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Gankyrin Oncoprotein: Structure, Function, and Involvement in Cancer Junan Li*,1,2 and Yi Guo3 1
Division of Environmental Health Sciences, College of Public Health; 2Comprehensive Cancer Center; 3Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA Abstract: Gankyrin, a newly defined oncoprotein also known as PSMD10 and P28, functions as a dual-negative regulator of the two most prominent tumor suppressor pathways, the CDK/pRb and HDM2/P53 pathways. Its aberrant expression has been prevalently found in human hepatocellular carcinomas (HCC) and esophagus squamous cell carcinomas (ESCC), indicative of the potential of gankyrin as a rational diagnostic and therapeutic target in cancers. Here, we review the unique structural features and functional diversity of gankyrin, and discuss its implication in cancer diagnostics and therapeutics from the perspective of chemical biology.
Keywords: Structure and function, gankyrin, oncoprotein, cancer. INTRODUCTION Recently, a novel oncogenic protein, gankyrin has caused a lot of attention due to its roles in cell cycle progression, apoptosis and tumorigenesis. The gene encoding this protein was initially identified as a potential oncogene prevalently over-expressed in human hepatocellular carcinomas in Japan, and it was named gankyrin (gann ankyrin-repeat protein; gann means “cancer” in Japanese) due to the fact that this protein is composed of seven ankyrin repeats (ARs), conserved motifs first found in human ankyrin [1]. Shortly after the first report of the gankyrin cDNA sequence, an identical sequence was independently cloned and the protein product, namely P28 or PSMD10, was described as a novel regulatory subunit associated with the 26S proteasome, which could function in the proteasome-mediated degradation [2]. Later on, genes encoding fission yeast (NP_593722.1), mouse (BAA36869.1), rat (NP_446377.1), Golden hamster (AAN76708.1), wolf (XP_538135.1), and zebrafish (ABD43170.1) have been identified. As shown in Fig. (1), the sequence identities of gankyrin proteins vary among species. The sequence homologies between human and other mammals are higher than 90%, which is consistent with the fact that the proteasome system is highly conserved among mammals. While there is a homology of 72% between human and zebrafish gankyrin, the sequence homology between human and fission yeast is relatively low, 29%, however, most of residues important for the sketch structure of ankyrin repeats are conserved, implying that human and fission yeast gankyrin proteins could have a structural similarity higher than the sequence homology. THE STRUCTURE OF GANKYRIN The structure of human gankyrin has been determined by X-ray crystallography [3] and NMR spectroscopy [4] independently. As shown in Fig. (2A), gankyrin is exclusively composed of seven ARs (different from the previous predic-
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tion of six ARs), each of which exhibits a canonical helixturn-helix conformation with the two helices in an antiparallel fashion [5]. These seven ankyrin repeats are stacked together near linearly to form a helix bundle, and neighboring ankyrin repeats are linked by loops of varied size, which orientate perpendicularly to the axes of the helices of ankyrin repeats. While it is generally described as “linearly stacked”, a slight bending of the repeat stack toward the -hairpin loop can be clearly discerned for gankyrin [5]. Like most of ankyrin repeat proteins, there is no disulfide bond or longrange intramolecular interaction present in gankyrin, and the elongated structure is mainly stabilized through inter- and intra-ankyrin repeat hydrophobic interactions predominantly associated with conserved nonpolar residues in the helical regions as well as hydrogen bonding interactions between polar residues and the main chain atoms from adjacent ankyrin repeats [6, 7]. The crystal structures of mouse gankyrin [8] and yeast Nas6 (the fission yeast version of gankyrin) [9, 10] have also been reported. No significant difference has been found in the sketch structures of these gankyrin proteins, especially at the helical regions, even though the sequence homology between human gankyrin and yeast Nas6 is about 29%. Moreover, the solution structure of gankyrin is almost superimposable to that of gankyrin in complex with the Cterminal domain of S6 ATPase [11], implying that free gankyrin is in a biologically active conformation. DIVERSE FUNCTIONS OF GANKYRIN As described earlier, gankyrin belongs to the ankyrin repeat protein class, and proteins in this class are involved in numerous physiological processes exclusively through mediating protein/protein interactions [6, 7]. A number of important proteins have been identified as physiological targets for gankyrin binding and modulating, some of which, such as MDM2 [12], pRb [1], cyclin-dependent kinase (CDK) 4 [13, 14], play pivotal roles in cell cycle progression, apoptosis, and tumorigenesis. Gankyrin and Rb, P53 In their previous study [1], Higashitsuji and his colleagues demonstrated that gankyrin binds to pRb through a © 2010 Bentham Science Publishers Ltd.
