Mechanistic target of rapamycin complex 1 (mTORC1) is a conserved serine/threonine kinase that integrates various environmental signals to regulate cell growth and metabolism. mTORC1 activation requires tethering to lysosomes by the Ragulator-Rag complex. However, the dynamic regulation of the interaction between Ragulator and Rag guanosine triphosphatase (GTPase) remains unclear. In this study, that LAMTOR1, an essential component of Ragulator, is dynamically ubiquitinated depending on amino acid abundance is reported. It is found that the E3 ligase TRAF4 directly interacts with LAMTOR1 and catalyzes the K63-linked polyubiquitination of LAMTOR1 at K151. Ubiquitination of LAMTOR1 by TRAF4 promoted its binding to Rag GTPases and enhanced mTORC1 activation, K151R knock-in or TRAF4 knock-out blocks amino acid-induced mTORC1 activation and accelerates the development of inflammation-induced colon cancer. This study revealed that TRAF4-mediated LAMTOR1 ubiquitination is a regulatory mechanism for mTORC1 activation and provides a therapeutic target for diseases involving mTORC1 dysregulation.

Nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. Despite continuous improvement in treatment strategies, recurrence or persistence of cancer after radiotherapy is still inevitable, highlighting the need to identify therapeutic resistance factors and develop effective methods for NPC treatment. Herein, we found that TRAF4 is overexpressed in NPC cells and tissues. Knockdown TRAF4 significantly increased the radiosensitivity of NPC cells, possibly by inhibiting the Akt/Wee1/CDK1 axis, thereby suppressing survivin phosphorylation and promoting its degradation by FBXL7. TRAF4 is positively correlated with p-Akt and survivin in NPC tissues. High protein levels of TRAF4 were observed in acquired radioresistant NPC cells, and knockdown of TRAF4 overcomes radioresistant in vitro and the xenograft mouse model. Altogether, our study highlights the TRAF4-survivin axis as a potential therapeutic target for radiosensitization in NPC.

Background Pathological cardiac hypertrophy is a major cause of heart failure morbidity. The complex mechanism of intermolecular interactions underlying the pathogenesis of cardiac hypertrophy has led to a lack of development and application of therapeutic methods. Methods and Results Our study provides the first evidence that TRAF4, a member of the tumor necrosis factor receptor-associated factor (TRAF) family, acts as a promoter of cardiac hypertrophy. Here, Western blotting assays demonstrated that TRAF4 is upregulated in cardiac hypertrophy. Additionally, TRAF4 deletion inhibits the development of cardiac hypertrophy in a mouse model after transverse aortic constriction surgery, whereas its overexpression promotes phenylephrine stimulation-induced cardiomyocyte hypertrophy in primary neonatal rat cardiomyocytes. Mechanistically, RNA-seq analysis revealed that TRAF4 promoted the activation of the protein kinase B pathway during cardiac hypertrophy. Moreover, we found that inhibition of protein kinase B phosphorylation rescued the aggravated cardiomyocyte hypertrophic phenotypes caused by TRAF4 overexpression in phenylephrine-treated neonatal rat cardiomyocytes, suggesting that TRAF4 may regulate cardiac hypertrophy in a protein kinase B-dependent manner. Conclusions Our results revealed the regulatory function of TRAF4 in cardiac hypertrophy, which may provide new insights into developing therapeutic and preventive targets for this disease.

Tumor necrosis factor receptor-associated factor 4 (TRAF4) is an important regulator of type 2 responses in the airway; however, the underlying cellular and molecular mechanisms remain elusive. Herein, we generated T cell-specific TRAF4-deficient (CD4-cre Traf4fl/fl) mice and investigated the role of TRAF4 in memory Th2 cells expressing IL-33 receptor (ST2, suppression of tumorigenicity 2) (ST2+ mTh2 cells) in IL-33-mediated type 2 airway inflammation. We found that in vitro-polarized TRAF4-deficient (CD4-cre Traf4fl/fl) ST2+ mTh2 cells exhibited decreased IL-33-induced proliferation as compared with TRAF4-sufficient (Traf4fl/fl) cells. Moreover, CD4-cre Traf4fl/fl mice showed less ST2+ mTh2 cell proliferation and eosinophilic infiltration in the lungs than Traf4fl/fl mice in the preclinical models of IL-33-mediated type 2 airway inflammation. Mechanistically, we discovered that TRAF4 was required for the activation of AKT/mTOR and ERK1/2 signaling pathways as well as the expression of transcription factor Myc and nutrient transporters (Slc2a1, Slc7a1, and Slc7a5), signature genes involved in T cell growth and proliferation, in ST2+ mTh2 cells stimulated by IL-33. Taken together, the current study reveals a role of TRAF4 in ST2+ mTh2 cells in IL-33-mediated type 2 pulmonary inflammation, opening up avenues for the development of new therapeutic strategies.

