Research ArticleTRB3 protects cells against the growth inhibitory and cytotoxic effect of ATF4
Introduction
TRB3 (also known as NIPK, SKIP3, TRIB3 and SINK) was first described as a protein encoded by a gene which is activated during the neuronal cell death induced by nerve growth factor deprivation and the exposure to calcium ionophore A23187 [1]. Further studies have revealed that TRB3 expression is upregulated in many cell types in response to various stresses, including nutrient deficiency, endoplasmic reticulum stress, hypoxia and oxidative stress [2], [3], [4], [5], [6], [7], [8], [9], and that TRB3 is overexpressed in multiple primary tumors and cancer cell lines [5], [10]. TRB3, as well as Drosophila protein Tribbles and its two other mammalian homologs, TRB1 and TRB2, belong to a group of proteins named pseudokinases or kinase-like proteins, because they contain a region with a considerable similarity to subdomains VI-XI of the common catalytic core structure of the protein kinase family [11], but due to strong deviations from the kinase active site consensus sequence, it is likely that Tribbles and its homologs have no kinase activity [12], [13], [14]. TRB3 interacts with a number of proteins and has been implicated in several processes. TRB3 associates with the E3 ubiquitin ligase COP1 and recruits it to acetyl-coenzyme A carboxylase (ACC), triggering the degradation of ACC and stimulating lipolysis [15]. It has been reported that TRB3 is involved in the regulation of insulin signal transduction [16], [17] and muscle differentiation [18] by binding to serine–threonine kinase Akt/PKB and blocking its activation. However, recent articles question the role of TRB3 in the insulin signaling pathway [19], [20]. Kiss-Toth et al. [21], [22] have reported that TRB3 controls mitogen-activated protein kinase (MAPK) cascades by binding to MAPK kinases, and Selim et al. [23] have suggested a role for TRB3 in the cell cycle arrest and the cellular depletion of lymphocytes. We and other researchers have shown that TRB3 interacts with ATF4 and inhibits its transcriptional activation activity, and that ATF4 participates in the transcriptional upregulation of TRB3 [3], [4], [5], [6]. Therefore, TRB3 may serve as a negative feedback regulator of ATF4 [4], [6]. Recently, Jousse et al. [24], studying the regulation of gene expression by amino acid limitation, have reached the same conclusion.
ATF4, a basic leucine-zipper transcription activator, carries out a variety of functions. It plays a major role in the control of gene expression during the integrated stress response (ISR) [25], [26] and is crucial for the long-term memory formation [27], the proliferation of fetal hematopoietic progenitors [28], the differentiation of osteoblasts [29] and the survival of lens cells at the embryonic stage [30], [31]. The ISR is activated by divergent cellular stresses (protein malfunction in endoplasmic reticulum, amino acid starvation, viral infection, heme deficiency) through the signaling pathways that converge on a single event—phosphorylation of the translation initiation factor eIF2α, resulting in general translational pause with selective increase in ATF4 mRNA translation and the subsequent stimulation of expression of ATF4 target genes [26]. The ISR facilitates the cells to adapt to the stresses, as revealed by the increased sensitivity of cells with inactivated ISR signaling to glucose starvation [32] and hypoxia [33], and ATF4-deficient cells to amino acid depletion and oxidative stress [25]. However, the target genes of ATF4 that are upregulated in the conditions of stress also include in addition to cytoprotective genes (such as heme oxygenase-1 [34]) genes known to be proapoptotic (such as CHOP [35]), suggesting that ATF4 may contribute to the initiation of cell death program.
In the present paper, we characterize the growth and viability properties of ATF4 and its negative regulator TRB3 in proliferating and postmitotic cells. The results indicate that the uncontrolled or excessive expression of ATF4 is growth inhibitory or cytotoxic, and that the coexpression of TRB3 suppresses the deleterious effect of ATF4.
Section snippets
Plasmids
To make plasmid ATF4-pcDNA4/TO, the tetracycline (tet)-inducible expression construct for human ATF4, the full-length coding region of ATF4 cDNA was amplified by PCR and inserted into vector pcDNA4/TO (Invitrogen, Carlsbad, CA) between BamHI and XhoI sites. To make plasmid TRB3-pcDNA4/TO, the tet-inducible expression construct for human TRB3, the full-length coding region of TRB3 cDNA was inserted into vector pcDNA4/TO between KpnI and BamHI sites. The tet-inducible expression construct for
Overexpression of ATF4 decreases the growth rate of HEK293 cells, and coexpression of TRB3 suppresses the deleterious effect of ATF4
We used T-REx conditional expression system to generate HEK293 cells which, upon addition of tet to the culture medium, start to express human ATF4 or human TRB3 (the cells were designated as ATF4-293 and TRB3-293, respectively). As shown in Fig. 1A, ATF4 and TRB3 proteins are undetectable in the cells grown in the absence of tet, but after the addition of tet, the proteins accumulate rapidly and are maintained at the approximately constant level for at least up to 48 h. To examine the effect
Discussion
In this report, we study the roles of ATF4 and TRB3 in the regulation of cell growth and viability in normal conditions and under the nutrient deprivation stress. The short half-life of ATF4 [52], [53] and the existence of the complex mechanism regulating ATF4 synthesis and transcriptional activity (including the expression control at the translational level [54], two negative feedback loops mediated by GADD34 [55], [56] and TRB3 [4], [6], [24], posttranslational modification [29], and the
Acknowledgments
We thank Drs. Andres Merits, Mart Ustav, Einari Aavik and Anne Kalling for advice and help, and Dr. Aksel Soosaar for critically reading the manuscript. This work was supported in part by Grant 6000 from the Estonian Science Foundation.