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Fig. (1). Sequence alignments of human, wolf, mouse, rat, hamster, zebrafish, and yeast gankryin proteins. The identification codes for these gankyrin proteins are NM_002814, NCBI XP_538135.1, GenBank BAA36869.1, NCBI NP_446377.1, GenBank AAN76708.1, GenBank ABD43170.1, and NCBI NP_593722.1, respectively. The alignment was performed using the Clustal W2 software provided by EMBL_EBI (www.expasy.ch). Rectangles represent helices.
conserved pRb-binding motif LxCxE at its C-terminus, and such binding is essential for gankyrin-induced transformation of NIH 3T3 fibroblasts. More importantly, overexpression of gankyrin led to increased pRb hyperphosphorylation (loss of suppressor activity), activation of the E2F transcription factor (activates the expression of DNA synthesis genes) and accelerated the degradation of pRb, suggesting that increased expression of gankyrin could promote tumorigenicity by targeting pRb to the proteasome (Fig. 3) [1]. Interestingly, it has been shown that gankyrin is able to modulate the pRb pathway through an alternative mechanism. That is, gankyrin competes with P16 as well as other INK4 proteins for binding to CDK4 and preludes the latter’s inhibition to the kinase activity of CDK4, resulting in enhanced pRb phosphorylation and concomitant deregulation of E2F1mediated transcription and cell cycle progression [14]. Evidently, these studies indicate that gankyrin deactivates the pRb pathway at multiple levels, including by direct binding to pRb and facilitating its degradation and by ensuring its inactivation through the maintenance of CDK4 kinase activity. More recently, the role of gankyrin in tumorigenesis has been expanded to the disruption of the P53 tumor suppressor
pathway [12, 15]. It has been shown that gankyrin binds to HDM2, an E3 ubiquitin ligase, enhances the ability of HDM2 to ubiquitinate P53 [12, 16, 17]. Consequently, gankyrin recruits the HDM2 and P53 complex to the protease and fosters the turnover of P53 in an HDM2-dependent manner. Moreover, silencing gankyrin expression by RNA interference led to increased P53 protein and activity, thus promoting apoptosis. In addition, it has been reported that pRb inhibits MDM2-mediated P53 ubiquitination in a gankyrin-dependent manner and the Rb-gankyrin interaction is critical for pRb-induced P53 stabilization [18]. Furthermore, acute ablation of pRb facilitates gankyrin-mediated P53 destablization and desensitizes cancer cells for chemotherapyinduced apoptosis, indicating that Rb antagonizes gankyrin to inhibit MDM2-mediated P53 ubiquitination in cancer cells and the status of both P53 and Rb is important for efficacy of cancer chemotherapy [18]. Taken together, gankyrin functions as a dual-negative regulator in both pRb and P53 pathways (Fig. 3). Gankyrin and the Proteasome The 26S proteasome is the primary component of a major non-lysosomal proteolytic system that is responsible for the degradation of a wide variety of intracellular proteins, in-
Structure/Function Relationship of Gankyrin
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Fig. (2). Solution structure of human gankyrin. A, Structure of gankyrin with identified target-binding domains; B, Tertiary structures of CDK4-interacting AR proteins, gankyrin, P16, P18, and IB67-302. The first four ARs of P18, IB67-302, and gankyrin are superimposed with the corresponding ARs in P16. While the coordinates of IB67-302 (yellow) are derived from the crystal structure of IB/NF B complex, the structures of P16 (green), P18 (blue), and gankyrin (magenta) are solution structures solved by NMR in our laboratory. In both A and B, N and C represent the N- and C- termini, respectively.
