Phosphorylation of Skp2 regulated by CDK2 and Cdc14B protects it from degradation by APC(Cdh1) in G1 phase

The p27(Kip1) ubiquitin ligase receptor Skp2 is often overexpressed in human tumours and displays oncogenic properties. The activity of SCF(Skp2) is regulated by the APC(Cdh1), which targets Skp2 for degradation. Here we show that Skp2 phosphorylation on Ser64/Ser72 positively regulates its function in vivo. Phosphorylation of Ser64, and to a lesser extent Ser72, stabilizes Skp2 by interfering with its association with Cdh1, without affecting intrinsic ligase activity. Cyclin-dependent kinase (CDK)2-mediated phosphorylation of Skp2 on Ser64 allows its expression in mid-G1 phase, even in the presence of active APC(Cdh1). Reciprocally, dephosphorylation of Skp2 by the mitotic phosphatase Cdc14B at the M --> G1 transition promotes its degradation by APC(Cdh1). Importantly, lowering the levels of Cdc14B accelerates cell cycle progression from mitosis to S phase in an Skp2-dependent manner, demonstrating epistatic relationship of Cdc14B and Skp2 in the regulation of G1 length. Thus, our results reveal that reversible phosphorylation plays a key role in the timing of Skp2 expression in the cell cycle.     

Nuclear translocation of Skp2 facilitates its destruction in response to TGFβ signaling

Skp2, a F-box protein that determines the substrate specificity for SCF ubiquitin ligase, has recently been demonstrated to be degraded by Cdh1/APC in response to TGFβ signaling. The TGFβ-induced Skp2 proteolysis results in the stabilization of p27 that is necessary to facilitate TGFβ cytostatic effect. Previous observation from immunocytochemistry indicates that Cdh1 principally localizes in the nucleus while Skp2 mainly localizes in the cytosol, which leaves us a puzzle on how Skp2 is recognized and then ubiquitylated by Cdh1/APC in response to TGFβ stimulation. Here, we report that Skp2 is rapidly translocated from the cytosol to the nucleus upon the cellular stimulation with TGFβ. Using a combinatorial approach of immunocytochemistry, biochemical-fraction-coupled immunoprecipitation, mutagenesis as well as protein degradation assay, we have demonstrated that the TGFβ-induced Skp2 nucleus translocation is critical for TGFβ cytostatic effect that allows physical interaction between Cdh1 and Skp2 and in turn facilitates the Skp2 ubquitylation by Cdh1/APC. Disruption of nuclear localization motifs on Skp2 stabilizes Skp2 in the presence of TGF-β signaling, which attenuates TGFβ-induced p27 accumulation and antagonizes TGFβ-induced growth inhibition. Our finding reveals a cellular mechanism that facilitates Skp2 ubiquitylation by Cdh1/APC in response to TGFβ.     

Nuclear translocation of Skp2 facilitates its destruction in response to TGFβ signaling

Skp2, a F-box protein that determines the substrate specificity for SCF ubiquitin ligase, has recently been demonstrated to be degraded by Cdh1/APC in response to TGFβ signaling. The TGFβ-induced Skp2 proteolysis results in the stabilization of p27 that is necessary to facilitate TGFβ cytostatic effect. Previous observation from immunocytochemistry indicates that Cdh1 principally localizes in the nucleus while Skp2 mainly localizes in the cytosol, which leaves us a puzzle on how Skp2 is recognized and then ubiquitylated by Cdh1/APC in response to TGFβ stimulation. Here, we report that Skp2 is rapidly translocated from the cytosol to the nucleus upon the cellular stimulation with TGFβ. Using a combinatorial approach of immunocytochemistry, biochemical-fraction-coupled immunoprecipitation, mutagenesis as well as protein degradation assay, we have demonstrated that the TGFβ-induced Skp2 nucleus translocation is critical for TGFβ cytostatic effect that allows physical interaction between Cdh1 and Skp2 and in turn facilitates the Skp2 ubquitylation by Cdh1/APC. Disruption of nuclear localization motifs on Skp2 stabilizes Skp2 in the presence of TGFβ signaling, which attenuates TGFβ-induced p27 accumulation and antagonizes TGFβ-induced growth inhibition. Our finding reveals a cellular mechanism that facilitates Skp2 ubiquitylation by Cdh1/APC in response to TGFβ.Key words: Skp2, nuclear translocation, ubiquitylation, TGFβ     

