Phosphorylation of Ser72 does not regulate the ubiquitin ligase activity and subcellular localization of Skp2

Skp2 is the substrate binding subunit of the SCFSkp2 ubiquitin ligase, which plays a key role in the regulation of cell cycle progression. The activity of Skp2 is regulated by the APCCdh1, which targets Skp2 for degradation in early G1 and prevent premature S phase entry. Overexpression of Skp2 leads to dysregulation of the cell cycle and is commonly observed in human cancers. We have previously shown that Skp2 is phosphorylated on Ser64 and Ser72 in vivo, and that these modifications regulate its stability. Recently, two studies have proposed a role for Ser72 phosphorylation in the cytosolic relocalization of Skp2 and in the assembly and activity of SCFSkp2 ubiquitin ligase complex. We have revisited this question and analyzed the impact of Ser72 phosphorylation site mutations on the biological activity and subcellular localization of Skp2. We show here that phosphorylation of Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCFSkp2 ubiquitin ligase activity, and does not affect the subcellular localization of Skp2 in a panel of cell lines.     

The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway, allowing CDK activity to drive cells into S phase. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5-8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate.     

The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCFSkp2 ubiquitin ligase

The Skp2 oncoprotein belongs to the family of F-box proteins that function as substrate recognition factors for SCF (Skp1, cullin, F-box protein) E3 ubiquitin-ligase complexes. Binding of the substrate to the SCFSkp2 complex catalyzes the conjugation of ubiquitin molecules to the bound substrate, resulting in multi-ubiquitination and rapid degradation by the 26 S proteasome. Using Skp2 as bait in a yeast two-hybrid screen, we have identified UBP43 as a novel substrate for Skp2. UBP43 belongs to the family of ubiquitin isopeptidases and specifically cleaves ISG15, a ubiquitin-like molecule that is induced by cellular stresses, such as type 1 interferons (IFN), nephrotoxic damage, and bacterial infection. UBP43 was originally identified as an up-regulated gene in knock-in mice expressing an acute myelogenous leukemia fusion protein, AML1-ETO, as well as in melanoma cell lines treated with IFN-beta. The phenotype of UBP43 knockout mice includes shortened life span, hypersensitivity to IFN, and neuronal damage, suggesting that tight regulation of ISG15 conjugation is critical for normal cellular function. In this study, we demonstrate that UBP43 is ubiquitinated in vivo and accumulates in cells treated with proteasome inhibitors. We also show that Skp2 promotes UBP43 ubiquitination and degradation, resulting in higher levels of ISG15 conjugates. In Skp2-/- mouse cells, levels of UBP43 are consistently up-regulated, whereas levels of ISG15 conjugates are reduced. Our results demonstrate that the SCFSkp2 is involved in controlling UBP43 protein levels and may therefore play an important role in modulating type 1 IFN signaling.     

Activin A induces phosphorylation of Smad2 but not complex formation of Smad2 with Smad4 in human colon cancer cell line HT-29

Western blotting coupled with immunoprecipitation showed that activin A treatment induced phosphorylation of Smad2 but not complex formation of Smad2/4 in human colon cancer-derived HT-29 cells. Because HT-29 cells expressed neither Smad4 mRNA nor Smad4 protein, it is suggested that deletion of Smad4 leads to a defect of formation of Smad2/4 complex upon activin A stimulation in HT-29 cells.     

Phosphorylation of Ser72 is dispensable for Skp2 assembly into an active SCF ubiquitin ligase and its subcellular localization

F-box proteins are the substrate recognition subunits of SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes. Skp2 is a nuclear F-box protein that targets the CDK inhibitor p27 for ubiquitin- and proteasome-dependent degradation. In G0 and during the G1 phase of the cell cycle, Skp2 is degraded via the APC/CCdh1 ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G0/G1 state. APC/CCdh1 binds Skp2 through an N-terminal domain (amino acids 46-94 in human Skp2). It has been shown that phosphorylation of Ser69 and Ser72 in this domain dissociates Skp2 from APC/C. More recently, it has instead been proposed that phosphorylation of Skp2 on Ser72 by Akt/PKB allows Skp2 binding to Skp1, promoting the assembly of an active SCFSkp2 ubiquitin ligase, and Skp2 relocalization/retention into the cytoplasm, promoting cell migration via an unknown mechanism. According to these reports, a Skp2 mutant in which Ser72 is substituted with Ala is unable to promote cell proliferation and loses its oncogenic potential. Given the contrasting reports, we revisited these results and conclude that phosphorylation of Skp2 on Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCFSkp2 ubiquitin ligase activity, and does not affect the subcellular localization of Skp2.     

Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.     

A negatively charged amino acid in Skp2 is required for Skp2-Cks1 interaction and ubiquitination of p27Kip1

Proteolysis of cyclin-dependent kinase inhibitor p27 occurs predominantly in the late G1 phase of the cell cycle through a ubiquitin-mediated protein degradation pathway. Ubiquitination of p27 requires the SCFSkp2 ubiquitin ligase and Skp2 F-box binding protein Cks1. The mechanisms by which Skp2 recognizes Cks1 to ubiquitylate p27 remain obscure. Here we show that Asp-331 in the carboxyl terminus of Skp2 is required for its association with Cks1 and ubiquitination of p27. Mutation of Asp-331 to Ala disrupts the interaction between Skp2 and Cks1. Although Asp-331 mutation negates the ability of the Skp1-Cullin-F-box protein (SCF) complex to ubiquitylate p27, such a mutation has no effect on Skp2 self-ubiquitination. A conservative change from Asp to Glu at position 331 of Skp2 does not affect Skp2-Cks1 interaction. Our results revealed a unique requirement for a negatively charged residue in the carboxyl-terminal region of Skp2 in recognition of Cks1 and ubiquitination of p27.     

Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation

Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Here, we identify Smurf2, a C2-WW-HECT domain ubiquitin ligase and show that Smurf2 associates constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGF beta receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFN gamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGF beta receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGF beta receptor for degradation.     

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

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.     

Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase

The cyclin-dependent kinase inhibitor p21Cip1 has important roles in the control of cell proliferation, differentiation, senescence, and apoptosis. It has been observed that p21 is a highly unstable protein, but the mechanisms of its degradation remained unknown. We show here that p21 is a good substrate for an SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, which contains the F-box protein Skp2 (S phase kinase-associated protein 2) and the accessory protein Cks1 (cyclin kinase subunit 1). A similar ubiquitin ligase complex has been previously shown to be involved in the degradation of a related cyclin-dependent kinase inhibitor, p27Kip1. The levels of Skp2 oscillate in the cell cycle, reaching a maximum in S phase. The ubiquitylation of p21 in vitro required the supplementation of all components of the SCF complex as well as of Cks1 and Cdk2-cyclin E. The protein kinase Cdk2-cyclin E acts both by the phosphorylation of p21 on Ser-130 and by the formation of a complex with p21, which is required for its presentation to the ubiquitin ligase. As opposed to the case of p27, the phosphorylation of p21 stimulates its ubiquitylation but is not absolutely required for this process. Levels of p21 are higher in Skp2-/- mouse embryo fibroblasts than in wild-type fibroblasts in the S phase, and the rates of the degradation of p21 are slower in cells that lack Skp2. It is suggested that SCFSkp2 participates in the degradation of p21 in the S phase.     

The papillomavirus E7 oncoprotein is ubiquitinated by UbcH7 and Cullin 1- and Skp2-containing E3 ligase

Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2(-/-) mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.     

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.     

Acetylation-dependent regulation of Skp2 function

Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.