cluding tumor suppressor genes, transcription factors, and proteins that regulate the cell cycle [2, 19-21]. The 26S proteasome is a large, multi-subunit, multi-catalytic protease found in the nucleus and cytosol of all eukaryotic cells, and it is composed of a 700 kD, 28 subunit protease called the 20S proteasome that is capped at one or both ends by the 900 kD 19S activator. The 19S activator consists of six AAA ATPase subunits that may form a ring structure to unfold proteins targeted for proteasome degradation and translocate them into the 20S proteasome’s central proteolytic chamber. Other 19S subunits include enzymes that perform processing or editing roles and structural proteins that serve as docking platforms for binding partners. Gankyrin is a subunit of the 19S activator by virtue of its interaction with the C-terminal domain of the 19S S6b ATPases [2, 11] and appears to be a shuttle protein for transporting ubiquitinated proteins, such as P53 to the proteasome for degradation [17]. Disruption of the possible p28 homolog in yeast (NAS6) resulted in nondisruption of the 26S proteasome, further implicating its supportive role in protein degradation [2]. Gankyrin and MAGE A4, RelA In addition to the aforementioned roles in the P53 and pRb pathways, gankyrin has been found to be able to interact with melanoma antigen (MAGE)-A4, a tumor specific antigen with potential in antitumor immunotherapy [22]. This interaction is mediated by the C-terminal half of MAGE-A4, and is very specific since other MAGE family proteins structurally similar to MAGE-A4, i.e. MAGE-A1, MAGE-A2, and MAGE-A12 do not bind to gankyrin. While it remains
yet to know how gankyrin binding influences the function of MAGE-A4, it has been shown that MAGE-A4 binding partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of gankyrinoverexpressing cells, indicating that MAGE-A4 could counteract against the oncogenic activity of gankyrin. Moreover, gankyrin is involved in the regulation of the IB/NF-B pathway [23, 24]. On one hand, gankyrin directly binds to NF-B/RelA and exports RelA from nucleus through a chromosomal region maintanence-1 (CRM-1) dependent pathway, thus suppressing the nuclear translocation of NFB/RelA as well as its activity [23]. On the other hand, gankyrin can bind to NF-B and negatively regulates its activity at the transcription level through modulating acetylation via SIRT1, a class III histone deacetylase [24]. Last but not least, it has been shown that overepxression of gankyrin in human hepatocellular cell line Huh-7 up-regulated expression of insulin-like growth factor binding protein 5 (IGFBP5), which subsequently promoted cell proliferation [25]. The structural basis for gankyrin functioning In general, ankyrin repeat is a relatively conserved motif with a consistent pattern of key residues to retain the characteristic helix-turn-helix topology, and there exists a certain sequence homology as well as a structural similarity among most ankyrin repeat proteins [6, 7]. From this point of view, gankyrin has a simple, repetitive sketch structure of seven helix-turn-helix units. The question is how this “small and simple” protein can bind to multiple targets that are significantly different in structure and function. Previous structural
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Fig. (3). The dual-negative regulatory roles of gankyrin in the pRb and P53 pathways. Arrows represent positive modulation, and bars represent negative regulation.