Regulation of Skp2 Levels by the Pim-1 Protein Kinase

The Pim-1 protein kinase plays an important role in regulating both cell growth and survival and enhancing transformation by multiple oncogenes. The ability of Pim-1 to regulate cell growth is mediated, in part, by the capacity of this protein kinase to control the levels of the p27, a protein that is a critical regulator of cyclin-dependent kinases that mediate cell cycle progression. To understand how Pim-1 is capable of regulating p27 protein levels, we focused our attention on the SCF^Skp2 ubiquitin ligase complex that controls the rate of degradation of this protein. We found that expression of Pim-1 increases the level of Skp2 through direct binding and phosphorylation of multiple sites on this protein. Along with known Skp2 phosphorylation sites including Ser⁶⁴ and Ser⁷², we have identified Thr⁴¹⁷ as a unique Pim-1 phosphorylation target. Phosphorylation of Thr⁴¹⁷ controls the stability of Skp2 and its ability to degrade p27. Additionally, we found that Pim-1 regulates the anaphase-promoting complex or cyclosome (APC/C complex) that mediates the ubiquitination of Skp2. Pim-1 phosphorylates Cdh1 and impairs binding of this protein to another APC/C complex member, CDC27. These modifications inhibit Skp2 from degradation. Marked increases in Skp2 caused by these mechanisms lower cellular p27 levels. Consistent with these observations, we show that Pim-1 is able to cooperate with Skp2 to signal S phase entry. Our data reveal a novel Pim-1 kinase-dependent signaling pathway that plays a crucial role in cell cycle regulation.     

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

We previously reported that aberrant TGF-β/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-β 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-β 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-β-mediated inhibition of proliferation. Protein synthesis was not required for TGF-β-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-β-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-β signaling.     

Notch3 overexpression causes arrest of cell cycle progression by inducing Cdh1 expression in human breast cancer cells

Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27^Kip accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27^Kip at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27^Kip accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

Dual mode of degradation of Cdc25 A phosphatase

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G(1) is mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment.     

Regulation of APC/CCdh1 ubiquitin ligase in differentiation of human embryonic stem cells

We have recently shown that Skp2 levels are high in undifferentiated human embryonic stem cells, but decline rapidly following induction of differentiation, thereby leading to accumulation of p27. Changes in Skp2 levels were found to be caused mainly by its rate of degradation. Here we show that the activity of APC/C^Cdh1, the ubiquitin ligase that targets Skp2 for degradation, increases markedly during the differentiation process of human embryonic stem cells. APC/C^Cdh1 is present but inactive in undifferentiated embryonic stem cells and becomes active in the differentiated state. The rise in APC/C^Cdh1 activity with differentiation appears to be due, at least in part, to a dramatic decline in the levels of its inhibitor Emi1. In addition, protein kinase activity also appears to contribute to the suppression of APC/C^Cdh1 activity in undifferentiated stem cells, possibly by inhibitory phosphorylation of Cdh1.     

Rapamycin potentiates transforming growth factor beta-induced growth arrest in nontransformed,oncogene-transformed,and human cancer cells

Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.     

Hsl1p, a Swe1p inhibitor, is degraded via the anaphase-promoting complex

Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events. The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome. Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates. Hsl1p is absent in G(1), accumulates as cells begin to bud, and disappears in late mitosis. Hsl1p is stabilized by mutations in CDH1 and CDC23, both of which result in compromised APC activity. Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G(1)-arrested cells containing active APC. Mutation of a destruction box motif within Hsl1p (Hsl1p(db-mut)) stabilized Hsl1p. Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies. These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination. Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCF(Met30) in a bud morphogenesis checkpoint. Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events.     

Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p.

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).     

The anaphase-promoting complex or cyclosome supports cell survival in response to endoplasmic reticulum stress

The anaphase-promoting complex or cyclosome (APC/C) is a multi-subunit ubiquitin ligase that regulates exit from mitosis and G1 phase of the cell cycle. Although the regulation and function of APC/C(Cdh1) in the unperturbed cell cycle is well studied, little is known of its role in non-genotoxic stress responses. Here, we demonstrate the role of APC/C(Cdh1) (APC/C activated by Cdh1 protein) in cellular protection from endoplasmic reticulum (ER) stress. Activation of APC/C(Cdh1) under ER stress conditions is evidenced by Cdh1-dependent degradation of its substrates. Importantly, the activity of APC/C(Cdh1) maintains the ER stress checkpoint, as depletion of Cdh1 by RNAi impairs cell cycle arrest and accelerates cell death following ER stress. Our findings identify APC/C(Cdh1) as a regulator of cell cycle checkpoint and cell survival in response to proteotoxic insults.