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

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 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 (1Tirone F. J. Cell. Physiol. 2001; 187: 155-165Crossref PubMed Scopus (194) Google Scholar, 2Matsuda S. Rouault J. Magaud J. Berthet C. FEBS Lett. 2001; 497: 67-72Crossref PubMed Scopus (208) Google Scholar) consisting of Tob (3Matsuda S. Kawamura-Tsuzuku J. Ohsugi M. Yoshida M. Emi M. Nakamura Y. Onda M. Yoshida Y. Nishiyama A. Yamamoto T. Oncogene. 1996; 12: 705-713PubMed Google Scholar), Tob2 (4Ikematsu N. Yoshida Y. Kawamura-Tsuzuku J. Ohsugi M. Onda M. Hirai M. Fujimoto J. Yamamoto T. Oncogene. 1999; 18: 7432-7441Crossref PubMed Scopus (125) Google Scholar), BTG1 (5Rouault J.P. Rimokh R. Tessa C. Paranhos G. Ffrench M. Duret L. Garoccio M. Germain D. Samarut J. Magaud J.P. EMBO J. 1992; 11: 1663-1670Crossref PubMed Scopus (264) Google Scholar), BTG2/Tis21/PC3 (6Rouault J.P. Falette N. Guéhenneux F. Guillot C. Rimokh R. Wang Q. Berthet C. Moyret-Lalle C. Savatier P. Pain B. Shaw P. Berger R. Samarut J. Magaud J.P. Ozturk M. Samarut C. Puisieux A. Nat. Genet. 1996; 14: 482-486Crossref PubMed Scopus (343) Google Scholar, 7Fletcher B.S. Lim R.W. Varnum B.C. Kujubu D.A. Koski R.A. Herschman H.R. J. Biol. Chem. 1991; 266: 14511-14518Abstract Full Text PDF PubMed Google Scholar, 8Bradbury A. Possenti R. Shooter E.M. Tirone F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3353-3357Crossref PubMed Scopus (176) Google Scholar), PC3B (9Buanne P. Corrente G. Micheli L. Palena A. Lavia P. Spadafora C. Lakshmana M.K. Rinaldi A. Banfi S. Quarto M. Bulfone A. Tirone F. Genomics. 2000; 68: 253-263Crossref PubMed Scopus (58) Google Scholar), and ANA/BTG3 (10Yoshida Y. Matsuda S. Ikematsu N. Kawamura-Tsuzuku J. Inazawa J. Umemori H. Yamamoto T. Oncogene. 1998; 16: 2687-2693Crossref PubMed Scopus (95) Google Scholar, 11Guehenneux F. Duret L. Callanan M.B. Bouhas R. Hayette S. Berthet C. Samarut C. Rimokh R. Birot A.M. Wang Q. Magaud J.P. Rouault J.P. Leukemia. 1997; 11: 370-375Crossref PubMed Scopus (106) Google Scholar) in mammalian cells, AF177464 in Drosophila, and FOG-3 in Caenorhabditis elegans (12Ellis R.E. Kimble J. Genetics. 1995; 139: 561-577Crossref PubMed Google Scholar). 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 (13Yoshida Y. Nakamura T. Komoda M. Satoh H. Suzuki T. Tsuzuku J.K. Miyasaka T. Yoshida E.H. Umemori H. Kunisaki R.K. Tani K. Ishii S. Mori S. Suganuma M. Noda T. Yamamoto T. Genes Dev. 2003; 17: 1201-1206Crossref PubMed Scopus (99) Google Scholar). 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 (14Yoshida Y. Tanaka S. Umemori H. Minowa O. Usui M. Ikematsu N. Hosoda E. Imamura T. Kuno J. Yamashita T. Miyazono K. Noda M. Noda T. Yamamoto T. Cell. 2000; 103: 1085-1097Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 15Yoshida Y. von Bubnoff A. Ikematsu N. Blitz I.L. Tsuzuku J.K. Yoshida E.H. Umemori H. Miyazono K. Yamamoto T. Cho K.W. Mech. Dev. 2003; 120: 629-637Crossref PubMed Scopus (53) Google Scholar), and via association with SMAD 2 and 4 in anergic quiescent T cells (16Tzachanis D. Freeman G.J. Hirano N. van Puijenbroek A.A. Delfs M.W. Berezovskaya A. Nadler L.M. Boussiotis V.A. Nat. Immunol. 2001; 2: 1097-1098Crossref PubMed Scopus (223) Google Scholar). Tob/BTG family proteins also bind to protein arginine methyltransferase, which regulates chromatin assembly by histone methylation (17Lin W.J. Gary J.D. Yang M.C. Clarke S. Herschman H.R. J. Biol. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Much evidence has been accumulated to suggest that CCR4-associated factor 1 (Caf1), 2The abbreviations used are: Caf1, 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 protein; 2The abbreviations used are: Caf1, 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 protein; also as and involved in the CCR4-Not deadenylase complex, is a of the Tob/BTG family proteins (4Ikematsu N. Yoshida Y. Kawamura-Tsuzuku J. Ohsugi M. Onda M. Hirai M. Fujimoto J. Yamamoto T. Oncogene. 1999; 18: 7432-7441Crossref PubMed Scopus (125) Google Scholar, C. D.A. J.K. J. 1998; PubMed Scopus Google Scholar, J.P. D. Berthet C. Birot A.M. M. Magaud J.P. L. