The two human TOB proteins, encoded by paralogous genes (TOB1 and TOB2) (20
), belong to a group of antiproliferative factors, the BTG/TOB family (26
). In addition to roles in cell proliferation, BTG/TOB proteins have also been implicated in embryonic development, cellular differentiation, cancer suppression, and apoptosis (21
). 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
, and 39
), the detection of direct interaction between BTG/TOB proteins and the CAF1 deadenylase (13
) suggests a role of BTG/TOB proteins in deadenylation, a critical posttranscriptional step that regulates cytoplasmic mRNA levels (8
). CAF1 associates with CCR4 to form a deadenylase complex that plays a predominant role in shortening the mRNA poly(A) tail in eukaryotes (2
). TOB proteins interact with CAF1 via their N-terminal domains (18
). 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
; see also Fig. S1 in the supplemental material). Recently, we showed that TOB proteins can interact with CAF1 and PABP simultaneously (13
). Overexpression of TOB promotes general deadenylation (13
). We further demonstrated that the deadenylation-enhancing effect of TOB proteins is dependent upon their ability to bind PABP (13
). Based on these results, we proposed that the interaction of TOB with CAF1 and PABP promotes deadenylation by recruiting the CAF1-CCR4 deadenylase complex to the 3′ end of mRNAs with a poly(A) tail (13
). Nevertheless, the exact mechanism by which TOB promotes deadenylation remains unclear.
In mammals, TOB proteins are substrates for posttranslational modification, including phosphorylation by mitogen-activated protein kinases (MAPKs) (24
) and ubiquitination, which targets them for degradation (32
). Upon serum or growth factor stimulation, TOB1 is rapidly phosphorylated by extracellular signal-regulated kinase 1 (ERK1) and ERK2 MAP kinases at serine residues 152, 154, and 164 (34
; see also Fig. S1 in the supplemental material). When these serine residues were phosphorylated or mutated to glutamate, TOB's inhibitory effect on cell growth was blunted (24
). Our finding that TOB proteins promote mRNA deadenylation (13
) raises important questions as to whether the antiproliferative action of TOB is linked to its ability to promote deadenylation and whether the deadenylation-enhancing ability of TOB is regulated through phosphorylation when cells reenter the cell cycle. Moreover, although ectopically overexpressed TOB accelerates deadenylation (13
), it remains unknown whether TOB proteins promote the degradation of the mRNA body and reduce the cognate protein levels. Furthermore, as most studies thus far have focused on TOB1, it is unclear whether TOB2 differs mechanistically from TOB1.
In this study, we addressed these critical issues regarding human TOB1 and TOB2 using transcriptional pulse-chase and RNA-tethering approaches (7
). The results from these functional assays provide crucial new insights into the mechanism by which TOB proteins downregulate gene expression. They also reveal a link between the TOB proteins' antiproliferative and mRNA deadenylation/decay-promoting actions.