation in separase specifically abolished separase hyperphosphorylation in Smad3-deficient cells. The present results unveil a new function for the TGF-beta pathway in the regulation of APC to mediate chromatid separation during mitosis.

| S-EPMC2592668 | biostudies-literature

APC(CDH1) targets MgcRacGAP for destruction in the late M phase.

Project description:BackgroundMale germ cell RacGTPase activating protein (MgcRacGAP) is an important regulator of the Rho family GTPases--RhoA, Rac1, and Cdc42--and is indispensable in cytokinesis and cell cycle progression. Inactivation of RhoA by phosphorylated MgcRacGAP is an essential step in cytokinesis. MgcRacGAP is also involved in G1-S transition and nuclear transport of signal transducer and activator of transcription 3/5 (STAT3/5). Expression of MgcRacGAP is strictly controlled in a cell cycle-dependent manner. However, the underlying mechanisms have not been elucidated.Methodology/principal findingsUsing MgcRacGAP deletion mutants and the fusion proteins of full-length or partial fragments of MgcRacGAP to mVenus fluorescent protein, we demonstrated that MgcRacGAP is degraded by the ubiquitin-proteasome pathway in the late M to G1 phase via APC(CDH1). We also identified the critical region for destruction located in the C-terminus of MgcRacGAP, AA537-570, which is necessary and sufficient for CDH1-mediated MgcRacGAP destruction. In addition, we identified a PEST domain-like structure with charged residues in MgcRacGAP and implicate it in effective ubiquitination of MgcRacGAP.Conclusions/significanceOur findings not only reveal a novel mechanism for controlling the expression level of MgcRacGAP but also identify a new target of APC(CDH1). Moreover our results identify a C-terminal region AA537-570 of MgcRacGAP as its degron.

| S-EPMC3656054 | biostudies-literature

Ama1p-activated anaphase-promoting complex regulates the destruction of Cdc20p during meiosis II.

Project description:The execution of meiotic divisions in Saccharomyces cerevisiae is regulated by anaphase-promoting complex/cyclosome (APC/C)-mediated protein degradation. During meiosis, the APC/C is activated by association with Cdc20p or the meiosis-specific activator Ama1p. We present evidence that, as cells exit from meiosis II, APC/C(Ama1) mediates Cdc20p destruction. APC/C(Ama1) recognizes two degrons on Cdc20p, the destruction box and destruction degron, with either domain being sufficient to mediate Cdc20p destruction. Cdc20p does not need to associate with the APC/C to bind Ama1p or be destroyed. Coimmunoprecipitation analyses showed that the diverged amino-terminal region of Ama1p recognizes both Cdc20p and Clb1p, a previously identified substrate of APC/C(Ama1). Domain swap experiments revealed that the C-terminal WD region of Cdh1p, when fused to the N-terminal region of Ama1p, could direct most of Ama1p functions, although at a reduced level. In addition, this fusion protein cannot complement the spore wall defect in ama1? strains, indicating that substrate specificity is also derived from the WD repeat domain. These findings provide a mechanism to temporally down-regulate APC/C(Cdc20) activity as the cells complete meiosis II and form spores.

| S-EPMC3031463 | biostudies-literature

Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis.

Project description:Ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, and catalyzes the rate-limiting step in DNA precursor synthesis: the reduction of ribonucleotides to deoxyribonucleotides. A strictly balanced supply of deoxyribonucleotides is essential for both accurate DNA replication and repair. Therefore, ribonucleotide reductase activity is under exquisite control both transcriptionally and posttranscriptionally. In proliferating mammalian cells, enzyme activity is regulated by control of R2 protein stability. This control, which responds to DNA damage, is effective until cells pass into mitosis. We demonstrate that the mitotic degradation and hence the overall periodicity of R2 protein levels depends on a KEN box sequence, recognized by the Cdh1-anaphase-promoting complex. The mouse R2 protein specifically binds Cdh1 and is polyubiquitinated in an in vitro ubiquitin assay system. Mutating the KEN signal stabilizes the R2 protein during mitosisG(1) in R2 protein-overexpressing cells. The degradation process, which blocks deoxyribonucleotide production during G(1), may be an important mechanism protecting the cell against unscheduled DNA synthesis. The newly discovered p53-induced p53R2 protein that lacks a KEN box may supply deoxyribonucleotides for DNA repair during G(0)G(1).

| S-EPMC153024 | biostudies-literature

Estrogen inhibits transforming growth factor beta signaling by promoting Smad2/3 degradation.

Project description:Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.

| S-EPMC2863224 | biostudies-literature

Acm1 is a negative regulator of the CDH1-dependent anaphase-promoting complex/cyclosome in budding yeast.

Project description:Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/CCdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Delta strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Delta cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Delta and hsl1Delta resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.

| S-EPMC1698549 | biostudies-literature

STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1.

Project description:The novel oncogene STYK1/NOK plays critical roles in cancer development. However, its regulation during cell division is less defined. In this paper, we show that over-expression of STYK1/NOK caused mitotic arrest and cytokinesis defects. The protein level of STYK/NOK fluctuated during the cell cycle, with a peak at mitosis and a quick reduction upon mitotic exit. The cell cycle-related expression pattern of STYK1/NOK resembled the one of aurora kinases and polo-like kinase 1. Depletion of APC3 led to accumulation of STYK1/NOK and to the G2/M arrest. Co-immunoprecipitation experiment demonstrated the direct interaction of STYK1/NOK with CDH1. Overexpression of CDH1 shortened the half-life of STYK1/NOK. The kinase domain, but not the five D boxes, of STYK1/NOK was responsible for the interaction with CDH1. Altogether, our data demonstrated for the first time that STYK1/NOK could affect cell division, probably by directly targeting key components of APC/C such as CDH1 at late mitosis. Current study may provide a vital mechanistic clue for understanding the roles of STYK1/NOK in mitosis and cytokinesis during STYK1NOK mediated genomic instability and oncogenesis.

| S-EPMC9732331 | biostudies-literature

The anaphase-promoting complex/cyclosome activator Cdh1 modulates Rho GTPase by targeting p190 RhoGAP for degradation.

Project description:Cdh1 is an activator of the anaphase-promoting complex/cyclosome and contributes to mitotic exit and G(1) maintenance by targeting cell cycle proteins for degradation. However, Cdh1 is expressed and active in postmitotic or quiescent cells, suggesting that it has functions other than cell cycle control. Here, we found that homozygous Cdh1 gene-trapped (Cdh1(GT/GT)) mouse embryonic fibroblasts (MEFs) and Cdh1-depleted HeLa cells reduced stress fiber formation significantly. The GTP-bound active Rho protein was apparently decreased in the Cdh1-depleted cells. The p190 protein, a major GTPase-activating protein for Rho, accumulated both in Cdh1(GT/GT) MEFs and in Cdh1-knockdown HeLa cells. Cdh1 formed a physical complex with p190 and stimulated the efficient ubiquitination of p190, both in in vitro and in vivo. The motility of Cdh1-depleted HeLa cells was impaired; however, codepletion of p190 rescued the migration activity of these cells. Moreover, Cdh1(GT/GT) embryos exhibited phenotypes similar to those observed for Rho-associated kinase I and II knockout mice: eyelid closure delay and disruptive architecture with frequent thrombus formation in the placental labyrinth layer, respectively. Furthermore, the p190 protein accumulated in the Cdh1(GT/GT) embryonic tissues. Our data revealed a novel function for Cdh1 as a regulator of Rho and provided insights into the role of Cdh1 in cell cytoskeleton organization and cell motility.

| S-EPMC2916440 | biostudies-literature

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