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. T. M.C. Rimokh R. Magaud J.P. L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (106) Google Scholar, Y. Hosoda E. Nakamura T. Yamamoto T. J. 2001; PubMed Scopus Google Scholar). the of Caf1 in been in two are because of a of the cells that to a in T. R. T. Suzuki T. C. N. K. R. Miyasaka T. Yoshida Y. A. K. T. Yamamoto T. Noda T. Nat. Genet. PubMed Scopus (99) Google Scholar, C. A.M. F. Magaud J.P. P. Rouault J.P. Cell. Biol. PubMed Scopus Google Scholar). of the C. elegans also that with the C. elegans of Caf1, and that this is germ cells to L. A. PubMed Scopus Google Scholar). and human Caf1 and as of yeast a component of the CCR4-Not complex C. D.A. J.K. J. 1998; PubMed Scopus Google Scholar, C. Cell. Biol. 1995; PubMed Scopus Google Scholar). Pop2 activity but the of the of by the CCR4-Not deadenylase complex M. J. R. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M.C. F. B. 2001; PubMed Scopus Google Scholar, J. EMBO J. PubMed Scopus Google Scholar, M. D. R. EMBO J. PubMed Scopus Google Scholar). The structure of mammalian Caf1 is to that of the ribonuclease D (RNase D) and all of the are conserved A. 1997; PubMed Scopus Google Scholar). both and hCaf1 deadenylase activity J. Biol. 2003; PubMed Scopus Google Scholar, P. T. Y. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. F. S. B. L. 11: Scholar). the cell cycle and is not and are in the cell cycle Cell. Full Text PDF PubMed Scopus Google Scholar). The in a cell a of of is by of the the structure is and the of the is The of is at of the cell Tob was reported to associate with poly(A)-binding protein and to the of in K. Suzuki T. J. Matsuda S. Yamamoto T. Genes PubMed Scopus (58) Google Scholar). also that Tob and with the CCR4-Not deadenylase complex using the domain and the cytoplasmic poly(A)-binding protein using the C-terminal and N. Yamashita A. Yamashita Y. D. Cell. Biol. PubMed Scopus Google Scholar, Y. Y. Hosoda N. N. M. M. Suzuki T. T. S. Genes Dev. Scopus Google Scholar, F. C. B. EMBO J. PubMed Scopus Google Scholar). the the antiproliferative activity of Tob and the of the we determined the crystal structure of the complex. that hCaf1 has a structure to yeast Pop2 and human of and the of In Tob has a novel Box A and Box B the Tob and Cell growth assays using the and mutant proteins, with the revealed that the complex formation is crucial to cell growth inhibition. and of the antiproliferative region of human Tob the N-terminal is and hCaf1 in E. The of was a the the and the of hCaf1 was the These in E. proteins and to of at and with 1 cell the protein was to and with a of in and 1 The complex was the using a with and The was a with The was using a of to 1 containing the and and at to The protein complex was to and used In the 1 of complex and was with of a DTT, and The was with of at and the 1 of containing was with of the and with a in a 1 of the of was with 1 of protein and at with of at of at in and and containing 1 1 The crystal of the was by the crystal at in a but with 1 to by in a containing and the and in Data and the complex using with a at the in at using the and using in the and The structure was by multiple isomorphous using the crystallography and NMR The of the by of the and of by The had a of of at a of and was by using the A was the using the and with CNS, several of with and with with the to a of all at a of The crystal structure of the complex with was determined by using the of the structure a of all at a of was The and are in and in a In The of 1 2 in the of 1, 2 and 1 at The was by the of of of the 8 and by The and using and In wild-type hCaf1 and the to mutant of the of hCaf1 expression of and in E. and by to the For of was with of the of with at 1 at The by of and tob and by tob to and to by The and also by which was to tob and which was to The the by and to the expression by The the of and by using These also the and to the expression Cell cells embryonic cells, and cells in growth with at in a of expression cells. h, cells with and with containing a The by and with The with and the by in the in 1 the The proteins by and by the with and with by and of and Cell Tob and hCaf1 proteins the