Structural Basis for the Antiproliferative Activity of the Tob-hCaf1 Complex

Toru Suzuki,Takahisa Nakamura,Nobuyuki Muroya,Kosei Takeuchi,Fuyuhiko Inagaki,Masataka Horiuchi,Nobuo Noda,Junko Kawamura-Tsuzuku,Tadashi Yamamoto,Kiyohiro Takahasi

doi:10.1074/jbc.m809250200

Abstract

The Tob/BTG family is a group of antiproliferative proteins containing two highly homologous regions, Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D) family of deadenylases and is a component of the CCR4-Not deadenylase complex. Here we determined the crystal structure of the complex of the N-terminal region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A and Box B of Tob. Cell growth assays using both wild-type and mutant proteins revealed that deadenylase activity of Caf1 is not critical but complex formation is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers several factors at its C-terminal region, such as poly(A)-binding proteins, to exert antiproliferative activity.

Highlights

The antiproliferative activities of the Tob/BTG family proteins are due to their association with target proteins in cells
Much evidence has been accumulated to suggest that CCR4-associated factor 1 (Caf1),2 known as Cnot7 and involved in the CCR4-Not deadenylase complex, is a common binding partner of the Tob/BTG family proteins (4, 18 –21)
It was difficult to purify intact Tob because of degradation. As both Box A and Box B of BTG/Tob family proteins are sufficient to associate with human Caf1 (hCaf1), we coexpressed an antiproliferative region of human Tob comprising the N-terminal 138 residues and intact hCaf1 in E. coli BLR(DE3)

Summary

The abbreviations used are

CCR4-associated factor 1; m, mouse; h, human; PABP, poly(A)-binding protein; PABPC1, cytoplasmic poly(A)-binding protein; CNS, crystallography and NMR system; MIR, multiple isomorphous replacement; PARN, poly(A)-specific ribonuclease; FITC, fluorescein isothiocyanate; DTT, dithiothreitol; GST, glutathione S-transferase; HEK, human embryonic kidney; HRP, horseradish peroxidase; PDB, Protein Data Bank; PARN, poly(A)-specific ribonuclease; ERK, extracellular signal-regulated kinase; NES, nuclear export signal; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein. Structural Basis for the Tob-Caf Complex the remaining portion of the mRNA is rapidly degraded. Tob was reported to associate with inducible poly(A)-binding protein (iPABP) and to abrogate the translation of interleukin-2 mRNA in vitro [35]. Recent reports showed that Tob and BTG2 interact with the CCR4-Not deadenylase complex using the Tob/BTG2 domain and the cytoplasmic poly(A)-binding protein (PABPC1) using the C-terminal tail and enhanced mRNA degradation (36 –38). To help elucidate the relationship between the antiproliferative activity of Tob and the degradation of the poly(A) tail, we determined the crystal structure of the Tob-hCaf complex. Box A and Box B mediate the interaction between Tob and hCaf. Cell growth assays using the wild and mutant proteins, together with the structural studies, revealed that the complex formation is crucial to cell growth inhibition

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION