Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia and Africa, where hepatitis virus infection and exposure to specific liver carcinogens are prevalent. Although inactivation of some tumor suppressor genes such as p53 and p16INK4Ahas been identified, no known oncogene is commonly activated in hepatocellular carcinomas. Here we have isolated genes overexpressed in hepatocellular carcinomas by cDNA subtractive hybridization, and identified an oncoprotein consisting of six ankyrin repeats (gankyrin). The expression of gankyrin was increased in all 34 hepatocellular carcinomas studied. Gankyrin induced anchorage-independent growth and tumorigenicity in NIH/3T3 cells. Gankyrin bound to the product of the retinoblastoma gene (RB1), increasing its phosphorylation and releasing the activity of the transcription factor E2F-1. Gankyrin accelerated the degradation of RB1 in vitro and in vivo, and was identical to or interacted with a subunit of the 26S proteasome. These results demonstrate the importance of ubiquitin-proteasome pathway in the regulation of cell growth and oncogenic transformation, and indicate that gankyrin overexpression contributes to hepatocarcinogenesis by destabilizing RB1.

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Gankyrin is an ankyrin repeat oncoprotein commonly overexpressed in hepatocellular carcinomas. Gankyrin interacts with the S6 proteasomal ATPase and accelerates the degradation of the tumor suppressor Rb. We show here that gankyrin has an antiapoptotic activity in cells exposed to DNA damaging agents. Downregulation of gankyrin induces apoptosis in cells with wild-type p53. In vitro and in vivo experiments revealed that gankyrin binds to Mdm2, facilitating p53-Mdm2 binding, and increases ubiquitylation and degradation of p53. Gankyrin also enhances Mdm2 autoubiquitylation in the absence of p53. Downregulation of gankyrin reduced amounts of Mdm2 and p53 associated with the 26S proteasome. Thus, gankyrin is a cofactor that increases the activities of Mdm2 on p53 and probably targets polyubiquitylated p53 into the 26S proteasome.

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A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein.Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle. Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin. Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability. Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4. The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis.The 26 S proteasome is an exquisitely regulated protease responsible for most of the non-lysosomal degradation of intracellular proteins (1). The particle is responsible for the degradation of regulatory proteins including tumor suppressors (2, 3), transcription factors (4, 5), and proteins that regulate the cell cycle (6, 7).The 26 S proteasome consists of a cylindrical catalytic "core" containing 28 subunits (␣ 7 ␤ 7 ␤ 7 ␣ 7 ) with a regulatory complex (RC) 1 attached to each end of the proteolytic core containing at least 15 subunits (1). The 20 S cylinder contains three chambers: two distal antechambers and a central chamber containing the catalytic threonine residues (8, 9). The ends of the cylindrical core appear closed in the yeast 20 S proteasome (8); the ends of these antechambers need to be opened for substrate proteins to enter into the catalytic core for proteolysis. The RC can be subdivided into a "base" and a "lid" complex (10). The base contains six ATPases, which belong to the AAA (ATPases associated with diverse cellular activities) superfamily of ATPases together with the non-ATPase RPN1 and RPN2 subunits. The lid subcomplex contains the remaining non-ATPase subunits of the RC. The AAA ATPase superfamily controls events as diverse as 26 S proteasomal functions, peroxisomal biogenesis (11), membrane docking and fusion (12), protein egress from the endoplasmic reticulum (13), nuclear transport (14), and transcription factor regulation (15). The ATPases of the RC may be involved in substrate unfolding for entry into the catalytic core of the 26 S proteasome. It has recently been demonstrated that the base subcomplex of the RC exhibits chaperone-like activity (16); and both the ClpX and ClpA ATPases of Escherichia coli, multisubunit complexes similar to the base subcomplex of the 26 S proteasome, can mediate protein unfolding (17, 18) and refolding events (19,20).The proteasomal regulatory ATPases have in addition to the Walker A and B motifs involved in ATP binding and hydrolysis, sequence patterns shared with DNA/RNA helicases...