References (63)
- et al.
Identification of a novel kinase-like gene induced during neuronal cell death
Biochem. Biophys. Res. Commun.
(1999) - et al.
TRB3 is a PI 3-kinase dependent indicator for nutrient starvation
Cell. Signal.
(2006) - et al.
Mouse NIPK interacts with ATF4 and affects its transcriptional activity
Exp. Cell Res.
(2003) - et al.
Characterization of human NIPK (TRB3, SKIP3) gene activation in stressful conditions
Biochem. Biophys. Res. Commun.
(2005) - et al.
TRB3 interacts with CtIP and is overexpressed in certain cancers
Biochim. Biophys. Acta
(2007) - et al.
Emerging roles of pseudokinases
Trends Cell Biol.
(2006) - et al.
Tribbles: a family of kinase-like proteins with potent signalling regulatory function
Cell. Signal.
(2007) - et al.
TRB3 modulates C2C12 differentiation by interfering with Akt activation
Biochem. Biophys. Res. Commun.
(2007) - et al.
Human tribbles, a protein family controlling mitogen-activated protein kinase cascades
J. Biol. Chem.
(2004) - et al.
Regulation of expression and signalling modulator function of mammalian tribbles is cell-type specific
Immunol. Lett.
(2006)
Fibrates upregulate TRB3 in lymphocytes independent of PPARα by augmenting CCAAT/enhancer-binding proteinβ (C/EBPβ) expression
Mol. Immunol.
TRB3 inhibits the transcriptional activation of stress-regulated genes by a negative feedback on the ATF4 pathway
J. Biol. Chem.
An integrated stress response regulates amino acid metabolism and resistance to oxidative stress
Mol. Cell
All roads lead to ATF4
Dev. Cell
Targeted disruption of the activating transcription factor 4 gene results in severe fetal anemia in mice
Blood
ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology: implication for Coffin-Lowry syndrome
Cell
Microphthalmia due to p53-mediated apoptosis of anterior lens epithelial cells in mice lacking the CREB-2 transcription factor
Dev. Biol.
Translational control is required for the unfolded protein response and in vivo glucose homeostasis
Mol. Cell
Differential transcriptional activation by Oct-1 and Oct-2: interdependent activation domains induce Oct-2 phosphorylation
Cell
PC12 pheochromacytoma cells on neurobiological research
Adv. Cell. Neurobiol.
ATF4 is a mediator of the nutrient-sensing response pathway that activates the human asparagine synthatase gene
J. Biol. Chem.
Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells
J. Biol. Chem.
A comparative study of apoptosis and necrosis in HepG2 cells: oxidant-induced caspase inactivation leads to necrosis
Biochem. Biophys. Res. Commun.
Regulated translation initiation controls stress-induced gene expression in mammalian cells
Mol. Cell
Delineation of a negative feedback regulatory loop that controls protein translation during endoplasmic reticulum stress
J. Biol. Chem.
Mitosin/CENP-F as a negative regulator of activating transcription factor-4
J. Biol. Chem.
Activating transcription factor 4 overexpression inhibits proliferation and differentiation of mammary epithelium resulting in impaired lactation and accelerated involution
J. Biol. Chem.
Tribbles coordinates mitosis and morphogenesis in Drosophila by regulating String/CDC25 proteolysis
Cell
Tribbles, a cell-cycle brake that coordinates proliferation and morphogenesis during Drosophila gastrulation
Curr. Biol.
SINK is a p65-interacting negative regulator of NF-κB-dependent transcription
J. Biol. Chem.
SKIP3, a novel Drosophila tribbles ortholog, is overexpressed in human tumors and is regulated by hypoxia
Oncogene
Cited by (79)
The endoplasmic reticulum: Homeostasis and crosstalk in retinal health and disease
2024, Progress in Retinal and Eye ResearchThe functions and molecular mechanisms of Tribbles homolog 3 (TRIB3) implicated in the pathophysiology of cancer
2023, International ImmunopharmacologyTRIB3 limits FGF21 induction during in vitro and in vivo nutrient deficiencies by inhibiting C/EBP–ATF response elements in the Fgf21 promoter
2018, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms