Novel insights into the INK4-CDK4/6-Rb pathway: Counter action of gankyrin against INK4 proteins regulates the CDK4-mediated phosphorylation of Rb.
[Cell Cycle 4:10, 1335-1337, October 2005]; ©2005 Landes Bioscience
The Oncoprotein Gankyrin Negatively Regulates Both p53 and RB by Enhancing Proteasomal Degradation Extra View
ABSTRACT Ubiquitin-dependent proteolysis mediates selective destruction of various cell cycle regulators, transcription factors and tumor suppressors. Gankyrin, a seven ankyrin-repeat protein, was originally identified as an oncoprotein commonly overexpressed in hepatocellular carcinomas and independently as a protein associated with the 19S regulatory complex of the 26S proteasome. Gankyrin also binds to CDK4 and the tumor suppressor RB, and accelerates phosphorylation and proteasomal degradation of RB. Recently, we have shown that gankyrin has an anti-apoptotic activity in cells exposed to DNA-damaging agents. Gankyrin binds to MDM2, a major E3 ubiquitin ligase for p53, and increases ubiquitylation and degradation of p53. Gankyrin increases activities of CDK4 and MDM2, and facilitates targeting of polyubiquitylated proteins to the 26S proteasome. Furthermore, inhibition of gankyrin induces apoptosis in cancer cells. Therefore, gankyrin is a promising target for potential anticancer therapeutic agents.
of Intracellular Proteolysis; School of Biomedical Sciences; University of Nottingham Medical School; Nottingham, UK
*Correspondence to: Jun Fujita; Department of Clinical Molecular Biology; Graduate School of Medicine; Kyoto University; 54 Shogoin Kawaharacho; Sakyo-ku; Kyoto 606-8507, Japan; Tel.: 81.75.751.3751; Fax: 81.75.751.3750; Email: jfujita@ virus.kyoto-u.ac.jp Received 08/15/05; Accepted 08/17/05
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gankyrin, cancer therapy, liver cancer, Mdm2, oncogene, proteasome, p53, RB, tumor suppressor, ubiquitin
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Hepatocellular carcinoma (HCC) is a malignancy of worldwide significance.1 HCC is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. Although screening of high risk populations by ultrasonography and measurement of the serum α-fetoprotein level has facilitated the early detection of HCC, a majority (70 to 85%) of patients present with advanced or unresectable disease. Even for those patients who undergo surgical resection, the recurrence rates can be as high as 50% at 2 years, and nonsurgical therapies are ineffective or minimally effective at best. It is, therefore, important to identify molecules that can be used to develop novel diagnostic, preventive or therapeutic strategies. We have identified 19 genes overexpressed in HCCs by constructing subtracted cDNA libraries including 2 novel genes.2 One of these genes was named HSCO (Hepatoma Subtracted-cDNA library Clone One).3 HSCO was overexpressed in 20 of 30 HCCs examined, accelerates nuclear export of NF-κB, and suppresses p53-induced apoptosis. The other gene was named gankyrin (gann ankyrin-repeat protein; “Gann” is the Japanese word for cancer).4 Independently, gankyrin was identified as the p28 component of the 26S proteasome and a protein that specifically interacts with the S6b/TBP7 ATPase of the PA700/19S regulatory complex of the 26S proteasome.5,6 Gankyrin/PSMD10 consists of 226 amino acids that encode a 25-kDa protein with seven ankyrin repeats.4,7 The ankyrin repeat is a functional domain involved in protein-protein interactions. Gankyrin is highly conserved throughout evolution (40% identity to yeast Nas6P)5 and is localized on human chromosome Xq22.3. Pseudogenes have been identified on chromosome 3 and 20. There are four isoforms of gankyrin expressed in human cells (Fig. 1 and data not shown) but most work has been done on the wild type (isoform 1). Amazingly, gankyrin expression is increased in all (34 of 34) HCCs compared with noncancerous liver tissues,4 and similar findings were subsequently made by others.8 In a rodent model of chemical hepato-carcinogenesis, gankyrin expression was induced from the earliest stage of tumor development, preceding the loss of RB protein and adenoma formation.9 Given that almost all HCCs overexpress gankyrin, what roles does it play in carcinogenesis? An initial hint was provided by the finding that gankyrin contains a domain consisting of the RB-recognition motif LxCxE (178LACDE182). As expected, gankyrin, but not RB related proteins p107 or p130, bound RB in vitro and in vivo.4 Overexpression of gankyrin in immortalized mouse fibroblasts and human tumor cell lines conferred ability to grow in soft agar. This activity of gankyrin was correlated with the ability to bind RB (e.g., the LxCxE point mutant E182A is inactive). Gankyrin facilitates the phosphorylation and degradation of RB, suggesting that increased expression of gankyrin promotes tumorigenicity by targeting RB to the proteasome.4 Gankyrin inhibits the RB tumor suppressor pathway at another level. Gankyrin also binds cyclin-dependent
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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2107
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of Clinical Molecular Biology; Graduate School of Medicine; Kyoto University; Kyoto, Japan
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Hiroaki Higashitsuji1 Yu Liu1 R. John Mayer2 Jun Fujita1,*
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This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Japan Society for the Promotion of Science, and the Smoking Research Foundation of Japan. The work in the UK was sponsored by EU Framework IV, the BBSRC and the University of Nottingham.
