Plasma exosomal DOK3 reflects immunological states in lung tumor and predicts prognosis of gefitinib treatment.

To identify liquid biomarkers that predict clinical outcomes of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), we enrolled patients with EGFR gene mutation-positive non-small-cell lung cancer who were intended to receive gefitinib treatment. Using plasma samples obtained prior to gefitinib treatment from 12 enrolled patients, we performed comprehensive proteomic analysis of plasma exosomes to explore proteins correlating with tumor reduction rate (TRR), progression-free survival (PFS), or overall survival (OS). Of the detected 1769 proteins, 119, 130, or 119 proteins demonstrated a strong correlation (|r| > 0.5) with TRR, PFS, or OS, respectively. Interestingly, 34 (29%), 41 (32%), or 27 (23%) of them, respectively, were functionally involved in the regulation of the immune response. CD8α chain was consistently listed as a molecule positively correlated with PFS and OS, suggesting that the long-lasting effects of gefitinib may be due to the antitumor effects of CD8+ T cells, as well as the induction of immunogenic apoptosis of tumor cells by blocking the EGFR signaling pathway. Notably, Doking Protein 3 (DOK3), a molecule involved in B-cell receptor signaling, and some immunoglobulin and complement molecules exhibited a clear correlation with PFS longevity of gefitinib treatment. Indeed, the strong expression of DOK3 in B cells was confirmed within tertiary lymphoid structures of lung cancer tissues derived from patients with long PFS. These findings suggest that the patients with active B-cell and T-cell immunity as a host immunological feature are more likely to benefit from gefitinib therapy. Circulating exosomal DOK3 has the potential as a predictive marker of response to gefitinib indicating this immunological feature.

Dok3 is involved in cisplatin-induced acute kidney injury via regulation of inflammation and apoptosis.

Cisplatin-induced acute kidney injury (AKI) is associated with high morbidity and mortality worldwide, but the underlying mechanisms are not fully understood. Downstream-of-kinase 3 (Dok3), a member of the Dok family of adaptor proteins plays a critical role in inflammatory response and immune regulation; however, the role of Dok3 in cisplatin-induced AKI remains unclear. This study explored the effect and potential molecular mechanisms of Dok3 in cisplatin-induced AKI using Dok3 knockout (Dok3-/-) and control mice (129S) with or without administration of a single intraperitoneal injection of cisplatin. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, lactate dehydrogenase (LDH) release, and Hoechst staining. Inflammatory factors were measured using ELISA kits. Protein and gene expression levels were measured by western blot analysis and real-time PCR, respectively. The results showed that Dok3 was expressed in renal tubular epithelial cells. Dok3 expression was decreased in kidneys of mice treated with cisplatin and cisplatin-treated HK2 cells. Dok3-/- mice showed lower creatinine levels and NGAL expression, and increased survival rates compared to 129S mice. Cisplatin-induced production of TNF-α and IL-6, and renal tubular cell apoptosis was attenuated in Dok3-/- mice. In vitro experiments demonstrated that HK2 cells overexpressing Dok3 exhibited exacerbated cisplatin-induced apoptosis and production of TNF-α and IL-6. These findings demonstrate that Dok3 regulates cisplatin-induced AKI by regulating apoptosis and inflammation.

The Dok-3/Grb2 adaptor module promotes inducible association of the lipid phosphatase SHIP with the BCR in a coreceptor-independent manner.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) plays a key role in preventing autoimmune phenomena by limiting antigen-mediated B cell activation. SHIP function is thought to require the dual engagement of the BCR and negative regulatory coreceptors as only the latter appear capable of recruiting SHIP from the cytosol to the plasma membrane by the virtue of phosphorylated immunoreceptor tyrosine-based inhibitory motifs. Here, we demonstrate a coreceptor-independent membrane recruitment and function of SHIP in B cells. In the absence of coreceptor ligation, SHIP translocates to sites of BCR activation through a concerted action of the protein adaptor unit Dok-3/Grb2 and phosphorylated BCR signaling components. Our data reveal auto-inhibitory SHIP activation by the activated BCR and suggest an unexpected negative-regulatory capacity of immunoreceptor tyrosine-based activation motifs in Igα and Igß.

Expression of DOK1, 2, and 3 genes in HTLV-1-infected T cells.

