Human Tribbles 3 Protects Nuclear DNA from Cytidine Deamination by APOBEC3A

The human polydeoxynucleotide cytidine deaminases APOBEC3A, APOBEC3C, and APOBEC3H are capable of mutating viral DNA in the nucleus, whereas APOBEC3A alone efficiently edits nuclear DNA. Deamination is rapidly followed by excision of uracil residues and can lead to double-stranded breaks. It is not known to which protein networks these DNA mutators belong. Using a yeast two-hybrid screen, we identified the human homolog of Drosophila Tribbles 3, TRIB3, as an interactor for APOBEC3A and APOBEC3C. The interaction was confirmed by co-affinity purification. Co-transfection of APOBEC3A with a TRIB3 expression vector reduced nuclear DNA editing whereas siRNA knockdown of TRIB3 increased the levels of nuclear DNA editing, indicating that TRIB3 functioned as a repressor of A3A. It also repressed A3A-associated γH2AX positive double-stranded breaks. The interaction results in degradation of A3A in a proteasome-independent manner. TRIB3 has been linked to cancer and via its own interactors and links the A3A DNA mutators to the Rb-BRCA1-ATM network. TRIB3 emerges as an important guardian of genome integrity.     

Mixed Lineage Kinase-3 Stabilizes and Functionally Cooperates with TRIBBLES-3 to Compromise Mitochondrial Integrity in Cytokine-induced Death of Pancreatic Beta Cells

Mixed lineage kinases (MLKs) have been implicated in cytokine signaling as well as in cell death pathways. Our studies show that MLK3 is activated in leukocyte-infiltrated islets of non-obese diabetic mice and that MLK3 activation compromises mitochondrial integrity and induces apoptosis of beta cells. Using an ex vivo model of islet-splenocyte co-culture, we show that MLK3 mediates its effects via the pseudokinase TRB3, a mammalian homolog of Drosophila Tribbles. TRB3 expression strongly coincided with conformational change and mitochondrial translocation of BAX. Mechanistically, MLK3 directly interacted with and stabilized TRB3, resulting in inhibition of Akt, a strong suppressor of BAX translocation and mitochondrial membrane permeabilization. Accordingly, attenuation of MLK3 or TRB3 expression each prevented cytokine-induced BAX conformational change and attenuated the progression to apoptosis. We conclude that MLKs compromise mitochondrial integrity and suppress cellular survival mechanisms via TRB3-dependent inhibition of Akt.     

Nanobodies identify an activated state of the TRIB2 pseudokinase

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Tribbles coordinates mitosis and morphogenesis in Drosophila by regulating string/CDC25 proteolysis

Morphogenesis and cell differentiation in multicellular organisms often require accurate control of cell divisions. We show that a novel cell cycle regulator, tribbles, is critical for this control during Drosophila development. During oogenesis, the level of tribbles affects the number of germ cell divisions as well as oocyte determination. The mesoderm anlage enters mitosis prematurely in tribbles mutant embryos, leading to gastrulation defects. We show that Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination.     

Tribbles: a family of kinase-like proteins with potent signalling regulatory function

The recent identification of tribbles as regulators of signal processing systems and physiological processes, including development, together with their potential involvement in diabetes and cancer, has generated considerable interest in these proteins. Tribbles have been reported to regulate activation of a number of intracellular signalling pathways with roles extending from mitosis and cell activation to apoptosis and modulation of gene expression. The current review summarises our current understanding of interactions between tribbles and various other proteins. Since our understanding on the molecular basis of tribbles function is far from complete, we also describe a bioinformatic analysis of various segments of tribbles proteins, which has revealed a number of highly conserved peptide motifs with potentially important functional roles.     

Tribbles homolog 3-mediated targeting the AKT/mTOR axis in mice with retinal degeneration

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3^−/− retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.Subject terms: Translation, Diseases     

Tribbles, a cell-cycle brake that coordinates proliferation and morphogenesis during Drosophila gastrulation

