Inhibition of DNA damage-induced apoptosis through Cdc7-mediated stabilization of Tob.

BACKGROUND: Preventing unnecessary cell death is essential for DNA-damaged cells to carry out the DNA repair process. RESULTS: Cdc7 inhibits the Cul4-DDB1(Cdt2)-dependent Tob degradation. CONCLUSION: Cdc7 enables mild DNA-damaged cells to keep their viability by competing with the Tob degradation system. SIGNIFICANCE: Cells deal with moderate DNA damage not only by cessation of the cell cycle but also through direct mediated pro-survival signaling. Cells respond to DNA damage by activating alternate signaling pathways that induce proliferation arrest or apoptosis. The correct balance between these two pathways is important for maintaining genomic integrity and preventing unnecessary cell death. The mechanism by which DNA-damaged cells escape from apoptosis during DNA repair is poorly understood. We show that the DNA replication-initiating kinase Cdc7 actively prevents unnecessary death in DNA-damaged cells. In response to mild DNA damage, Tob levels increase through both a transcriptional mechanism and protein stabilization, resulting in inhibition of pro-apoptotic signaling. Cells lacking Cdc7 expression undergo apoptosis after mild DNA damage, where Cul4-DDB1(Cdt2) induces Tob ubiquitination and subsequent degradation. Cdc7 phosphorylates and interacts with Tob to inhibit the Cul4-DDB1(Cdt2)-dependent Tob degradation. Thus, Cdc7 defines an essential pro-survival signaling pathway by contributing to stabilization of Tob, thereby the viability of DNA-damaged cells being maintained.

TRAF-interacting protein with a forkhead-associated domain B (TIFAB) is a negative regulator of the TRAF6-induced cellular functions.

Tumour necrosis factor receptor-associated factor (TRAF)-interacting protein with a forkhead-associated domain (TIFA) activates TRAF6 to induce NF-kappaB activation. TIFA-related protein, TIFAB, is highly expressed in the spleen and inhibits TIFA-mediated TRAF6 activation. However, little is known about cell types that express TIFAB and its function in those cells. Here, we show that TIFAB is mainly expressed in B cells rather than T cells in the spleen and that the expression level was much higher in dendritic cells (DCs) and macrophages than that in splenic lymphocytes. TIFAB expression was downregulated when B cells, DCs or macrophages were stimulated by TRAF6-mediated proliferative or maturation signals including those emanating from CD40, sIgM and TLRs. Furthermore, microinjection experiments using NIH3T3 cells revealed that TIFAB inhibited entry into the S phase of the cell cycle. Our results suggest that TIFAB could act as a negative regulator of the TRAF6-induced cellular function such as B cell proliferation and maturation of DCs and macrophages.

Structural basis for the antiproliferative activity of the Tob-hCaf1 complex.

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.

Nuclear localization of Tob is important for regulation of its antiproliferative activity.

TOB: is a member of an antiproliferative gene family that includes btg1, pc3/tis21/btg2, pc3b, ana/btg3, and tob2. Exogenous overexpression of the family proteins suppresses cell proliferation. These proteins participate in transcriptional regulation of several genes. Here, we show that Tob is a nuclear protein that is imported into the nucleus through a nuclear localization signal (NLS)-mediated mechanism. Mutation in the NLS sequence of Tob affects its nuclear localization and impairs antiproliferative activity. Additionally, Tob contains a nuclear export signal (NES). In oncogenic ErbB2-transformed cells, nuclear export of Tob is facilitated by NES-mediated mechanism, resulting in decrease of its antiproliferative activity. These results indicate that regulation of nuclear localization of Tob is important for its antiproliferative activity.

Alteration of expression or phosphorylation status of tob, a novel tumor suppressor gene product, is an early event in lung cancer.

Tob is a member of the Tob/BTG family, a novel class of anti-proliferative proteins. To investigate the involvement of tob as a tumor suppressor gene in human lung cancer, we analyzed the expression of tob mRNA and protein in lung cancer tissue and adjacent normal lung tissue. Immunohistochemical analysis using anti-Tob antibody showed decreased expression of Tob in 72% (31/43) of lung cancer tissues. Furthermore, 95% (19/20) of squamous cell carcinoma patients showed an apparent decrease in Tob in cancer tissues, associated with smoking status. The phosphorylated form of Tob, an inactive form of Tob, was detected in 76% (16/21) of cancer tissues of adenocarcinoma patients, but not in normal alveolar epithelial cells. Either a decrease in Tob expression or an accumulation of phosphorylated Tob was observed from early clinical stages, even in bronchial dysplasia, a premalignant lesion of squamous cell carcinoma. The above findings suggest that the disruption of anti-proliferative Tob plays a distinct part in the early stage of lung carcinogenesis.

Tob proteins enhance inhibitory Smad-receptor interactions to repress BMP signaling.

Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts. Here we provide evidence that Tob also interacts with the inhibitory Smads 6 and 7. A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob. Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors. Furthermore, we have isolated Xenopus Tob2, and show that it cooperates with Smad6 in inducing secondary axes when expressed in early Xenopus embryos. Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells. Our results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions.

Mice lacking a transcriptional corepressor Tob are predisposed to cancer.

tob is a member of antiproliferative family genes. Mice lacking tob are prone to spontaneous formation of tumors. The occurrence rate of diethylnitrosamine-induced liver tumors is higher in tob(-/-) mice than in wild-type mice. tob(-/-)p53(-/-) mice show accelerated tumor formation in comparison with single null mice. Expression of cyclin D1 mRNA is increased in the absence of Tob and is reduced by Tob. Tob acts as a transcriptional corepressor and suppresses the cyclin D1 promoter activity through an interaction with histone deacetylase. Levels of tob mRNA are often decreased in human cancers, implicating tob in cancer development.

Phosphorylation of three regulatory serines of Tob by Erk1 and Erk2 is required for Ras-mediated cell proliferation and transformation.

tob is a member of an emerging family of genes with antiproliferative function. Tob is rapidly phosphorylated at Ser 152, Ser 154, and Ser 164 by Erk1 and Erk2 upon growth-factor stimulation. Oncogenic Ras-induced transformation and growth-factor-induced cell proliferation are efficiently suppressed by mutant Tob that carries alanines but not glutamates, mimicking phosphoserines, at these sites. Wild-type Tob inhibits cell growth when the three serine residues are not phosphorylated but is less inhibitory when the serines are phosphorylated. Because growth of Rb-deficient cells was not affected by Tob, Tob appears to function upstream of Rb. Intriguingly, cyclin D1 expression is elevated in serum-starved tob(-/-) cells. Reintroduction of wild-type Tob and mutant Tob with serine-to-alanine but not to glutamate mutations on the Erk phosphorylation sites in these cells restores the suppression of cyclin D1 expression. Finally, the S-phase population was significantly increased in serum-starved tob(-/-) cells as compared with that in wild-type cells. Thus, Tob inhibits cell growth by suppressing cyclin D1 expression, which is canceled by Erk1- and Erk2-mediated Tob phosphorylation. We propose that Tob is critically involved in the control of early G(1) progression.