Human T-cell leukemia virus type 1 (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATLL). The Tax protein encoded by the pX region of the HTLV-1 genome appears to be a key element in the early stage of ATLL development. In this study, we examined the expression of the downstream of tyrosine kinase (DOK) family members DOK1, DOK2 and DOK3, recently reported to be tumor suppressors, in HTLV-1-transformed T cells (MT-2 and HUT-102) and TL-Om1 cells derived from ATLL leukemic cells. DOK2 and DOK3 expression was significantly reduced in MT-2, HUT-102, and TL-Om1 cells compared with their expression in uninfected T cells, and the expression of DOK3 was reduced by the induction of Tax expression in T cells.

Disrupting the Dok3-Card9 Interaction with Synthetic Peptides Enhances Antifungal Effector Functions of Human Neutrophils.

Invasive fungal disease is an emerging and serious public health threat globally. The expanding population of susceptible individuals, together with the rapid emergence of multidrug-resistant fungi pathogens, call for the development of novel therapeutic strategies beyond the limited repertoire of licensed antifungal drugs. Card9 is a critical signaling molecule involved in antifungal defense; we have previously identified Dok3 to be a key negative regulator of Card9 activity in neutrophils. In this study, we identified two synthetic peptides derived from the coiled-coil domain of Card9, which can specifically block Dok3-Card9 binding. We showed that these peptides are cell-permeable, non-toxic, and can enhance antifungal cytokine production and the phagocytosis of human neutrophils upon fungal infection. Collectively, these data provide a proof of concept that disrupting the Dok3-Card9 interaction can boost the antifungal effector functions of neutrophils; they further suggest the potential utility of these peptide inhibitors as an immune-based therapeutic to fight fungal infection.

Dok3 restrains neutrophil production of calprotectin during TLR4 sensing of SARS-CoV-2 spike protein.

Increased neutrophils and elevated level of circulating calprotectin are hallmarks of severe COVID-19 and they contribute to the dysregulated immune responses and cytokine storm in susceptible patients. However, the precise mechanism controlling calprotectin production during SARS-CoV-2 infection remains elusive. In this study, we showed that Dok3 adaptor restrains calprotectin production by neutrophils in response to SARS-CoV-2 spike (S) protein engagement of TLR4. Dok3 recruits SHP-2 to mediate the de-phosphorylation of MyD88 at Y257, thereby attenuating downstream JAK2-STAT3 signaling and calprotectin production. Blocking of TLR4, JAK2 and STAT3 signaling could prevent excessive production of calprotectin by Dok3-/- neutrophils, revealing new targets for potential COVID-19 therapy. As S protein from SARS-CoV-2 Delta and Omicron variants can activate TLR4-driven calprotectin production in Dok3-/- neutrophils, our study suggests that targeting calprotectin production may be an effective strategy to combat severe COVID-19 manifestations associated with these emerging variants.

DOK3 is involved in microglial cell activation in neuropathic pain by interacting with GPR84.

Adaptor molecule downstream of kinase-3 (DOK3) is a vital regulator of innate immune responses in macrophages and B cells, and G-protein-coupled receptor 84 (GPR84) is significant in mediating the biosynthesis and maintenance of inflammatory mediators that are induced by neuropathic pain in microglia. In the present study, we determined the role of DOK3 in activating microglia-induced neuropathic pain and investigated the underlying mechanisms associated with GPR84. We found that knockdown of DOK3 in microglial cells dramatically reduced the levels of inflammatory factors, and we uncovered a physical association between DOK3 and GPR84 in the induction of inflammatory responses. We also observed that neuropathic pain and inflammatory responses induced by chronic constriction injury (CCI) of the sciatic nerve or intrathecal injection of a GPR84 agonist were compromised in DOK3-/- mice in vivo. Finally, enforced expression of DOK3 provoked inflammatory responses, and administration of pregabalin relieved neuropathic pain via inhibition of DOK3 expression. In conclusion, DOK3 induced neuropathic pain in mice by interacting with GPR84 in microglia. We hypothesize that targeting the adaptor protein DOK3 may open new avenues for pharmaceutical approaches to the alleviation of neuropathic pain in the spinal cord.

Emerging Roles of Downstream of Kinase 3 in Cell Signaling.

