Oncogene database

6502

SKP2

S-phase kinase-associated protein 2, E3 ubiquitin protein ligase;SKP2;S-phase kinase-associated protein 2, E3 ubiquitin protein ligase

CDK2/cyclin A-associated protein p45|F-box/LRR-repeat protein 1|S-phase kinase-associated protein 2|S-phase kinase-associated protein 2 (p45)|p45skp2

5p13

protein-coding

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin-protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G(1) progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-Ras(G12V) to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

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.

OBJECTIVE: To examine whether oncogenic Ras affects transforming growth factor (TGF)-beta-mediated cell-cycle arrest in hematopoietic cells and the downstream signal transduction pathway involved in the interference with TGF-beta-induced cell-cycle arrest. MATERIALS AND METHODS: Two leukemic cell lines bearing N-Ras(L61) mutations; HL-60 and TF-1, and the M1 cell line with wt Ras were investigated for their response to TGF-beta. Signal transduction inhibitors, overexpression and RNA interference studies were performed to investigate the involvement of the various proteins. RESULTS: Although TGF-beta signal transduction was not affected, G0-G1 arrest was absent in HL-60 and TF-1 cells due to the absence of p27. Overexpression of p27 restored TGF-beta-induced cell-cycle arrest, as well as interfering in Ras-mediated signaling. The farnesyl transferase inhibitor L744832 and the MEK inhibitor U0126 both restored p27 levels and cell-cycle arrest in response to TGF-beta. The absence of p27 protein is due to elevated levels of the ubiquitin ligase SKP2, which complexes with and targets p27 for degradation. RNA interference for SKP2 and treatment of these cells with the proteasome inhibitor MG132 restored p27 levels, corresponding with decreasing SKP2 levels after interfering in N-Ras signal transduction. P27, phosphorylated at threonine 187, is nuclear localized in N-Ras-containing cells. mutation of this residue to alanine rendered p27 insensitive to degradation. CONCLUSION: N-Ras(L61) transformed cells lack a G0-G1 arrest upon TGF-beta treatment due to absence of p27. p27 is degraded through a MapK-, and SKP2-dependent pathway. Overexpression of p27 results in restoration of cell-cycle arrest upon TGF-beta treatment.

S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex. Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpress ERBB2/HER2, additional FOXP3 targets may be involved in its tumor suppressor activity. Here, we show that mammary carcinomas from mice heterozygous for a Foxp3 mutation exhibited increased Skp2 expression. Ectopic expression of FOXP3 in mouse mammary cancer cells repressed SKP2 expression with a corresponding increase in p27 and polyploidy. Conversely, siRNA silencing of the FOXP3 gene in human mammary epithelial cells increased SKP2 expression. We also show that Foxp3 directly interacted with and repressed the Skp2 promoter. Moreover, the analysis of over 200 primary breast cancer samples revealed an inverse correlation between FOXP3 and SKP2 levels. Finally, we demonstrated that downregulation of SKP2 was critical for FOXP3-mediated growth inhibition in breast cancer cells that do not overexpress ERBB2/HER2. Our data provide genetic, biochemical, and functional evidence that FOXP3 is a novel transcriptional repressor for the oncogene SKP2.

Skp2 is an F-box protein that forms the SCF complex with Skp1 and Cullin-1 to constitute an E3 ligase for ubiquitylation. Ubiquitylation and degradation of the p27 are critical for Skp2-mediated entry to the cell cycle, and overexpression and cytosolic accumulation of Skp2 have been clearly associated with tumorigenesis, although the functional significance of the latter is still unknown. Here we show that Akt/protein kinase B (PKB) interacts with and directly phosphorylates Skp2. We find that Skp2 phosphorylation by Akt triggers SCF complex formation and E3 ligase activity. A phosphorylation-defective Skp2 mutant is drastically impaired in its ability to promote cell proliferation and tumorigenesis. Furthermore, we show that Akt-mediated phosphorylation triggers 14-3-3beta-dependent Skp2 relocalization to the cytosol, and we attribute a specific role to cytosolic Skp2 in the positive regulation of cell migration. Finally, we demonstrate that high levels of activation of Akt correlate with the cytosolic accumulation of Skp2 in human cancer specimens. Our results therefore define a novel proto-oncogenic Akt/PKB-dependent signalling pathway.

