Gankyrin Is an Ankyrin-repeat Oncoprotein That Interacts with CDK4 Kinase and the S6 ATPase of the 26 S Proteasome

Abstract

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...

Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome

“…1A), except Rpn3, Rpn5, Rpn7, and Rpn12, which were not resolved, perhaps because of poor solubility of these proteins (13). We also confirmed the proteasome association of several known cofactors, including PA28 (27), Hsp70 (28), gankyrin (29), and Usp14/Ubp6 (28,30). In addition to these proteins, Txnl1, a thioredoxinlike protein implicated in endocytosis (31) and reactions to glucose deprivation (32), was consistently found in the proteasome preparations (Fig.…”

Gankyrin oncoprotein overexpression as a critical factor for tumor growth in human esophageal squamous cell carcinoma and its clinical significance

“…However, the role of gankyrin in cell cycle progression in ESCC is still unclear. Moreover, based on the studies on apoptotic growth factors that have suggested that G1/S stages of the cell cycle may be apoptotic insensitive stages 37 and that S6 ATPase-gankyrin-Rb-E2F1 interactions may control E2F1 stability in relation to the G1/S transition, 38 we demonstrated that the down-regulation of gankyrin expression inhibited proliferation in an ESCC cell line, inducing an accumulation of cells in the G1/S phase in vitro. Furthermore, we clearly demonstrated that the down-regulation of gankyrin inhibited tumor growth in vivo.…”

The α4 and α7 subunits and assembly of the 20S proteasome

“…For binding studies with radiolabelled subunits, equal amounts (9 lg) of GST or GST-a-subunit were immobilised onto (GSH)-Sepharose beads and processed as described [17]. Protein fractions were subjected to SDS-PAGE followed by Western blot analyses, Coomassie staining or phosphor-imager analyses (Fujifilm FLA-2000R).…”

“…Strain SFY526 has only a lacZ reporter gene, but it is controlled by an intact GAL1 UAS and therefore can be expressed at $10Â the level obtained with the UAS G17-mer ðx3Þ promoter. For qualitative analysis of Y2H interactions, activity of the b-galactosidase reporter gene was assessed as described previously [17].…”