Downstream of kinase (Dok) 3 is a member of the Dok family of adaptor proteins known to regulate signaling pathways downstream of various immunoreceptors. As Dok-3 lacks intrinsic catalytic activity, it functions primarily as a molecular scaffold to facilitate the nucleation of protein complexes in a regulated manner and hence, achieve specificity in directing signaling cascades. Since its discovery, considerable progress has been made toward defining the role of Dok-3 in limiting B cell-receptor signaling. Nonetheless, Dok-3 has since been implicated in the signaling of Toll-like and C-type lectin receptors. Emerging data further demonstrate that Dok-3 can act both as an activator and inhibitor, in lymphoid and non-lymphoid cell types, suggesting Dok-3 involvement in a plethora of signal transduction pathways. In this review, we will focus on the structure and expression profile of Dok-3 and highlight its role during signal transduction in B cells, innate cells as well as in bone and lung tissues.

Dok-3 deficient mice display different immune clustering and Tim-3 expression.

BACKGROUND: Dok-3 has been shown to play an important role in immune system. Tim-3 also has been recognized as an important immune regulator which involves in many diseases. The relationship of them is still unclear. METHODS: We detected the expression of Tim-3 on spleen immune cells from Dok-3 deficient mice and control mice by flow cytometry. RESULTS: In this article, we found that Dok-3-/- mice display almost entirely different immune clustering characteristics compared with wild type 129 mice. The CD4 T cells and CD8 T cells decreased and DC cells, macrophages, MDSCs increased when the Dok-3 gene knocked-out. The Tim-3 expression on CD4 T cells, CD8 T cells, NK cells, DC cells increased when the Dok-3 gene knocked-out. The macrophages and MDSCs just display the opposite results. CONCLUSIONS: Although Dok-3-/- mice display different immune clustering and Tim-3 expression, the mechanism still needs further study.

DOK3 Modulates Bone Remodeling by Negatively Regulating Osteoclastogenesis and Positively Regulating Osteoblastogenesis.

Osteoclastogenesis is essential for bone remodeling and normal skeletal maintenance. Receptor activator of NF-κB ligand (RANKL) promotes osteoclast differentiation and function but requires costimulation of immunoreceptor tyrosine-based activation motif (ITAM)-coupled immunoreceptors. Triggering receptor expressed on myeloid cells-2 (TREM2) coupled to ITAM-adaptor protein DNAX activation protein 12kDA (DAP12) provides costimulation of intracellular calcium signaling during osteoclastogenesis. Previously, we found that downstream of kinase-3 (DOK3) physically associates with DAP12 to inhibit toll-like receptor (TLR)-induced inflammatory signaling in macrophages. However, whether and how DOK3 modulates DAP12-dependent osteoclastogenesis is unknown and the focus of this study. Bone microarchitecture and histology of sex- and age-matched wild-type (WT) and DOK3-deficient (DOK3-/- ) mice were evaluated. Male and female DOK3-/- mice have significantly reduced trabecular bone mass compared with WT mice with increased TRAP+ osteoclasts in vivo. In vitro, DOK3-/- bone marrow-derived macrophages (BMMs) have increased macrophage colony-stimulating factor (M-CSF)-induced proliferation and increased sensitivity to RANKL-induced osteoclastogenesis. Compared with WT, DOK3-/- osteoclasts are significantly larger with more nuclei and have increased resorptive capacity. Mechanistically, DOK3 limits osteoclastogenesis by inhibiting activation of Syk and ERK in response to RANKL and M-CSF. DOK3 is phosphorylated in a DAP12-dependent manner and associates with Grb2 and Cbl. Compared with DAP12-/- mice with high bone mass, DOK3- and DAP12- doubly deficient mice (DKO) have normalized bone mass, indicating that DOK3 also limits DAP12-independent osteoclastogenesis in vivo. In vitro osteoclasts derived from DKO mice are mononuclear with poor resorptive capacity similar to DAP12-/- osteoclasts. Histomorphometry reveals that DOK3-/- mice also have reduced osteoblast parameters. DOK3-/- osteoblasts have reduced in vitro osteoblastogenesis and increased osteoprotegerin (OPG) to RANKL expression ratio compared with WT osteoblasts. Co-culture of WT and DOK3-/- osteoblasts with pre-osteoclasts reveals a reduced capacity of DOK3-/- osteoblasts to support osteoclastogenesis. These data indicate that DOK3 regulates bone remodeling by negatively regulating M-CSF- and RANKL-mediated osteoclastogenesis and positively regulating osteoblastogenesis. © 2017 American Society for Bone and Mineral Research.