and biochemical studies in our laboratory and other groups have provided insights about the structural basis for the functional diversity of gankyrin [1, 4, 6, 11, 13, 14]. The pRb-binding motif has been mapped to 178LxCxE182 in the fifth ankyrin repeat of gankyrin (Fig. 2A), and E182 has been shown to be critical for binding with pRb since mutations in E182 completely disrupts gankyrin/pRb association [1, 14]. As revealed in the crystal structure of the gankyrin/S6-C terminal domain complex [11], E182 is located at the edge of the S6-C terminal domain interface, hence it is very likely that gankyrin binds to pRb and form a pRb/gankyrin/S6-C terminal domain complex, thus locating pRb to the vicinity of the ATPases for processing and degradation. It has been also demonstrated that CDK4 binding is distinct from pRb binding. On one hand, an Rb-binding peptide, 176LHLACDEERN185, can eliminate the binding of gankyrin to pRb but does not influence the gankyrin/CDK4 association [14]. On the other hand, truncated gankyrin protein with the first four ankyrin repeats remaining is substantially potent in binding to CDK4 and counteracting the inhibitory function of P16 [14]. Interestingly, the structure of first four ankyrin repeats of gankyrin is almost superimposable with the structure of P16 (as well as the structures of the first four ankyrin repeats in another two CDK4-interacting ankyrin repeat proteins, P18 and IB), especially in the helical regions where most contacts between CDK4 and P16 are located (Fig. 2B) [26]. Such high structural resemblance between P16 and gankyrin provides the basis for their potential similarity in binding to CDK4. Indeed, besides residues universally conserved for the formation of the helix-turn-helix conformation of an ankyrin repeat, residues mainly contributing to CDK4 binding, such as E26, D74, and D92 of P16, are also conserved in gankyrin, indicating that gankyrin interacts with CDK4 in a way similar to that P16 does [26, 27]. This notion is further supported by the following observation that a single substitution, I79D of gankyrin enables gankyrin
to inhibit CDK-mediated phosphorylation of pRb as potently as P16 does, suggesting that the difference between tumor suppressing and oncogenic functions of P16 and gankyrin, respectively, mainly resides in a single residue, D84 of P16, which is located in the vicinity of the active site of CDK4. As revealed in the crystal structures of P16/CDK6 [28], P19/CDK6 [29], and P18/CDK6/viral cyclin [30] complexes, most of CDK4-intercating residues are located within the second and third ankyrin repeats while the first and fourth ankyrin repeats are required to form a stable structure, which is consistent with our previous observation that the first four ankyrin repeats of gankyrin are responsible for CDK4 binding. Similarly, the structural resemblance between gankyrin and IB in the first four ankyrin repeats enables gankyrin to compete with IB in binding to NF-B/RelA thus modulating NF-B-mediated transactivation [23, 24, 26]. In addition, deletion of the last ankyrin repeat of gankyrin eliminates its binding to MDM2, implying that the last ankyrin repeat is critical for MDM2 binding (Fig. 2B) [12]. While the domain responsible for MAGE-A4 binding remains to be further investigated [22], the association with the C-terminal S6 ATPase involves a number of residues discontinuously dispersed in all of the seven ankyrin repeats of gankyrin, including residues at the tips (defined as the first residue of an ankyrin repeat and the last residue of the preceding ankyrin repeat), adjacent to the tips, and in the helical regions [11]. Such discontinuous, multiple-residue-interacting patterns have been found in all crystal structures of complexes containing ankyrin repeat proteins, such as the 53BP2-P53 complex [31] and the IB/NF-B complex [32]. GANKYRIN AND CANCER Aberrant Expression of Gankyrin is Prevalent in Human Cancers Gankyrin appears to be one of few oncogenic proteins negatively modulating both pRb and P53 tumor suppressive
Structure/Function Relationship of Gankyrin
pathways. Considering the fact that more than 90% of cancer cells have inactivated pRB and P53 pathways either directly or indirectly [15], the status of gankyrin in cells could be associated with the development of human cancers. In an endeavor to explore the potential involvement of gankyrin in human cancer, Higashitsuji and his colleagues evaluated the expression of gankyrin mRNA in primary hepatocellular carcinomas [1]. Increased expression of gankyrin mRNA was found in 34 of 34 cases (100%) compared to corresponding histologically normal tissues. In another independent study [33], the expression of gankyrin mRNA was markedly increased in 57 of 64 HCC specimens and moderately increased in another 5 HCC specimens in the same cohort. In comparison with liver cirrhosis and para-carcinomas liver tissues, the average expression of gankyrin mRNA in HCC was increased by 3.6- and 5.2-fold, respectively. In chemically-induced rodent HCC [34], gankyrin mRNA was overexpressed in all tested HCC specimens in comparison with the matched “histologically normal” samples. Taken together, these results demonstrate that overexpression of gankyrin plays an important role in the development of HCC. Additionally, a recent study on human esophageal SCC (ESCC) [35] showed that all 30 tested tumor specimens and 11 ESCC cell lines exhibited high levels of gankyrin expression, and gankyrin