Structure of the oncoprotein gankyrin in complex with S6 ATPase of the 26S proteasome

RIKEN Genomic Sciences Center* RIKEN GSC, Riken Harima Inst., Grad. Sch. of Sci., Univ. of Tokyo**
â—‹Yoshihiro Nakamura* Kazumi Nakano* Takashi Umehara* Eru Adachi* Yoshihide Hayashizaki* Balasundaram Padmanabhan* Shigeyuki Yokoyama**

Tumorigenesis is mainly regulated by both oncoproteins and tumor-suppressors in G1-S transition during the cell cycle. In this regulatory pathway, one of the key factors is the retinoblastoma tumor-suppressor protein (pRb). pRb binds to the transcription factor E2F which is required for the expression of genes involved in G1-S transition and inhibits its transactivation function. The hyperphosphorylation of pRb by cyclin-dependent kinase 4 and 6 (CDK4/6) leads to the release of E2F from the Rb-E2F complex and degradation of Rb by the proteasome via a ubiquitin-dependent pathway. Rb phosphorylation by CDK4/6 is regulated mainly by proteins containing ankyrin repeats: gankyrin (Gann ankyrin: Gann means cancer in Japanese) for positive regulation and INK4 (CDK4/6 inhibitor) for negative regulation, in addition to cyclin for activating CDK4/6. Recent studies have proposed that gankyrin is also one of the subunit of 19S non-regulatory particle of the proteasome, and is likely to interact with the S6 ATPase subunit. To understand the functional role of gankyrin in the proteasome, we have initiated the project on structure and functional analysis of gankyrin complexed with S6 ATPase.
The protein complex, gankyrin- C-term domain of S6 ATPase, was co-expressed, purified to homogeneity, crystallized and determined its tertiary structure at 2.4 A resolution. The gankyrin contains seven ankyrin repeats with crescent shape like structure. The C-terminal domain of S6 ATPase which possess mainly alpha-helices, binds to gankyrin at the region containing the ankyrin repeats 3, 4, 5 and 6. Biochemical studies based on the crystal structure revealed that gankyrin recognizes pRb both in the presence or absence of S6 ATPase.