M. S. Y. A. J. A. T. 1996; Google Scholar, S. T. T. 2000; PubMed Scopus Google Scholar). cells with and in the of and 1 cells in by using the For the we used the cell to the cell and in at a of 1 of cell with of and the a in The was with using cell the cells in The cells in cells by and at the cell in was using a The cells the of their protein Tob and protein Caf1 The cells and cells their with and the the of regulation by Tob cell cells at and the of cell cells by and in using a and a of the expression of the proteins was by in the cell For the of cells, was with the of at and 2 and in the of and the of wild-type of also in the expression of levels of proteins the cell growth of the Tob hCaf1 in the growth complex, we determined the crystal structure of the complex. we to Tob in E. was to Tob because of both Box A and Box B of BTG/Tob family proteins are to associate with we antiproliferative region of human Tob the N-terminal we to as and hCaf1 in E. The crystal of the complex in the of in determined the crystal structure of at by The has of and of 1; the region 1 to and the hCaf1 region to in the that is structure with consisting of by a containing two and hCaf1 is structure with of by The association and hCaf1 is of and and and of with and and and of hCaf1 The and hCaf1 is Interestingly, Box A in and Box B of Tob the association with The containing and are by two and These that are conserved in the Tob/BTG family and and are at the with the that Box B to The of complex the of which was as the 1, B and of and hCaf1 by of the of the complex, we the of hCaf1 and to the that hCaf1 is to Pop2 of and of and PubMed Scopus Google Scholar) and S. F. B. D. EMBO 2003; PubMed Scopus Google ribonuclease of of M. M. H. Y. E. H. EMBO J. PubMed Scopus Google the N-terminal domain of the of E. of S. Full Text Full Text PDF PubMed Scopus Google of E. of Nat. Biol. 2000; PubMed Scopus Google and the of of C. 1997; Full Text Full Text PDF PubMed Scopus Google the two to by the of the BTG/Tob domain of Tob of and that of of that is a domain the BTG/Tob the of the of the we Tob and Tob/BTG family proteins and hCaf1 and D family proteins of hCaf1 structure of hCaf1 is of the catalytic domain of the D which is by the These conserved are involved in the crystal structure of hCaf1 was in the of we two in the of the in a used For this we the activity of hCaf1 in the of and hCaf1 and of and with of the and at in the of and but not and hCaf1 higher catalytic activity with than with as the activity in the of was higher than in the of is hCaf1 to activity. also that of the that at are to the of that mammalian Caf1 has poly(A)-specific deadenylase activity J. Biol. 2003; PubMed Scopus Google Scholar, C. F. S. B. L. 11: Scholar, N. Yamashita A. Yamashita Y. D. Cell. Biol. PubMed Scopus Google Scholar). hCaf1 used in the has poly(A)-specific deadenylase we assays in the of using and to the of a that of by hCaf1 was than of and B and The of hCaf1 to was not because was to hCaf1 a to a a as a we that hCaf1 is with deadenylase activity in the of activity of hCaf1 is by BTG/Tob domain in assays of in the of was the activity of hCaf1 in the of 4 of was of the of the of hCaf1 and is the catalytic activity of we the crystal of with a containing The crystal structure was determined at with of and of The structure of the complex is in with that of deadenylase to the M. M. H. Y. E. H. EMBO J. PubMed Scopus Google The complex two and at to the two of the domain of The of and associate with whereas of and associate with of the of D family been the and of hCaf1 with the of the D family proteins These of hCaf1 bind to and as the in the the conserved of hCaf1 as catalytic we a and of hCaf1 to and of with in the of not in conserved are critical catalytic of In yeast the to and of hCaf1 are by and in deadenylase activity with The at the is by in which to in The of in which to in is by the and the the is with of In this is by suggest that the and the catalytic of hCaf1 are to of Tob and and hCaf1 of both Box A and Box B and that the conserved in Tob/BTG family proteins are crucial their and in Box B in the with and with and its the in The is by the of in Box A and in Box B with and In mediated by