overexpression was positively correlated with lower survival rate, extent of the primary tumor, lymph node metastasis, distant lymph node metastasis and stage, indicating that gankyrin might be important during the development of malignancy potential in ESCC, and may play an important role in its progression. In an ongoing study in our laboratory, we investigated the incidence of aberrant gankyrin expression in human oral SCC cell lines and SCC tumors. Our results showed that gankyrin was overexpressed in 84% and 71% of tested oral SCC specimens (n=32) at the mRNA and protein levels, respectively, while all six tested oral SCC cell lines exhibited overexpressed gankyrin at both mRNA and protein levels, indicating that aberrant gankyrin expression is a prevalent event in human oral SCCs (unpublished data). Aberrant Expression of Gankyrin could be an Early Event in the Development of Human cancers With regard to cancer diagnosis and prevention, it is very important to identify those molecular events occurring at the earliest stage of cancer development, whereas emerging evidence demonstrates that gankyrin overexpression could be one of such events in the development of hepatocellular carcinoma [1, 36-38]. In the aforementioned chemicallyinduced rodent hepatocarcinogenesis model, Lim et al. [36] found that aberrant molecular events occurred sequentially in association with the development of HCC. Hypermethylation of the p16 gene and p53 mutation appear at the late stage (the HCC stage), whereas gankyrin is overexpressed early just after carcinogen treatment (the liver fibrosis stage), preceding the loss of pRb (the cirrhosis stage) and hepatocellular adenoma formation (the HCA stage). Accordingly, Tan et al. [37] reported that the frequencies of gankyrin overexpression in Edmondson’s grade I to II, III, and IV human HCCs were 82%, 63%, and 22%, respectively. Similarly, Fujita et al. [38] observed positivity in 81% and 35% of low and high TNM stage of human HCCs. These results indicate that gan-
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kyrin overexpression is associated with the early clinical stage of human HCCs. In our present study on oral SCCs, we also observed overexpression of gankyrin protein in 50% (6 out of 12) of preneoplastic oral lesions (unpublished data). Hence, gankyrin may play an important role in the early stage of liver and oral cancer progression, and serve as a biomarker with potentials in cancer diagnosis and therapeutics. Moreover, it has been reported recently that overexpression of gankyrin confers multi-drug resistance of gastric cancer cells [39], indicating that down-regulation of gankyrin could provide a novel approach to combat against drug resistance, a challenge frequently found in cancer chemotherapy. Potential Mechanisms Underlying the Involvement of Gankyrin in Cancer The detailed molecular mechanisms underlying the contribution of aberrant gankyrin expression to cancer progression remain to be further explored. However, three distinct possibilities are now emerging with regard to the incorporation of gankyrin into the well-known cell cycle control machinery (Fig. 4). First, gankyrin overexpression may deregulate the pRb pathway of cell cycle control by mechanisms similar to those associated with the HPV 16 E7 protein [1, 40, 41]. Over abundant gankyrin would bind to hypo- and hyper-phosphorylated pRb, targeting both for proteasomal degradation. Second, P21 functions as a universal inhibitor to CDKs, such as CDK2 and CDK4, and its inhibition to CDKs is modulated by P53 [42]. Hence, gankyrin overexpression may indirectly deregulate the cell cycle progression through facilitating and enhancing ubiquitination and degradation of P53. Third, in normal cells, there exists a dynamic balance between gankyrin and P16 in controlling the activity of CDK4 as well as cell cycle progression [14]. Apparently, gankyrin overexpression could move this balance in the direction favoring gankyrin/CDK4 interaction thus compromising the tumor suppressor activity of P16. Since multiple molecular events, including the activation of protooncogenes and the inactivation of tumor suppressor genes, such as P53, P16, are involved in tumor development, the question is whether it is essential to have two functionallyrelated molecular events, gankyrin overexpression and P16 inactivation, especially when gankyrin overexpression or P16 inactivation alone is sufficient to “functionally” inactivate the P16/CDK/pRb pathway. The reasons lie in the following facts. First, as mentioned earlier [36], gankyrin overexpression likely occurs prior to genetic alterations of the p16 gene. Second, not all p16 gene alteration events result in functionally inactivated P16 proteins. Some P16 missense mutations led to P16 proteins with only moderately reduced CDK4-inhibitory activities [6]. Third, the loss of P16 activity via p16 alterations could be rescued by the gene redundancy and the upregulation of other INK family members (e.g., p15, p18 and p19) [43, 44], whereas gankyrin counteracts against the CDK4-inhibitory activities of all INK4 proteins. Fourth, it is always important to know that gankyrin can de-regulate the cell cycle progression in mechanisms other than competition with P16 (Fig. 3) [1, 11], while P16's cell cycle deregulating properties could be manifest through its inactivation in other critical nonCDK/pRB regulatory pathways (e.g. cell senescence, anoikis, cell spreading, and angiogenesis) [45-47].