DOK3 Degradation is Required for the Development of LPS-induced ARDS in Mice.

It has been reported that DOK3 protein negatively regulates LPS responses and endotoxin tolerance in mice. However, the role of DOK3 in the development of acute respiratory distress syndrome (ARDS) remains unknown. In this study, we showed that DOK3 is degraded in the lung tissues of LPS-induced ARDS. Through lentivirus transduction containing DOK3(K27R) via the intranasal route, we created a mice model, in which DOK3 maintains stable expression. We found that the forced DOK3 expression significantly attenuated LPS-induced pulmonary histological alterations, inflammatory cells infiltration, lung edema, as well as the generation of inflammatory cytokines TNFα, IL- 1ß and IL-6 in BALF of LPS-induced ARDS mice. In addition, DOK3 expression apparently suppressed LPS-induced NF-κB and ERK activation. These data suggested that DOK3 expression negatively regulates the development of LPS-induced ARDS in mice.

Impact of Genetic Variation in MicroRNA-binding Site on Susceptibility to Colorectal Cancer.

The present study analyzed single nucleotide polymorphisms (SNPs) located at putative microRNA(miRNA)-binding sites of the 3'-untranslated region (UTR) in different genes and investigated their impact on the susceptibility to colorectal cancer (CRC). Ninety-two SNPs were selected using an in silico analysis of 3'-UTR SNPs in an SNP database and their miRNA binding efficiency was calculated using several miRNA databases and the HapMap database. Two independent study sets were used: 380 healthy controls and 371 patients with colorectal adenocarcinoma for the discovery set, and 521 healthy controls and 524 patients with colorectal adenocarcinoma for the validation set. The SNP genotyping was performed using a Sequenom MassARRAY. In addition, a luciferase assay was used to investigate whether miR-370 modulated docking protein 3 (DOK3) gene expression when rs2279398G>A was included in the DOK3 3'-UTR region. For the discovery set, 16 out of 92 SNPs were significantly associated with the risk of CRC in at least one of the genetic models. The validation set showed that among these 16 SNPs, DOK3 rs2279398G>A was significantly associated with reduced risk of CRC in a recessive model [adjusted odds ratio (aOR)=0.65, 95% confidence interval (CI)=0.44-0.97, p=0.03]. In a combined analysis, DOK3 rs2279398G>A was associated with a significantly reduced risk of CRC in a co-dominant and recessive model (aOR=0.84, 95% CI=0.73-0.96, p=0.012; aOR=0.65, CI=0.49-0.88, p=0.004, respectively). Significantly lower Renilla activity was also observed with the rs2279398 AA construct when compared to the rs2279398 GG construct (p<0.001). DOK3 rs2279398G>A may affect the expression of DOK3 by altering the miRNA binding efficiency at the miRNA-binding sites of the 3'-UTR in DOK3, thereby impacting CRC tumorigenesis.

TRAF6-mediated degradation of DOK3 is required for production of IL-6 and TNFα in TLR9 signaling.

Our previous study showed that the downstream of kinase 3 (DOK3) is degraded during macrophage stimulation with CpG. However, the underlying mechanism and role in Toll-like receptor 9 (TLR9) signaling remains elusive. In this study, we demonstrate that CpG treatment leads to ubiquitin-mediated degradation of DOK3 via interaction with an E3 ligase TNFR-associated factor 6 (TRAF6). We also identified the 27th amino acid (lysine) of DOK3 is responsible for Ly48 polyubiquitination of DOK3. Furthermore, reintroduction of DOK3 (K27R) into DOK3-deficient macrophages abolishes DOK3 degradation induced by CpG and suppresses the production of IL-6 and TNFα. More importantly, our study uncovers a novel role of an E3 ligase TRAF6, namely, TRAF6 is also able to catalyse Lys 48 polyubiquitylation of target protein except for Lys 63 polyubiquitylation.