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The Oncoprotein Gankyrin Negatively Regulates Both p53 and RB by Enhancing Proteasomal Degradation
kinase 4 (CDK4), competing with p16INK4A, an inhibitor of cyclin kinases, for binding to CDK4.6,10 Thus, by inhibiting p16 INK4A, gankyrin activates CDK4, resulting in the hyperphosphorylation of RB, enhancement of E2F1-mediated transcription and cell cycle progression. Gankyrin seems to play a role in cell cycle progression in normal cells as well. The upregulation of gankyrin correlates with cell cycle progression in normal rat primary hepatocytes and the liver tissue of patients with fulminant hepatic failure, a model of active proliferation of human hepatocytes associated with liver regeneration.11 Structural and biochemical analyses have suggested that the N-terminal 3 or 4 ankyrin repeats of gankyrin are involved in binding to CDK4, and the 6th ankyrin repeat in binding to RB.7,12,13 Interestingly, gankyrin Figure 1. Binding characteristics of isoforms and mutants of gankyrin. Wild type gankyrin isoforms 2 and 3 lack the putative RB-binding region, (isoform1) consists of seven ankyrin repaeats (AK1 to AK7). Presence (+) or absence (-) of and isoforms 3 and 4 have deletions in the putative binding to MDM2, RB, S6b subunit of the 26S proteasome, MAGE-A4 and CDK4 are shown CDK4-binding region (Fig. 1). However, the physio- on the right (data from refs. 4, 6, 10, 15, and 25). The isoform 1 in ref. 15 has been logic role of these isoforms and how expression of renamed to isoform 4 here. *, results with mutants lacking AK6 and AK7; **, results with mutants lacking AK5, AK6 and AK7; ND, not determined. gankyrin and its isoforms is regulated in HCCs and in cells in general have not yet been explored. The 26S proteasome is a large multi-protein complex that selectively degrades proteins conjugated to ubiquitin.14 Ubiquitin is first activated in an ATP-dependent manner by a ubiquitin-activating enzyme (E1). It is then transferred to an ubiquitin-conjugating enzyme (E2), and to an ubiquitin protein ligase (E3). An additional step involving the activity of an E4 enzyme may also be required for efficient polyubiquitylation. Recently, we have found that gankyrin suppresses p53-dependent apoptosis in tumor cells.15 This could be explained, at least partly, by the fact that gankyrin binds to and potentiates the activity of MDM2, an E3 ubiquitin ligase that negatively regulates p53. We have demonstrated that this interaction occurs between endogenous gankyrin and MDM2 in nonmalignant as well as malignant cells. Gankyrin facilitates binding of MDM2 to p53, and enhances the ability of MDM2 to mono- and poly-ubiquitylate p53. The data further suggests that, gankyrin recruits a MDM2/p53 complex to the 26S proteasome and accelerates the degradation of p53 in an MDM2-dependent manner.15 Several mechanisms are known by which polyubiquitylated substrates are delivered and recognized by the 26S proteasome.14,16 Polyubiquitin chains are specifically bound by the proteasomal subunits, Rpn10/S5a and Rpt5/S6’. One family of substrate-delivery proteins contains a ubiquitin-like (UBL) domain and a ubiquitin associated (UBA) domain (Fig. 2). The UBA domain is a polyubiquitin binding domain, and the UBL Figure 2. Delivering polyubiquitylated substrates to the 26S proteasome. domain interacts with the 26S proteasome. As exemplified by the Polyubiquitin chains ((Ub)n, where n ≥ 4) are specifically bound by the proteasomal subunits. One family of substrate-delivery factor contains a ubiquitin protein ligase parkin, that consists of an N-terminal UBL ubiquitin-like domain and a ubiquitin associated domain, with the former and a C-terminal RING finger-type E3 ligase, some E3s can deliver binding to the proteasome and the latter to polyubiquitins. If an E3 ubiquitin ubiquitylated substrates directly to the proteasome. Association with protein ligase has a ubiquitin-like domain, it may also work as a delivery gankyrin, an interactor with the S6b subunit of the 26S proteasome, factor. Gankyrin