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

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

doi:10.1074/jbc.m809250200

Cited by 91 publications

(143 citation statements)

References 51 publications

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“…Earlier studies found that knocking down CAF1 or ectopically expressing a CAF1 mutant unable to bind TOB1 compromised the ability of TOB1 to arrest cell growth or to reduce foci formation in cancer cells (18), suggesting that interaction between TOB1 and CAF1 is involved in the antiproliferative effect of TOB1. However, as efficient cell proliferation requires CAF1 (4), it is unclear whether the observed loss of cell growth inhibition was simply due to a loss of proper CAF1 function or whether binding to CAF1 is necessary for TOB1 to achieve its antiproliferative effect.…”

Section: Tethering Tobs To Mrnas Promotes Deadenylation and Decay Of mentioning

confidence: 99%

“…CAF1 associates with CCR4 to form a deadenylase complex that plays a predominant role in shortening the mRNA poly(A) tail in eukaryotes (2,9,11,35,36,38,41). TOB proteins interact with CAF1 via their N-terminal domains (18,27). Unlike other BTG/TOB family members, TOB proteins contain an extra-long C-terminal domain with two poly(A)-binding protein (PABP)-interacting motif 2 (PAM2) motifs (13,22,29; see also Fig.…”

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confidence: 99%

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Evidence Providing New Insights into TOB-Promoted Deadenylation and Supporting a Link between TOB's Deadenylation-Enhancing and Antiproliferative Activities

Ezzeddine

Chen

Shyu

2012

Molecular and Cellular Biology

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The mammalian TOB1 and TOB2 proteins have emerged as key players in repressing cell proliferation. Accumulating evidence indicates that TOBs regulate mRNA deadenylation. A recruitment model was proposed in which TOBs promote deadenylation by recruiting CAF1-CCR4 deadenylase complex to the 3= end of mRNAs by simultaneously binding CAF1 and PABP. However, the exact molecular mechanism underlying TOB-promoted deadenylation remains unclear. It is also unclear whether TOBs' antiproliferative and deadenylation-promoting activities are connected. Here, we combine biochemical analyses with a functional assay directly monitoring deadenylation and mRNA decay to characterize the effects of tethering TOBs or their mutant derivatives to mRNAs. The results provide direct evidence supporting the recruitment model and reveal a link between TOBs' antiproliferative and deadenylation-promoting activities. We also find that TOBs' actions in deadenylation are independent of the phosphorylation state of three serines known to regulate antiproliferative actions, suggesting that TOBs arrest cell growth through at least two different mechanisms. TOB1 and TOB2 were interchangeable in the properties tested here, indicating considerable functional redundancy between the two proteins. We propose that their multiple modes of modulating mRNA turnover and arresting cell growth permit the TOB proteins to coordinate their diverse roles in controlling cell growth and differentiation.T he two human TOB proteins, encoded by paralogous genes (TOB1 and TOB2) (20, 25), belong to a group of antiproliferative factors, the BTG/TOB family (26,39). In addition to roles in cell proliferation, BTG/TOB proteins have also been implicated in embryonic development, cellular differentiation, cancer suppression, and apoptosis (21,26,27,39). Levels of BTG/TOB proteins fluctuate during the cell cycle and can be induced by diverse stimuli, such as growth factors, tumor promoters, and genotoxic stress. Given the varied roles attributed to BTG/TOB proteins, the varied pathways controlling their expression, and the identification of varied associated proteins, the biological functions of BTG/ TOB proteins are rather complicated.In addition to the roles of BTG/TOB proteins in regulating mRNA production (reviewed in references 21, 26, 27, and 39), the detection of direct interaction between BTG/TOB proteins and the CAF1 deadenylase (13,20,28,31) suggests a role of BTG/TOB proteins in deadenylation, a critical posttranscriptional step that regulates cytoplasmic mRNA levels (8, 16). CAF1 associates with CCR4 to form a deadenylase complex that plays a predominant role in shortening the mRNA poly(A) tail in eukaryotes (2,9,11,35,36,38,41). TOB proteins interact with CAF1 via their N-terminal domains (18,27). Unlike other BTG/TOB family members, TOB proteins contain an extra-long C-terminal domain with two poly(A)-binding protein (PABP)-interacting motif 2 (PAM2) motifs (13,22,29; see also Fig. S1 in the supplemental material). Recently, we showed that TOB proteins can intera...

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Section: Tethering Tobs To Mrnas Promotes Deadenylation and Decay Of mentioning

confidence: 99%

mentioning

confidence: 99%

Evidence Providing New Insights into TOB-Promoted Deadenylation and Supporting a Link between TOB's Deadenylation-Enhancing and Antiproliferative Activities

Ezzeddine

Chen

Shyu

2012

Molecular and Cellular Biology

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“…To test this idea, we created mutations in Xenopus laevis CAF1b that were predicted to disrupt interaction with NOT1, CCR4, and TOB, each of which directly interacts with CAF1 ( Fig. 1A; Horiuchi et al 2009;Basquin et al 2012). The known human CAF1a structure was used to guide mutagenesis (Horiuchi et al 2009).…”