Excessive inflammation is a postulated cause of severe disease and death in respiratory virus infections. In response to severe influenza virus infection, adoptively transferred naïve hemagglutinin-specific CD4+ T cells from CD4+ TCR-transgenic 6.5 mice drive an IFN-γ-producing Th1 response in wild-type mice. It helps in virus clearance but also causes collateral damage and disease aggravation. The donor 6.5 mice have all the CD4+ T cells with TCR specificity toward influenza hemagglutinin. Still, the infected 6.5 mice do not suffer from robust inflammation and grave outcome. The initial Th1 response wanes with time, and a prominent Th17 response of recent thymic emigrants alleviates inflammation and bestows protection in 6.5 mice. Our results suggest that viral neuraminidase-activated TGF-β of the Th1 cells guides the Th17 evolution, and IL-17 signaling through the non-canonical IL-17 receptor EGFR activates the scaffold protein TRAF4 more than TRAF6 during alleviation of lung inflammation in severe influenza.

Castration-resistant prostate cancer (CRPC) poses a major clinical challenge with the androgen receptor (AR) remaining to be a critical oncogenic player. Several lines of evidence indicate that AR induces a distinct transcriptional program after androgen deprivation in CRPCs. However, the mechanism triggering AR binding to a distinct set of genomic loci in CRPC and how it promotes CRPC development remain unclear. We demonstrate here that atypical ubiquitination of AR mediated by an E3 ubiquitin ligase TRAF4 plays an important role in this process. TRAF4 is highly expressed in CRPCs and promotes CRPC development. It mediates K27-linked ubiquitination at the C-terminal tail of AR and increases its association with the pioneer factor FOXA1. Consequently, AR binds to a distinct set of genomic loci enriched with FOXA1- and HOXB13-binding motifs to drive different transcriptional programs including an olfactory transduction pathway. Through the surprising upregulation of olfactory receptor gene transcription, TRAF4 increases intracellular cAMP levels and boosts E2F transcription factor activity to promote cell proliferation under androgen deprivation conditions. Altogether, these findings reveal a posttranslational mechanism driving AR-regulated transcriptional reprogramming to provide survival advantages for prostate cancer cells under castration conditions.

The E3 ligase TNF receptor-associated factor 4 (TRAF4) is frequently overexpressed and closely related to poor prognosis in human malignancies. However, its effect on carcinogenesis and radiosensitivity in oral squamous cell carcinoma (OSCC) remains unclear. The present study found that TRAF4 was significantly upregulated in primary and relapsed OSCC tumor tissues. Depletion of TRAF4 markedly improved the sensitivity of OSCC cells to irradiation (IR) treatment, showing that tumor cell proliferation, colony formation and xenograft tumor growth were reduced. Mechanistically, IR promoted the interaction between TRAF4 and Akt to induce Akt K63-mediated ubiquitination and activation. TRAF4 knockout inhibited the phosphorylation of Akt and upregulated GSK3β activity, resulting in increased myeloid cell leukemia-1 (MCL-1) S159 phosphorylation, which disrupted the interaction of MCL-1 with Josephin domain containing 1 (JOSD1), and ultimately induced MCL-1 ubiquitination and degradation. Moreover, TRAF4 was positively correlated with MCL-1 in primary and in radiotherapy-treated, relapsed tumor tissues. An MCL-1 inhibitor overcame radioresistance in vitro and in vivo. Altogether, the present findings suggest that TRAF4 confers radioresistance in OSCC by stabilizing MCL-1 through Akt signaling, and that targeting TRAF4 may be a promising therapeutic strategy to overcome radioresistance in OSCC.