probably delivers ubiquitylated p53 to the proteasome by provides a novel mechanism for targeting a ubiquitylated substrate binding to MDM2 as well as the S6b subunit of the 26S proteasome. (p53) directly to the proteasome by an E3 (MDM2) (Fig. 2). Gankyrin does not bind to other E3s for p53 including E6-AP, COP1 or Pirh2.15 Whether gankyrin affects activity of other E3s, HPV E6 binds directly to p53, and targets it for degradation by especially those for RB, remains to be elucidated. recruiting the cellular HECT ubiquitin ligase E6-AP.18 Adenovirus Viral oncoproteins such as HPV E7 and adenovirus E1A block E1B 55K binds to p53 and helps target its degradation by the 26S the function of RB, which results in the production of the ARF, proteasome.19 Interestingly, HPV E7, adenovirus E1A, and gankyrin leading to stabilization of p53.17 The viruses counteract these cellular all interact with ATPases of the 19S regulatory complex of the 26S defenses by producing proteins that inhibit the function of p53. proteasome in addition to their effects on RB.20 The importance of 1336
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The Oncoprotein Gankyrin Negatively Regulates Both p53 and RB by Enhancing Proteasomal Degradation
the dual inactivation of both the RB and p53 pathways for oncogenesis has also been demonstrated in an animal model of malignant transformation.21 Gankyrin inhibits both RB and p53 and functions as a transforming oncoprotein. In this sense, gankyrin imitates the function of the Large T antigen of SV40 virus.22 Clinically, p53 mutation is not so frequent in HCCs, especially in low-grade or low-stage HCCs.23 Consistent with this, hypermethylation of the p16INK4A gene and p53 mutation appeared at a late stage in a rodent hepatocarcinogenesis model, whereas gankyrin overexpression was observed from early after carcinogen treatment.9 Taken together, these results suggest that gankyrin has important roles from early stages of hepatocarcinogenesis by inhibiting wild type p53, RB and p16INK4A. Moreover, in HCC cells, downregulation of gankyrin expression by RNAi increased p53 protein levels and activity, and promoted apoptosis.15,24 Overexpression of MAGE-A4, another gankyrin interactor of unknown function, suppresses the tumorigenic activity of gankyrin.25,26 Thus, gankyrin could be a good target for cancer therapy. Currently, response rates of HCCs to systemic chemotherapy is only 0 to 25%, and has never been shown to prolong survival of the patients.1 Blocking expression and/or function of gankyrin will be a valuable therapeutic and/or preventive strategy against human HCCs.
18. Weissman AM. Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2001; 2:169-78. 19. Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell 2002; 2:103-12. 20. Ferrell K, Wilkinson CR, Dubiel W, Gordon C. Regulatory subunit interactions of the 26S proteasome, a complex problem. Trends Biochem Sci 2000; 25:83-8. 21. Bardeesy N, Bastian BC, Hezel A, Pinkel D, DePinho RA, Chin L. Dual inactivation of RB and p53 pathways in RAS-induced melanomas. Mol Cell Biol 2001; 21:2144-53. 22. Pipas JM, Levine AJ. Role of T antigen interactions with p53 in tumorigenesis. Semin Cancer Biol 2001; 11:23-30. 23. Konishi M, Kikuchi-Yanoshita R, Tanaka K, Sato C, Tsuruta K, Maeda Y, Koike M, Tanaka S, Nakamura Y, Hattori N, et al. Genetic changes and histopathological grades in human hepatocellular carcinomas. Jpn J Cancer Res 1993; 84:893-9. 24. Li H, Fu X, Chen Y, Hong Y, Tan Y, Cao H, Wu M, Wang H. Use of adenovirus-delivered siRNA to target oncoprotein p28GANK in hepatocellular carcinoma. Gastroenterology 2005; 128:2029-41. 25. Nagao T, Higashitsuji H, Nonoguchi K, Sakurai T, Dawson S, Mayer RJ, Itoh K, Fujita J. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity. J Biol Chem 2003; 278:10668-74. 26. Sakurai T, Itoh K, Higashitsuji H, Nagao T, Nonoguchi K, Chiba T, Fujita J. A cleaved form of MAGE-A4 binds to Miz-1 and induces apoptosis in human cells. J Biol Chem 2004; 279:15505-14.
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