Section: Caf1 Requires Interaction With Not1 To Repress Translationmentioning

confidence: 99%

“…1A; Horiuchi et al 2009;Basquin et al 2012). The known human CAF1a structure was used to guide mutagenesis (Horiuchi et al 2009). We also mutated specific surface residues to alanine, reasoning that they too might mediate protein-protein contacts (Fig.…”

Section: Caf1 Requires Interaction With Not1 To Repress Translationmentioning

confidence: 99%

Xenopus CAF1 requires NOT1-mediated interaction with 4E-T to repress translation in vivo

Waghray

Williams

Coon

et al. 2015

RNA

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RNA-regulatory factors bound to 3 ′ UTRs control translation and stability. Repression often is associated with poly(A) removal. The deadenylase CAF1 is a core component of the CCR4-NOT complex. Our prior studies established that CAF1 represses translation independent of deadenylation. We sought the mechanism of its deadenylation-independent repression in Xenopus oocytes. Our data reveal a chain of interacting proteins that links CAF1 to CCR4-NOT and to Xp54 and 4E-T. Association of CAF1 with NOT1, the major subunit of CCR4-NOT, is required for repression by CAF1 tethered to a reporter mRNA. Affinity purification-mass spectrometry and coimmunoprecipitation revealed that at least five members of the CCR4-NOT complex were recruited by CAF1. The recruitment of these proteins required NOT1, as did the ability of tethered CAF1 to repress translation. In turn, NOT1 was needed to recruit Xp54 and 4E-T. We examined the role of 4E-T in repression using mutations that disrupted either eIF4E-dependent or -independent mechanisms. Expression of a 4E-T truncation that still bound eIF4E alleviated repression by tethered CAF1, NOT1, and Xp54. In contrast, a mutant 4E-T that failed to bind eIF4E did not. Repression of global translation was affected only by the eIF4E-dependent mechanism. Reporters bearing IRES elements revealed that repression via tethered CAF1 and Xp54 is cap-and eIF4E-independent, but requires one or more of eIF4A, eIF4B, and eIF4G. We propose that RNA-binding proteins, and perhaps miRNAs, repress translation through an analogous chain of interactions that begin with the 3 ′ UTR-bound repressor and end with the noncanonical activity of 4E-T.

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“…The affinity might be much greater at a physiological salt concentration because the high concentration of NaCl appears to impair the electrostatic interactions between Tob and Caf1. The crystal structure of the TobN138-Caf1 complex (PDB code: 2d5r) contains at least three intermolecular ion pairs (Lys-51-Glu-247, Asp-65-Lys-254, and Glu-98 -Lys-203) and a hydrogen bond (Glu-105-Tyr-255) at the relatively hydrophobic binding interface (20). On the other hand, the NMR and ITC analyses revealed that the PAM2-C sequence of Tob is the primary PABC-binding site, which contributes to the binding affinity with a K d value of 20 M ( Table 1).…”

Section: Structure Of Tob and Binding Of Tob To Caf1 And Pabpc1-thementioning

confidence: 99%

Quantitative Characterization of Tob Interactions Provides the Thermodynamic Basis for Translation Termination-coupled Deadenylase Regulation

Ruan

Osawa

Hosoda

et al. 2010

Journal of Biological Chemistry

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Translation termination-coupled deadenylation is the first and often the rate-limiting step of eukaryotic mRNA decay in which two deadenylases, Ccr4-Caf1 and Pan2, play key roles. One of the deadenylases, Caf1, associates with Tob, which recruits Caf1 to the poly(A) tail through interactions with a cytoplasmic poly(A)-binding protein 1 (PABPC1). We previously proposed that the competition between Tob and eRF3 (a translation termination factor that interacts with PABPC1) is responsible for the regulation of deadenylase activity. However, the molecular mechanism of the regulation should be addressed by investigating the binding affinity and the cellular levels of these proteins. In this work, we characterized the human Tob interactions with Caf1 and a C-terminal domain of PABPC1 (PABC). Nuclear magnetic resonance (NMR) and Western blot analyses revealed that Tob consists of a structured N-terminal BTG-Tob domain and an unstructured C-terminal region with two conserved PAM2 (PABPC1-interacting motif 2) motifs. The BTG-TOB domain associates with Caf1, whereas the C-terminal PAM2 motif binds to PABC, with a K d value of 20 M. Furthermore, we demonstrated that the levels of eRF3 and Tob in HeLa cells are 4 -5 M and less than 0.2 M, respectively. On the basis of these results, we propose a thermodynamic mechanism for the translation termination-coupled deadenylation mediated by the Tob-Caf1 complex.

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

Cited by 91 publications

References 51 publications

Evidence Providing New Insights into TOB-Promoted Deadenylation and Supporting a Link between TOB's Deadenylation-Enhancing and Antiproliferative Activities

Evidence Providing New Insights into TOB-Promoted Deadenylation and Supporting a Link between TOB's Deadenylation-Enhancing and Antiproliferative Activities

Xenopus CAF1 requires NOT1-mediated interaction with 4E-T to repress translation in vivo

Quantitative Characterization of Tob Interactions Provides the Thermodynamic Basis for Translation Termination-coupled Deadenylase Regulation

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