Structural Basis for the Antiproliferative Activity of the Tob-hCaf1
Complex |
| |
Authors: | Masataka Horiuchi Kosei Takeuchi Nobuo Noda Nobuyuki Muroya Toru Suzuki Takahisa Nakamura Junko Kawamura-Tsuzuku Kiyohiro Takahasi Tadashi Yamamoto and Fuyuhiko Inagaki |
| |
Institution: | ‡Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N12, W6, Kita-ku, Sapporo 060-0812, Japan, the §Division of Molecular and Cellular Biology, Graduate School of Medical and Dental Sciences, Niigata University, Asahi-machi, Chuo-ku, Niigata 951-8510, Japan, and the ¶Department of Oncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108-8639, Japan |
| |
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.The Tob/BTG family (also called the APRO family) is a group of
antiproliferative proteins (1,
2) consisting of Tob
(3), Tob2
(4), BTG1
(5), BTG2/Tis21/PC3
(6-8),
PC3B (9), and ANA/BTG3
(10,
11) in mammalian cells,
{"type":"entrez-nucleotide","attrs":{"text":"AF177464","term_id":"5916227","term_text":"AF177464"}}AF177464 in Drosophila, and FOG-3 in Caenorhabditis elegans
(12). A recent genome project
reported that the BTG/Tob family protein had already existed in
Choanoflagellida Monosiga brevicollis MX1. The N-terminal region of
the Tob/BTG family proteins is conserved and includes two highly homologous
regions, Box A and Box B. The Tob/BTG family proteins are involved in cell
cycle regulation in a variety of cells such as T lymphocytes, fibroblasts,
epithelial cells, and germ cells. In Tob-deficient mice, the incidence of
liver tumors is higher than in wild-type mice. Furthermore, because the levels
of tob expression are often repressed in human lung cancers,
suppression of its expression is thought to contribute to tumor progression
(13).The antiproliferative activities of the Tob/BTG family proteins are due to
their association with target proteins in cells. For example, Tob associates
with SMAD family proteins and acts as a negative regulator of SMAD signaling.
In osteoblasts, this negative regulation occurs via association with SMAD 1,
5, 6, and 8 (14,
15), and via association with
SMAD 2 and 4 in anergic quiescent T cells
(16). Tob/BTG family proteins
also bind to protein arginine methyltransferase, which regulates chromatin
assembly by histone methylation
(17). Much evidence has been
accumulated to suggest that CCR4-associated factor 1
(Caf1),2 also 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).
To reveal the functions of Caf1 in vivo, caf1-/- mice have
been generated in two groups. Male caf1-deficient mice are infertile
because of a malfunction of the testicular somatic cells that leads to a
defect in spermatogenesis (22,
23). Genetic analysis of the
nematode C. elegans also suggests that FOG3 (Tob orthologue)
interacts with CCF1, the C. elegans homologue of Caf1, and that this
interaction is essential for germ cells to initiate spermatogenesis
(24).Mouse and human Caf1 (mCaf1 and hCaf1) were found as homologues of yeast
Pop2, a component of the CCR4-Not complex
(18,
25). Yeast Pop2 displays weak
RNase activity but enhances the deadenylation of the poly(A) tail of mRNA by
the CCR4-Not deadenylase complex
(26-29).
The primary structure of mammalian Caf1 is related to that of the ribonuclease
D (RNase D) family, and all of the active site residues are well conserved
(30). Indeed, both mCaf1 and
hCaf1 have deadenylase activity
(31-33).Although the relationship between cell cycle repression and poly(A)
deadenylation is not well understood, mRNA degradation and synthesis are major
events in maintaining the cell cycle
(34). The mRNAs in a
eukaryotic cell have a wide range of half-lives. Degradation of mRNA is
initiated by shortening of the poly(A) tail. Thereafter, the 5′-cap
structure is removed and the remaining portion of the mRNA is rapidly
degraded. The degradation of eukaryotic mRNAs is regulated precisely at each
stage of the cell cycle. 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 also
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-hCaf1 complex. We found that hCaf1 has a structure
similar to yeast Pop2 and human PARN of deadenylases, exonuclease I, and the
Klenow fragment of DNA polymerase I from Escherichia coli. In
contrast, Tob has a novel structure. Specifically, Box A and Box B mediate the
interaction between Tob and hCaf1. 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. |
| |
Keywords: | |
|
|