SKP2 is the ubiquitin ligase subunit that targets p27(KIP1) (p27) for degradation. SKP2 is induced in the G(1)-S transit of the cell cycle, is frequently overexpressed in human cancer, and displays transformation activity in experimental models. Here we show that MYC induces SKP2 expression at the mRNA and protein levels in human myeloid leukemia K562 cells with conditional MYC expression. Importantly, in these systems, induction of MYC did not activate cell proliferation, ruling out SKP2 up-regulation as a consequence of cell cycle entry. MYC-dependent SKP2 expression was also detected in other cell types such as lymphoid, fibroblastic, and epithelial cell lines. MYC induced SKP2 mRNA expression in the absence of protein synthesis and activated the SKP2 promoter in luciferase reporter assays. With chromatin immunoprecipitation assays, MYC was detected bound to a region of human SKP2 gene promoter that includes E-boxes. The K562 cell line derives from human chronic myeloid leukemia. In a cohort of chronic myeloid leukemia bone marrow samples, we found a correlation between MYC and SKP2 mRNA levels. Analysis of cancer expression databases also indicated a correlation between MYC and SKP2 expression in lymphoma. Finally, MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated down-regulation of p27. Our data show that SKP2 is a direct MYC target gene and that MYC-mediated SKP2 induction leads to reduced p27 levels. The results suggest the induction of SKP2 oncogene as a new mechanism for MYC-dependent transformation.

The proto-oncogene c-Myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis. Myc accelerates the rate of cell proliferation, at least in part, through its ability to down-regulate the expression of the cell cycle inhibitor p27(Kip1). Moreover, p27(Kip1) protein levels are regulated by ubiquitin-mediated turnover, leading to destruction by the E3 ubiquitin ligase SCF(Skp2). Therefore, we hypothesize that a lack of Skp2 expression should lead to increased p27(Kip1) levels and further inhibition of Myc-mediated proliferation and tumorigenesis. Myc expression in epithelial tissues of transgenic mice (K5-Myc) led to increased keratinocyte proliferation and the development of spontaneous tumors within the oral cavity. We generated K5-Myc-transgenic mice in an Skp2-null background. Consistent with our hypothesis, we found that Myc-mediated keratinocyte hyperproliferation was abolished by the loss of Skp2. However, Skp2 ablation did not affect Myc-driven tumorigenesis because the incidence, latency, and degree of differentiation of oral tumors were identical between K5-Myc/Skp2(+/+) and K5-Myc/Skp2(-/-) mice. Altogether, these findings suggest that Skp2 and p27(Kip1) are critical for Myc-driven keratinocyte proliferation; however, Myc-mediated tumorigenesis in the oral epithelium is independent of the Skp2-p27(Kip1) axis.

Dysregulated Skp2 function promotes cell proliferation, which is consistent with observations of Skp2 over-expression in many types of human cancers, including cervical carcinoma (CC). However, the molecular mechanisms underlying elevated Skp2 expression have not been fully explored. Interleukin-6 (IL-6) induced Stat3 activation is viewed as crucial for multiple tumor growth and metastasis. Here, we demonstrate that Skp2 is a direct transcriptional target of Stat3 in the human cervical carcinoma cells. Our data show that IL-6 administration or transfection of a constitutively activated Stat3 in HeLa cells activates Skp2 mRNA transcription. Using luciferase reporter and ChIP assays, we show that Stat3 binds to the promoter region of Skp2 and promotes its activity through recruiting P300. As a result of the increase of Skp2 expression, endogenous p27 protein levels are markedly decreased. Thus, our results suggest a previously unknown Stat3-Skp2 molecular network controlling cervical carcinoma development.

