Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

BTG/TOB

  • Hibah Almasmoum
  • Rachel Doidge
  • Gerlof Sebastiaan Winkler
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_272

Synonyms

Historical Background

The human BTG/Tob proteins form a small family of six proteins, which share a conserved N-terminal domain and antiproliferative activity (Matsuda et al. 2001; Tirone 2001; Winkler 2010). BTG2 was discovered first by two laboratories: as the immediate/early response gene PC3 in rat PC12 cells stimulated with nerve growth factor (NGF) and as TIS21 in mouse 3T3 fibroblasts in response to treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). The discovery of BTG1 (B-cell translocation gene 1) as a gene involved in a chromosomal translocation associated with chronic lymphocytic leukemia suggested the presence of a new family of antiproliferative genes. These findings were extended by the discovery of TOB1, which was found as an interacting protein of the ErbB2 tyrosine-kinase receptor (HER2). The remaining three members BTG3 (ANA), BTG4 (PC3B), and TOB2 were identified based on sequence homology of the conserved N-terminal domain. The preferred gene names by the Human Genome Nomenclature Committee are BTG1, BTG2, BTG3, BTG4, TOB1, and TOB2.

Regulation of Gene Expression: mRNA Deadenylation

The conserved N-terminus is known as the BTG domain (Pfam number PF07742; also known as APRO domain) and comprises 104–106 amino acids. The C-terminal regions are less conserved and confer additional functions to the family members. Sequence analysis of both the BTG domain and the C-terminal regions suggests that Tob1 and Tob2 as well as BTG1 and BTG2 are highly similar, whereas BTG3 and BTG4 are more distantly related (Fig. 1). The BTG/Tob proteins are implicated in the regulation of gene expression by at least two distinct mechanisms.
BTG/TOB, Fig. 1

Schematic overview of the human BTG/Tob protein family. The approved gene names used by the human genome nomenclature committee are used. Indicated are the N-terminal BTG/Tob domains (dark gray) and PAM2 motifs (gray). The pair-wise percentage identities were determined using the Clustalw2 multiple sequence alignment program. The length of the proteins (amino acids) and the position of the BTG domain are also indicated

The best characterized role of the BTG/Tob proteins in gene expression is mediated via the BTG homology domain, which interacts with the Caf1 subunit of the Ccr4-Not complex, which is encoded by CNOT7 or CNOT8. The highly similar CNOT7 and CNOT8 proteins are deadenylase enzymes, which shorten and remove the poly(A) tails of cytoplasmic mRNA resulting in translational repression and mRNA degradation (Mauxion et al. 2009; Winkler 2010). The interaction of all BTG/Tob proteins with either CNOT7 and/or CNOT8 is experimentally confirmed, and a specific role in the regulation of deadenylation and mRNA degradation was demonstrated for all proteins except BTG4. BTG1, BTG2, TOB1, and TOB2 can interact with the poly(A)-binding protein PABPC1. In case of BTG1 and BTG2, this interaction is mediated by the BTG domain (Stupfler et al. 2016). By contrast, Tob1 and Tob2 contain PAM2 motifs in their C-terminal regions, which allow them to interact with poly(A) binding protein 1 (PABPC1) (Ezzeddine et al. 2007; Funakoshi et al. 2007). During termination of translation, several proteins containing a PAM2 motif are consecutively recruited to the mRNA by PABPC1: following binding of the translation termination complex eRF1-eRF3 and the PAN2-PAN3 deadenylase, Tob1 recruits the Ccr4-Not deadenylase via interactions between the conserved BTG domain and the CNOT7 and CNOT8 deadenylase subunits. This sequence of events implicates Tob1 – as well as the related Tob2 protein – in mRNA deadenylation coupled to termination of translation. Alternatively, Tob1 can be recruited to specific mRNAs by sequence specific RNA-binding proteins. For example, cytoplasmic polyadenylation element-binding protein 3 (CPEB3) binds Tob1 resulting in mRNA destabilization (Hosoda et al. 2011).