is at the in Box A and Box B than at the are crucial the Tob and For example, the of a with the of both of which are and by The is the of the conserved in Box A and Box B to the with the and hCaf1 in the crystal is also in we of in the of which was to of The that and exhibited a containing both and that a complex with in the the that and exhibited two consisting of and the of the complex exhibited in the crystal structure in the in the crystal structure is in we a of Tob with hCaf1 using a Tob was to and hCaf1 was to which to Tob and hCaf1 in in cells, and the cell the was to The proteins the by and with that Tob and to hCaf1 in 1 and the crystal structure of the complex, Tob was to that the BTG/Tob domain of Tob is complex Tob and the in which was by and by the to bind to Tob that is critical complex formation in as in the crystal we that the with hCaf1 in the crystal structure is also conserved in Tob in Tob and a mutant in deadenylase activity The complex formation of was that the deadenylase activity of hCaf1 is not to the complex formation is by the protein and both in and in T. Kawamura-Tsuzuku J. R. Nakamura T. Yoshida Y. Yamamoto T. Genes Dev. 16: PubMed Scopus Google Scholar, M. E. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Tob is the to the is and H. K. H. J. PubMed Scopus Google Scholar). the cell cycle to has been to bind to and Tob J. Suzuki T. Yoshida Y. Yamamoto T. Oncogene. PubMed Scopus Google Scholar) to the crystal are at the N-terminal of and are the of the by in the complex. the nuclear of Tob includes with and J. Suzuki T. Yoshida Y. Yamamoto T. Oncogene. PubMed Scopus Google Scholar) and the association of with the nuclear and Tob of of Tob cell cycle progression N. Yamashita A. Yamashita Y. D. Cell. Biol. PubMed Scopus Google Scholar, J. Suzuki T. Yoshida Y. Yamamoto T. Oncogene. PubMed Scopus Google Scholar). progression to Tob is the to the a the nuclear which has been as is a and is Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, M. Y. E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The two nuclear of Tob been at and In the crystal structure of the of are to a with and not to as that not as a nuclear whereas is M. Y. E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), which is with the crystal of the in Cell the antiproliferative activity of the complex, we cells with wild-type both using a expression The expression of Tob hCaf1 cell whereas cell growth was in the cells both Tob and hCaf1 with that of the cells, the expression of protein was A and For a of the antiproliferative we to the cell with both using a and expression we not the of both Tob and hCaf1 in the cell used because of the antiproliferative activities of the complex. the antiproliferative activity of the complex we the growth of cells and cells that Tob Tob hCaf1 hCaf1 For cells the cell growth was in the the of was the the of cells was not by the cell and was the of cells by and the cell at the In cells Tob the growth was with the cell and the growth of cell was of that of cell and in the cells Tob and the growth and growth the formation of the complex, we the growth of the cells. The growth of the cells Tob and was to that of the the association Tob and hCaf1 to crucial the of a antiproliferative activity D and Tob includes a C-terminal of the BTG/Tob the BTG/Tob domain antiproliferative we the growth of the cells of with hCaf1 had cell the BTG/Tob domain not antiproliferative activity D and In is the C-terminal region of Tob that the poly(A)-binding protein and the region of the SMAD family which and (14Yoshida Y. Tanaka S. Umemori H. Minowa O. Usui M. Ikematsu N. Hosoda E. Imamura T. Kuno J. Yamashita T. Miyazono K. Noda M. Noda T. Yamamoto T. Cell. 2000; 103: 1085-1097Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, K. Suzuki T. J. Matsuda S. Yamamoto T. Genes PubMed Scopus (58) Google Scholar, N. Yamashita A. Yamashita Y. D. Cell. Biol. PubMed Scopus Google Scholar, Y. Y. Hosoda N. N. M. M. Suzuki T. T. S. Genes Dev. Scopus Google Scholar). to the antiproliferative activity of the activity of we the growth of cells which is in activity and The growth of the cells was to that of wild-type Tob with also a cell growth