Tumor necrosis factor receptor-associated factors (TRAFs) are important adaptor molecules that play important roles in host immune regulation and inflammatory responses. Compared to other members of TRAFs, the function of TRAF4 in vertebrate immunity remains unclear, especially in teleosts. In the present study, TRAF4 ortholog was cloned and identified in large yellow croaker (Larimichthys crocea), named as Lc-TRAF4. The open reading frame (ORF) of Lc-TRAF4 is 1,413 bp and encodes a protein of 470 amino acids (aa), which is consisted of a RING finger domain, two zinc finger domains, and a MATH domain. The genome organization of Lc-TRAF4 is conserved in teleosts, amphibians, birds, and mammals, with 7 exons and 6 introns. Quantitative real-time PCR analysis revealed that Lc-TRAF4 was broadly distributed in various organs/tissues of healthy large yellow croakers and could be significantly up-regulated in the gill, intestine, spleen, head kidney, and blood under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations. Notably, luciferase assays showed that overexpression of Lc-TRAF4 could significantly induce the activation of IRF3, IRF7, and type I IFN promoters, with the RING finger and zinc finger domains function importantly in such promoter activation. Confocal microscopy revealed that Lc-TRAF4 is located in the cytoplasm, whereas the deletion of the RING finger, zinc finger or MATH domain showed little effect on the subcellular localization of Lc-TRAF4. Interestingly, Lc-TRAF4 overexpression could significantly enhance Lc-TRIF and Lc-TRAF6 medicated IRF3 and IRF7 promoter activation. In addition, co-expression of Lc-TRAF4 with Lc-TRIF or Lc-TRAF6 could significantly induce the expression of antiviral and inflammation-related genes, including IRF3, IRF7, ISG15, ISG56, Mx, RSAD2, TNF-α, and IL-1β compared to the only overexpression of Lc-TRAF4, Lc-TRIF or Lc-TRAF6. These results collectively imply that Lc-TRAF4 functions as an enhancer in Lc-TRIF and Lc-TRAF6 mediated antiviral and inflammatory signaling.

Tumor necrosis factor receptor-associated factors (TRAFs) play important roles in the biological processes of immune regulation, the inflammatory response, and apoptosis. TRAF4 belongs to the TRAF family and plays a major role in many biological processes. Compared with other TRAF proteins, the functions of TRAF4 in teleosts have been largely unknown. In the present study, the TRAF4 homologue (EcTRAF4) of the orange-spotted grouper was characterized. EcTRAF4 consisted of 1413 bp encoding a 471-amino-acid protein, and the predicted molecular mass was 54.27 kDa. EcTRAF4 shares 99.79% of its identity with TRAF4 of the giant grouper (E. lanceolatus). EcTRAF4 transcripts were ubiquitously and differentially expressed in all the examined tissues. EcTRAF4 expression in GS cells was significantly upregulated after stimulation with red-spotted grouper nervous necrosis virus (RGNNV). EcTRAF4 protein was distributed in the cytoplasm of GS cells. Overexpressed EcTRAF4 promoted RGNNV replication during viral infection in vitro. Yeast two-hybrid and coimmunoprecipitation assays showed that EcTRAF4 interacted with the coat protein (CP) of RGNNV. EcTRAF4 inhibited the activation of IFN3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). Overexpressed EcTRAF4 also reduced the expression of interferon (IFN)-related molecules and pro-inflammatory factors. Together, these results demonstrate that EcTRAF4 plays crucial roles in RGNNV infection.

Insulin-like growth factor (IGF) is a potent mitogen that activates the IGF receptor (IGFR)/insulin receptor substrate (IRS) axis, thus stimulating growth in normal cells and uncontrolled cell proliferation in cancer. Posttranslational modifications of IRS such as ubiquitination tightly control IGF signaling, and we previously identified IRS-1 as a potential substrate for the E3 ubiquitin ligase TRAF4 using an unbiased screen. Here we provide evidence that TRAF4-mediated ubiquitination of IRS-1 is physiologically relevant and crucial for IGF signal transduction. Through site-directed mutagenesis we found that TRAF4 promotes an atypical K29-linked ubiquitination at the C-terminal end of IRS-1. Its depletion abolishes AKT and ERK phosphorylation downstream of IGF-1 and inhibits breast cancer cell proliferation. Overexpression of TRAF4 enhances IGF1-induced IGFR-IRS-1 interaction, IRS-1 tyrosine phosphorylation, and downstream effector protein activation, whereas mutation of IRS-1 ubiquitination sites completely abolishes these effects. Altogether, our studies demonstrate that nonproteolytic ubiquitination of IRS-1 is a key step in conveying IGF-1 stimulation from IGFR to IRS-1.