Several protein structures illuminate the molecular details of the interaction between BTG/Tob proteins and the CNOT7/CNOT8 deadenylase enzymes. The BTG domain is characterized by two long antiparallel α-helices in the N-terminus of the domain that are part of a four-helix bundle and three β-sheets at the C-terminus of the domain (Fig. 2). Comparison of the structure of the free BTG domain of Tob1 with the domain in complex with the CNOT7 deadenylase indicates that the BTG domain does not undergo significant rearrangements upon binding. The RNA-binding, catalytic site of the CNOT7 deadenylase appears to be separated from the residues important for binding to the BTG domain. In agreement with this, binding of the BTG domain of Tob1 does not influence the catalytic activity of the CNOT7 deadenylase (Horiuchi et al. 2009).
BTG/TOB, Fig. 2

Structure of the BTG domain of Tob1 in complex with the Caf1/CNOT7 deadenylase enzyme. The representation was generated using structure 2d5r deposited in the Protein Data Bank (PDB) using Pymol (www.pymol.org). The BTG domain of Tob1 is represented by a multicolored cartoon. Indicated are the five α-helices and four β-sheets. The surface of the Caf1/CNOT7 deadenylase enzyme is represented in gray. Circled is a deep pocket that binds poly(A) RNA and corresponds to the catalytic center

Regulation of Gene Expression: Transcription

In addition to their role in mRNA deadenylation, BTG/Tob proteins can regulate gene expression at the level of transcription (Matsuda et al. 2001; Tirone 2001; Winkler 2010). Several reports point to the ability of the BTG/Tob proteins to interact with DNA-binding transcription factors and modulate their ability to bind their cognate DNA sequence elements. Both BTG1 and BTG2 can interact with Hoxb9, a homeobox DNA-binding transcription factor, through their extreme N-terminus (residues 1–14). This interaction enhances the ability of Hoxb9 to bind to its consensus DNA sequence. Thus, this may increase the transcription rates of Hoxb9 target genes, which may contribute to the antiproliferative function of BTG1 and BTG2. Tob1 and Tob2 have the most extensive C-terminal regions within the protein family. This region of Tob1 mediates interactions with a number of Smad transcription factors, altering their ability to bind to DNA. As a consequence, Tob1 regulates the expression of Smad target genes, such as the cytokine IL-2 promoter in quiescent T-cells. BTG3 presents a third example of this mode of action: BTG3 can interact with E2F1, a transcription factor important for S-phase entry and cell cycle progression. BTG3 binds E2F1 through its N-terminal region, which, in this case, inhibits DNA binding of the E2F1, thereby reducing the overall transcription rate of E2F1-responsive promoters and cell proliferation.

Finally, BTG1 and BTG2 can interact with protein arginine methyl-transferase 1 (PRMT1) through a short β-sheet region (also known as Box C) just outside the BTG domain, which is not conserved in other BTG/Tob proteins. PRMT1 specifically methylates the arginine 3 residue of histone H4 in vitro and in vivo, which facilitates subsequent acetylation of histone H4 tails by p300 and gene activation. Thus, this raises the possibility that BTG1 and BTG2 could be involved in the regulation of chromatin modifications.

Effectors of Signaling Pathways

There are a variety of different signaling pathways that exploit the antiproliferative properties of BTG/Tob proteins either positively or negatively by regulating the cellular levels of these proteins by transcriptional and post-translational mechanisms. Both Tob1 and BTG2 are phosphorylated upon stimulation with growth factors by the Erk1/Erk2 kinases at serine residues in the C-terminus. This results in subsequent deactivation, which – in the case of Tob1 – leads to increased cyclin D1 expression and enhanced activation of CDK4, driving cell cycle progression and cell proliferation.

In MCF7 cells (an estrogen receptor-expressing breast cancer cell line), BTG2 mRNA can be regulated both positively and negatively by signaling through nuclear receptor transcription factors. BTG2 expression is activated when MCF7 cells are treated with retinoic acid through direct binding of the retinoic acid receptor (RAR)/RXR heterodimers to three retinoic acid response elements (RARE) in the BTG2 promoter region. Conversely, BTG2 expression is reduced when MCF7 cells are treated with estrogen through estrogen receptor ERα and its corepressor REA.

BTG2 and BTG3 are both downstream targets of the p53 signaling pathway. Both proteins are direct transcriptional targets for p53 and play a role in the p53-mediated response to DNA damage (Rouault et al. 1996; Ou et al. 2007). In embryonic mouse fibroblasts, BTG2 plays critical role in suppressing transformation through oncogenic Ras by acting as a downstream effector of p53 (Boiko et al. 2006). BTG2 expression down-regulates cyclin D1, cyclin E1, and the phosphorylation of retinoblastoma (Rb) slowing cell cycle progression and preventing cellular transformation.