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Fig. (4). Proposed mechanisms of gankyrin-mediated deregulation of cell cycle control. Arrows represent positive regulation, and bars represent negative regulation. In B, two red crosses indicate that the inhibition of P16 and P21 to CDK4 can be “eliminated” by overexpressed gankyrin.
IMPLICATION IN CHEMICAL BIOLOGY In a relatively conserved AR motif, residues forming its helix-turn-helix framework are strictly conserved, whereas residues directly involved in target binding are less conserved and usually are located on the surfaces of the helices as well as in the flexible loops connecting neighboring ARs [6, 7]. Additionally, some residues in the loops, such as the TPLH tetrapeptide, are well conserved, but they are not directly involved in target binding or the formation of the sketch structure [4]. Instead, these residues mainly function to stabilize the global structure of an AR protein, thus indirectly impacting its function. These structural features enable us to use chemical biology approaches to modify the functioning of gankyrin or even generate novel gankyrin-like molecules with potentials in therapeutics. First, as described earlier, the four N-terminal ARs of gankyrin and P16 exhibit high structural similarities, and both proteins compete with each other for binding to CDK4 [14, 26]. Nevertheless, the impacts of gankyrin and P16 are totally different: P16 inhibits CDK4-mediated phosphorylation of pRb and acts as a tumor suppressor [27], while gankyrin enhances CDK4mediated phosphorylation of pRb [14]. Interstingly, gankyrin I79D mutant exhibits a CDK4-inhibitory activity comparable with that of P16, while other gankyrin functions remain intact. Tentatively, this gankyrin mutant has the potential to replace the tumor suppressive role of P16 in P16-inactivated cells [27]. Secondly, there are six TPLH tetrapeptides in the loop regions of gankyrin (Fig. 1 and Fig. 2) [4]. Results from ongoing studies in our laboratory have shown that introducing or eliminating TPLH tetrapeptides from different locations of gankyrin change the conformational stability of the global structure while the CDK4-binding ability remains unperturbed (manuscript in preparation). Thirdly, information from consensus analyses of AR proteins including gankyrin is used to generate a novel AR motif, in which residu-
als other than those essential for forming the helix-turn-helix framework are randomly chosen [6, 7]. This novel AR motif is used as a building block, and different copies of this AR motif are linked together to form a combinatorial library, whose members can form a simple, rod-like structure as gankyrin but retain potentials to bind to any chosen target protein. To date, a number of such “novel” AR proteins have been reported, including those specifically binding to aminoglycoside phosphotransferase (3’)-IIIa (APH) [48], maltose binding protein (MBP) [49], p38 AMP kinase [50], and JNK-2 [50]. From this perspective, consensus-based AR combinatorial libraries could be a very attractive approach in chemical biology to generate novel molecules with clinical potential. ACKNOWLEDGEMENTS Research work in the authors’ laboratory was supported by a grant from the National Institutes of Health (CA69472) to J. Li. REFERENCES [1] [2]
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Accepted: 00 00, 2009