BACKGROUND: The final shape and size of an organism is determined by both morphogenetic processes and cell proliferation and it is essential that these processes be properly coordinated. In particular, cell division is incompatible with certain types of morphogenetic cell behaviour, such as migration, adhesion and changes in cell shape. Mechanisms must therefore exist to ensure that one does not interfere with the other. RESULTS: We address here the coordination of proliferation and morphogenesis during the development of the mesoderm in Drosophila. We show that it is essential that mitosis be blocked in the mesoderm during early gastrulation, and identify the putative serine/threonine kinase Tribbles as controlling this block. In its absence, the mitotic block is lifted, resulting in severe defects during early gastrulation. Tribbles, a homologue of a group of vertebrate proteins of unknown function, acts in concert with another, as yet unidentified, factor to counteract the activity of the protein phosphatase Cdc25/String. CONCLUSIONS: In a finely tuned balance with Cdc25/String, Tribbles controls the timing of mitosis in the prospective mesoderm, allowing cell-shape changes to be completed. This mechanism for coordinating cell division and cell-shape changes may have helped Drosophila to evolve its mode of rapid early development.     

Involvement of PPARγ in the antitumoral action of cannabinoids on hepatocellular carcinoma

Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In this study, we examined whether the PPARγ-activated pathway contributed to the antitumor effect of two cannabinoids, Δ9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity and intracellular level of PPARγ mRNA and protein, which was abolished by the PPARγ inhibitor GW9662. Moreover, genetic ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARγ decreased the cannabinoid-induced cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC tumors in mice. In addition, PPARγ knockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62, suggesting that PPARγ is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARγ expression and induced p62 accumulation, which was counteracted by overexpression of PPARγ in TRIB3-knocked down cells. Taken together, we demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo, are modulated by upregulation of PPARγ-dependent pathways.     

Ubiquitin E3 ligase SCF regulates TRIB2 stability in liver cancer cells

Tribbles homolog 2 (TRIB2) is functionally important for liver cancer cell survival and transformation. Our previous study demonstrates TRIB2 is stable in liver cancer cells due to the impaired ubiquitination by Smurf1. However, overexpression of Smurf1 alone cannot completely abolish TRIB2 protein expression, whether other potential factors involved in the degradation of TRIB2 still remains unclear. In the present study, we reveal that the stability and ubiquitination of TRIB2 can also be controlled by ubiquitin E3 ligase SCF^β-TRCP. Depletion of either Cullin1 or β-TRCP up-regulates TRIB2 protein expression. Moreover, knockdown of β-TRCP extends the half-life, whereas reduces ubiquitylation of TRIB2. Similar to Smurf1, β-TRCP exerts its role through the TRIB2 Degradation Domain (TDD) at the N-terminus of the TRIB2 protein. Hence, we add TRIB2 to the substrate list of SCF^β-TRCP and the finding may be helpful in the treatment of TRIB2 dependent liver cancer.     

Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation.     

Impaired Phosphorylation and Ubiquitination by p70 S6 Kinase (p70S6K) and Smad Ubiquitination Regulatory Factor 1 (Smurf1) Promote Tribbles Homolog 2 (TRIB2) Stability and Carcinogenic Property in Liver Cancer

Tribbles homolog 2 (TRIB2) is critical for both solid and non-solid malignancies. Recently, TRIB2 was identified as a liver cancer-specific Wnt/β-catenin signaling downstream target and is functionally important for liver cancer cell survival and transformation. TRIB2 functions as a protein that interacts with E3 ubiquitin ligases and thereby modulates protein stability of downstream effectors. However, the regulation underlying TRIB2 protein stability per se has not yet been reported. In this study, we found that TRIB2 was up-regulated and exhibited high stability in liver cancer cells compared with other cells. We performed a structure-function analysis of TRIB2 and identified a domain (amino acids 1–5) at the N terminus that interacted with the E3 ubiquitin ligase Smurf1 and was critical for protein stability. Deletion of this domain extended TRIB2 half-life time accompanied with a more significant malignant property compared with wild type TRIB2. Furthermore, Smurf1-mediated ubiquitination required phosphorylation of TRIB2 by p70 S6 kinase (p70S6K) via another domain (amino acids 69–85) that is also essential for correct TRIB2 subcellular localization. Mutation of Ser-83 diminished p70S6K-induced phosphorylation of TRIB2. Moreover, the high stability of TRIB2 may be due to the fact that both p70S6K and Smurf1 were down-regulated and negatively correlated with TRIB2 expression in both liver cancer tissues and established liver cancer cell lines. Taken together, impaired phosphorylation and ubiquitination by p70S6K and Smurf1 increase the protein stability of TRIB2 in liver cancer and thus may be helpful in the development of diagnosis and treatment strategies against this malignant disease.