Finally, Tob1 and BTG2 are implicated in signaling of TGF-family members through Smad transcription factors. This was demonstrated in both quiescent T-cells activated by CD28, which impinges on TGF-β signaling, and in bone-forming osteoblast cells upon stimulation by bone morphogenic protein (BMP) 2, a TGF-family member (Fig. 3).
BTG/TOB, Fig. 3

Signaling pathways impinge on BTG/Tob proteins. Both antiproliferative and proliferative signals impinge on BTG/Tob family members by upregulation/activation or inhibition, respectively. In turn, BTG/Tob proteins can participate in the regulation of gene expression by deadenylation (left) or transcriptional mechanisms (right). See text for further details

Bone Formation: Tob1 and Tob2

The generation of mice containing null alleles of Tob1, Tob2, Btg2, and Btg3 uncovered a role for these proteins in bone formation and resorption (Yoshida et al. 2003; Park et al. 2004; Ajima et al. 2008; Miyai et al. 2009). The contrasting phenotypes observed in Tob1 and Tob2 knockout mice are of particular interest. Mice lacking Tob1 are apparently normal but display increased bone volume and bone density. Interestingly, in a mouse model for estrogen deficiency–induced osteoporosis, the increased bone mineralization in Tob1 null mice can compensate for bone loss associated with induced osteoporosis since ovariectomized Tob1 knockout mice have a bone mineral density and volume comparable to (sham operated) control mice (Usui et al. 2004). The increased bone density in Tob1−/− mice is due to enhanced bone formation, and osteoclast parameters are unchanged as compared to control mice. A similar increase in bone density is observed in mice lacking the Cnot7 deadenylase (Washio-Oikawa et al. 2007). As observed in Tob1 null mice, Cnot7 knockout mice do not display altered osteoclast parameters suggesting that the role of Tob1 in bone formation is mediated via its interactions with the CNOT7 deadenylase subunits of the Ccr4-Not complex.

By contrast, mice lacking Tob2 display decreased bone mass due to an increased number of differentiated osteoclast cells. Tob2 interacts with the vitamin D receptor and reduces expression of RANKL, a vitamin D-induced gene. In agreement with this notion and the observation that osteoclast parameters are unaltered in CNOT7 knockout mice, Tob2 is a repressor of vitamin D-induced osteoclast formation (Ajima et al. 2008).

Cancer and Tumorigenesis

The discovery of BTG2 as an effector of the tumor suppressor function of p53, as well as the critical role of Tob1 in Ras-mediated transformation, strongly implicates these BTG/Tob proteins as important cellular components that contribute to the prevention of tumorigenesis (Rouault et al. 1996; Suzuki et al. 2002; Boiko et al. 2006). In agreement with this notion, expression of BTG/Tob genes is reduced or undetectable in a variety of clinical cancer samples (Table 1). In particular, the presence of increased levels of phosphorylated, inactive Tob1 and the absence of Tob1 protein levels correlate with tumor grade in a panel of lung cancer samples. Similarly, expression of BTG3 is reduced in the majority of lung cancer cell lines and clinical samples derived from lung cancer patients. Furthermore, BTG1 and BTG2 are frequently found to be mutated in leukemia and non-Hodgkin lymphomas (Fig. 4; Table 1). Such mutations are seemingly present in a mutually exclusive manner as compared to p53 mutations, suggesting a causative role as a component of the p53 pathway in this type of cancer. It is yet unknown how the identified mutations in BTG1 and BTG2 interfere with the function of the encoded gene products.
BTG/TOB, Table 1

Relationship of BTG/Tob expression and cancer

Gene

Cancer

 

References

TOB1

Lung

Decreased expression/increased phosphorylationb

Iwanaga et al. 2003. Cancer Lett 202:71–79

Lung

Spontaneous tumor formationa

Yoshida et al. 2003. Genes Dev 17:1201–1206

Lymph node

Spontaneous tumor formationa

Ibid.

Liver

Spontaneous tumor formationa

Ibid.