to the cells Tob with the wild-type and the of hCaf1 in the antiproliferative activity is not due to its activity and D and also using cells not In the we determined the crystal structure of the complex. Interestingly, both Box A and Box B of the the BTG/Tob hCaf1 exhibited poly(A)-specific which was with Tob/BTG domain a complex with not the activity. of which is the of hCaf1 with Tob in the crystal complex formation in and in the of Tob and hCaf1 in antiproliferative we the growth of mammalian cells and their The that complex formation Tob and hCaf1 is crucial of antiproliferative whereas activity of hCaf1 is not Furthermore, the C-terminal region of Tob is in antiproliferative activity. Tob is of antiproliferative activity. In mammalian cells, of is by several poly(A)-specific the CCR4-Not deadenylase complex, and the mammalian deadenylase of catalytic the activity is associates with which with PABPC1, that the deadenylase activity of the CCR4-Not complex is in the of Tob and N. Yamashita A. Yamashita Y. D. Cell. Biol. PubMed Scopus Google Scholar, Y. Y. Hosoda N. N. M. M. Suzuki T. T. S. Genes Dev. Scopus Google Scholar, F. C. B. EMBO J. PubMed Scopus Google Scholar). the C-terminal region of Tob a that associates with PABPC1, the of to the CCR4-Not complex to of the is that which the not antiproliferative activity. These suggest that Tob a crucial in the CCR4-Not complex and and of the The complex as both a factor and poly(A)-specific deadenylase J. Biol. 2003; PubMed Scopus Google Scholar). SMAD family proteins are factors that bind to the C-terminal region of is evidence to SMAD family proteins to the complex. the are several that to Tob and the activity of the CCR4-Not complex K. Suzuki T. J. Matsuda S. Yamamoto T. Genes PubMed Scopus (58) Google Scholar, Y. Y. Hosoda N. N. M. M. Suzuki T. T. S. Genes Dev. Scopus Google Scholar). These to the Tob the complex to complex by to Caf1 as a to Tob and the complex than as a is by of the of cell growth is are to the is by formation of the complex and the to activity. a was reported in that the yeast protein which the yeast of hCaf1 and a component of the CCR4-Not deadenylase complex, of the of a Pop2 was to as a which the the complex to the target thereby activity M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, both the BTG/Tob and Caf1 are closely to germ cell In mice, is due to of cells T. R. T. Suzuki T. C. N. K. R. Miyasaka T. Yoshida Y. A. K. T. Yamamoto T. Noda T. Nat. Genet. PubMed Scopus (99) Google Scholar, C. A.M. F. Magaud J.P. P. Rouault J.P. Cell. Biol. PubMed Scopus Google Scholar). In C. and which are of hCaf1 and are germ (12Ellis R.E. Kimble J. Genetics. 1995; 139: 561-577Crossref PubMed Google Scholar, L. A. PubMed Scopus Google Scholar). The hCaf1 and Tob in the such as and are to of and of Furthermore, the of the BTG/Tob domain of FOG-3 the which to associate with and to the of FOG-3 by using Box A and which germ cell by In the we the of association of in The BTG/Tob family of antiproliferative proteins has been to associate with the SMAD family (13Yoshida Y. Nakamura T. Komoda M. Satoh H. Suzuki T. Tsuzuku J.K. Miyasaka T. Yoshida E.H. Umemori H. Kunisaki R.K. Tani K. Ishii S. Mori S. Suganuma M. Noda T. Yamamoto T. Genes Dev. 2003; 17: 1201-1206Crossref PubMed Scopus (99) Google Scholar, Y. Tanaka S. Umemori H. Minowa O. Usui M. Ikematsu N. Hosoda E. Imamura T. Kuno J. Yamashita T. Miyazono K. Noda M. Noda T. Yamamoto T. Cell. 2000; 103: 1085-1097Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 15Yoshida Y. von Bubnoff A. Ikematsu N. Blitz I.L. Tsuzuku J.K. Yoshida E.H. Umemori H. Miyazono K. Yamamoto T. Cho K.W. Mech. Dev. 2003; 120: 629-637Crossref PubMed Scopus (53) Google Scholar, D. Freeman G.J. Hirano N. van Puijenbroek A.A. Delfs M.W. Berezovskaya A. Nadler L.M. Boussiotis V.A. Nat. Immunol. 2001; 2: 1097-1098Crossref PubMed Scopus (223) Google Scholar), arginine (17Lin W.J. Gary J.D. Yang M.C. Clarke S. Herschman H.R. J. Biol. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar), and histone These are to and the of the BTG/Tob family proteins, and of the containing BTG/Tob family proteins and their are and all the of the of with also the of the