Substantial evidence supports the oncogenic role of the E3 ubiquitin ligase S-phase kinase-associated protein 2 (Skp2) in many types of cancers through its ability to target a broad range of signaling effectors for ubiquitination. Thus, this oncogenic E3 ligase represents an important target for cancer drug discovery. In this study, we report a novel mechanism by which CG-12, a novel energy restriction-mimetic agent (ERMA), down-regulates the expression of Skp2 in prostate cancer cells. Pursuant to our previous finding that upregulation of beta-transducin repeat-containing protein (beta-TrCP) expression represents a cellular response in cancer cells to ERMAs, including CG-12 and 2-deoxyglucose, we demonstrated that this beta-TrCP accumulation resulted from decreased Skp2 expression. Evidence indicates that Skp2 targets beta-TrCP for degradation via the cyclin-dependent kinase 2-facilitated recognition of the proline-directed phosphorylation motif (412)SP. This Skp2 downregulation was attributable to Sirt1-dependent suppression of COP9 signalosome (Csn)5 expression in response to CG-12, leading to increased cullin 1 neddylation in the Skp1-cullin1-F-box protein complex and consequent Skp2 destabilization. Moreover, we determined that Skp2 and beta-TrCP are mutually regulated, providing a feedback mechanism that amplifies the suppressive effect of ERMAs on Skp2. Specifically, cellular accumulation of beta-TrCP reduced the expression of Sp1, a beta-TrCP substrate, which, in turn, reduced Skp2 gene expression. This Skp2-beta-TrCP-Sp1 feedback loop represents a novel crosstalk mechanism between these two important F-box proteins in cancer cells with aberrant Skp2 expression under energy restriction, which provides a proof-of-concept that the oncogenic Csn5/Skp2 signaling axis represents a "druggable" target for this novel ERMA.

The Skp2 (S-phase kinase associated protein 2) oncoprotein is often highly expressed in various types of human cancers. However, the mechanistic basis of its oncogenic function, as well as the upstream regulatory pathway(s) that control Skp2 activities remains not fully understood. Recently, we reported that p300 acetylates Skp2 at two conserved lysine residues K68 and K71 within its NLS (Nuclear localization signal). This modification leads to increased Skp2 stability and cytoplasmic translocation, thus contributing to elevated Skp2 oncogenic potential. Moreover, we found that the SIRT3 tumor suppressor serves as the physiological deacetylase that antagonizes p300-mediated Skp2 acetylation. Furthermore, we showed that Skp2 governs E-cadherin ubiquitination and degradation in the cytosol. Consistent with this, we observed an inverse correlation between Skp2 and E-cadherin expression in clinical breast tumor samples. Therefore, our work elucidates a novel acetylation-dependent regulatory mechanism for Skp2 oncogenic functions.

Hepatitis B X-interacting protein (HBXIP) is a novel oncoprotein. In this study, we found that the expression levels of HBXIP were positively associated with those of S-phase kinase-associated protein 2 (Skp2) in clinical breast cancer tissues and cell lines. Moreover, we found that HBXIP was able to stimulate the promoter of Skp2 through binding to the -640/-443 region in Skp2 promoter involving activating E2F transcription factor 1 (E2F1). Skp2 plays crucial roles in HBXIP-enhanced proliferation of breast cancer cells in vitro and in vivo. We conclude that HBXIP up-regulates Skp2 via activating E2F1 to promote proliferation of breast cancer cells.

Altered DAB2IP gene expression often detected in prostate cancer (PCa) is due to epigenetic silencing. In this study, we unveil a new mechanism leading to the loss of DAB2IP protein; an oncogenic S-phase kinase-associated protein-2 (Skp2) as E3 ubiquitin ligase plays a key regulator in DAB2IP degradation. In order to unveil the role of Skp2 in the turnover of DAB2IP protein, both prostate cell lines and prostate cancer specimens with a variety of molecular and cell biologic techniques were employed. We demonstrated that DAB2IP is regulated by Skp2-mediated proteasome degradation in the prostate cell lines. Further analyses identified the N-terminal DAB2IP containing the ubiquitination site. Immunohistochemical study exhibited an inverse correlation between DAB2IP and Skp2 protein expression in the prostate cancer tissue microarray. In contrast, DAB2IP can suppressSkp2 protein expression is mediated through Akt signaling. The reciprocal regulation between DAB2IP and Skp2 can impact on the growth of PCa cells. This reciprocal regulation between DAB2IP and Skp2 protein represents a unique homeostatic balance between tumor suppressor and oncoprotein in normal prostate epithelia, which is apparently altered in cancer cells. The outcome of this study has identified new potential targets for developing new therapeutic strategy for PCa.