Breast

Increased expression associated with poor prognosis

Helms et al. 2009. Cancer Res 69:5049–5056

Thyroid

Decreased mRNA expressionb

Ito et al. 2005. Cancer Lett 220:237–242

Pancreatic

Induced expression inhibits tumorigenesis in nude mice

Yanagie et al. 2009. Biomed Pharmacother 3:275–286

BTG1

Leukemia

Reduced/undetectable expressionb

Cho et al. 2004. Proteomics 4:3456–3463

Waanders et al. 2012. PLoS Genet 8:e1002533

Xie et al. 2014. Cancer Genetics 207:226–230

Lymphoma

Somatic mutationsb

Morin et al. 2011. Nature 476:298–303

Lohr et al. 2012. PNAS 109:3879–3884

Zhang et al. 2013. PNAS 110:1398–1403

Waldenström macroglobulinemia

Somatic mutations and deletionb

Hunter et al. 2014. Blood 123:1637–1646

Gastric

Low expression associate with poor prognosisb

Kanda et al. 2014. Dig Dis Sci 60:1256–1264

BTG2

Breast

Reduced expression and relocalization (nuclear to cytoplasm) b

Kawakubo et al. 2006. Cancer Res 66:7075–7082

Renal

Reduced mRNA levelsb

Struckmann et al. 2004. Cancer Res 64:1632–1638

Prostate

Low/undetectable mRNA levelsb

Ficazzola et al. 2001. Carcinogenesis 22:1271–1279

Brain

Induced expression inhibits medulloblastomas (transgenic mice)

Farioli-Vecchioli et al. 2007. FASEB J 21:2215–2225

Lymphoma

Somatic mutationsb

Morin et al. 2011. Nature 476:298–303

Lohr et al. 2012. PNAS 109:3879–3884

Love et al. 2012. Nat Genet. 44:1321–1325

Zhang et al. 2013. PNAS 110:1398–1403

Fukumura et al. 2016 Acta Neuropathol 131:865–875

BTG3

Lung

Increased lung tumor formationa

Yoneda et al. 2009. Cancer Sci 100:225–232

Lung

Reduced expression in adenocarcinoma samplesb

Id.

Renal

Reduced mRNA expressionb

Majid et al. 2009. Carcinogenesis 30:662–670

BTG4

Colon

Reduced mRNA expressionb

Toyota et al. 2008. Cancer Res 68:4123–4132

Leukemia

Methylation associated with good prognosisb

Irving et al. 2011. Epigenetics 6:300–306

aObservations made using mouse knock-out models

bObservation made using human clinical cancer samples and biopsies

BTG/TOB, Fig. 4

Mutations identified in BTG1 and BTG2 in non-Hodgkin lymphoma. Mutations in BTG1 and BTG2 are identified by whole-exome and RNA sequencing data from over 100 non-Hodgkin lymphomas. In some cases, both alleles contained mutations. Indicated are schematic representations of BTG1 and BTG2, the location of the BTG domain, and the presence of secondary structure elements based on the crystal structure of BTG2 (PDB structures 3dju, 3djn and 3e9v)

An important role for BTG/Tob proteins in the suppression of tumorigenesis is further evident from mouse knockout models. Disruption of Tob1 in mice results in susceptibility to a variety of cancers, including lung tumors, which is also observed in mice lacking BTG3 (Table 1). Thus, a direct role of several BTG/Tob proteins in the suppression of tumorigenesis and cancer development has been demonstrated in a number of cases. However, there are a few notable exceptions. For example, TOB1 expression is increased in EGF- and HER2-positive breast cancer (Table 1). In this case, TOB1 was highly phosphorylated, which may counteract the antiproliferative function of the unphosphorylated protein.

Summary

The understanding of the function and mechanisms through which the BTG/Tob proteins act has rapidly advanced in the past few years. The best characterized role of the BTG/Tob proteins is mediated by the interaction of the BTG homology domain with the CNOT7 and CNOT8 deadenylase subunits of the Ccr4-Not complex, which impacts on mRNA deadenylation. In addition, BTG/Tob proteins are also involved in the regulation of transcription and, possibly, the establishment of histone H4 modifications through the interactions of BTG1 and BTG2 with the PRMT1 methyltransferase. Mouse models have uncovered the importance of these proteins in the biology of bone and cancer. Reduced expression of BTG/Tob proteins is observed in a variety of clinical samples, and mutations in BTG1 and BTG2 are found in non-Hodgkin lymphoma. It remains to be determined whether BTG/Tob proteins regulate cell proliferation through mRNA degradation or transcriptional mechanisms or both. Furthermore, there are still many questions with respect to unique and/or redundant roles of the individual BTG/Tob proteins.

References

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© Springer International Publishing AG 2018

Authors and Affiliations

  • Hibah Almasmoum
    • 1
    • 2
  • Rachel Doidge
    • 1
  • Gerlof Sebastiaan Winkler
    • 1
  1. 1.School of PharmacyUniversity of NottinghamNottinghamUK
  2. 2.School of Medicine and School of PharmacyUniversity of NottinghamNottinghamUK