Gankyrin: a new oncoprotein and regulator of pRb and p53

Gankyrin is a new oncoprotein with potent cell cycle and apoptotic properties that is overexpressed early in hepatocarcinogenesis and in hepatocellular carcinomas. Gankyrin regulates the phosphorylation of the retinoblastoma protein (pRb) by CDK4 and enhances the ubiquitylation of p53 by the RING ubiquitin ligase MDM2. Purified preparations of the 26S proteasome contain gankyrin, which specifically interacts with the S6b (Rpt3) ATPase of the 19S regulator. In conclusion, gankyrin is a small versatile cell cycle regulator that illustrates the essential interplay between the ubiquitin proteasome system and gene expression in the cell. Here, we discuss the activities of gankyrin and present a model for its function in the regulation of pRb and p53.     

Gankyrin promotes osteosarcoma tumorigenesis by forming a positive feedback loop with YAP

BackgroundAlthough gankyrin has been identified as a vital regulator of tumorigenesis, its role and regulatory mechanism in osteosarcoma (OS) remain unclear.MethodsQRT-PCR, western blot and IHC staining were conducted to detect the expression of gankyrin in OS. Pearson’s χ² test was adopted to examine the associations between gankyrin expression and clinicopathologic characteristics. Kaplan-Meier method was used to investigate the relationship between gankyrin expression and overall survival of patients with OS. Next, a series of in vitro and in vivo assays were performed to determine the positive feedback loop between gankyrin and YAP in OS.ResultsWe first reported that gankyrin is upregulated in human OS specimens and cell lines and predicts OS progression and poor prognosis. Furthermore, we demonstrated that gankyrin protects miR-200a-mediated yes-associated protein (YAP) downregulation through p53 and establishes a positive feedback loop to regulate YAP signaling in U2OS and MG63 cells. Intriguingly, gankyrin interacts with YAP to promote OS cell growth in vitro. In addition, our results showed that gankyrin promotes OS tumor growth and regulates YAP levels in vivo. Notably, we also observed a positive correlation between gankyrin and YAP expression in human OS tissues, and co-upregulation of gankyrin and YAP indicated a poor prognosis.ConclusionsOur results identify that gankyrin acts as an oncogene in OS by forming a positive feedback loop with YAP, and disrupting the gankyrin-YAP regulation may be beneficial for controlling OS tumorigenesis.     

Repression of Hedgehog signal transduction in T-lineage cells increases TCR-induced activation and proliferation

Hedgehog proteins signal for differentiation, survival and proliferation of the earliest thymocyte progenitors, but their functions at later stages of thymocyte development and in peripheral T-cell function are controversial. Here we show that repression of Hedgehog (Hh) pathway activation in T-lineage cells, by expression of a transgenic repressor form of Gli2 (Gli2δC2), increased T-cell differentiation and activation in response to TCR signalling. Expression of the Gli2δC2 transgene increased differentiation from CD4+CD8+ to single positive thymocyte, and increased peripheral T cell populations. Gli2δC2 T-cells were hyper-responsive to activation by ligation of CD3 and CD28: they expressed cell surface activation markers CD69 and CD25 more quickly, and proliferated more than wild-type T-cells. These data show that Hedgehog pathway activation in thymocytes and T-cells negatively regulates TCR-dependent differentiation and proliferation. Thus, as negative regulators of TCR-dependent events, Hh proteins provide an environmental influence on T-cell fate.     

Discovery of multiple interacting partners of gankyrin,a proteasomal chaperone and an oncoprotein—Evidence for a common hot spot site at the interface and its functional relevance

Gankyrin, a non‐ATPase component of the proteasome and a chaperone of proteasome assembly, is also an oncoprotein. Gankyrin regulates a variety of oncogenic signaling pathways in cancer cells and accelerates degradation of tumor suppressor proteins p53 and Rb. Therefore gankyrin may be a unique hub integrating signaling networks with the degradation pathway. To identify new interactions that may be crucial in consolidating its role as an oncogenic hub, crystal structure of gankyrin‐proteasome ATPase complex was used to predict novel interacting partners. EEVD, a four amino acid linear sequence seems a hot spot site at this interface. By searching for EEVD in exposed regions of human proteins in PDB database, we predicted 34 novel interactions. Eight proteins were tested and seven of them were found to interact with gankyrin. Affinity of four interactions is high enough for endogenous detection. Others require gankyrin overexpression in HEK 293 cells or occur endogenously in breast cancer cell line‐ MDA‐MB‐435, reflecting lower affinity or presence of a deregulated network. Mutagenesis and peptide inhibition confirm that EEVD is the common hot spot site at these interfaces and therefore a potential polypharmacological drug target. In MDA‐MB‐231 cells in which the endogenous CLIC1 is silenced, trans‐expression of Wt protein (CLIC1_EEVD) and not the hot spot site mutant (CLIC1_AAVA) resulted in significant rescue of the migratory potential. Our approach can be extended to identify novel functionally relevant protein‐protein interactions, in expansion of oncogenic networks and in identifying potential therapeutic targets. Proteins 2014; 82:1283–1300. © 2013 Wiley Periodicals, Inc.     

MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity

Hepatocellular carcinoma ranks among the most common malignancies in Southeast Asia and South Africa. Although there are many modalities of treatment, the recurrence and metastasis rates are high, and the prognosis is unsatisfactory. Gankyrin, a recently found oncoprotein, is a promising target for drug therapy because it is overexpressed in all studied hepatocellular carcinomas. Gankyrin contains six ankyrin repeats and interacts with Rb, Cdk4, and the S6 ATPase of the 26 S proteasome. In this study, a yeast two-hybrid screen with gankyrin has identified MAGE-A4 as another interacting protein. The interaction, mediated by the C-terminal half of MAGE-A4, was reproduced in mammalian cells. The interaction was specific to MAGE-A4, because other MAGE family proteins structurally similar to MAGE-A4, i.e. MAGE-A1, MAGE-A2, and MAGE-A12, did not bind to gankyrin. MAGE-A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of gankyrin-overexpressing cells. The ability of mutant MAGE-A4 to interact with gankyrin correlated with the ability to suppress the anchorage-independent growth. These results demonstrate that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity. So far, the major focus of studies on the MAGE proteins has been on their potential for cancer immunotherapy. Our results may also shed light on novel functions for MAGE-A proteins.