UHRF1P contributes to IL-17A-mediated systemic lupus erythematosus via UHRF1-MAP4K3 axis.

Inflammatory T cells contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Analysis of the T-cell transcriptomics data of two independent SLE patient cohorts by three machine learning models revealed the pseudogene UHRF1P as a novel SLE biomarker. The pseudogene-encoded UHRF1P protein was overexpressed in peripheral blood T cells of SLE patients. The UHRF1P protein lacks the amino-terminus of its parental UHRF1 protein, resulting in missing the proteasome-binding ubiquitin-like (Ubl) domain of UHRF1. T-cell-specific UHRF1P transgenic mice manifested the induction of IL-17A and autoimmune inflammation. Mechanistically, UHFR1P prevented UHRF1-induced Lys48-linked ubiquitination and degradation of MAP4K3 (GLK), which is a kinase known to induce IL-17A. Consistently, IL-17A induction and autoimmune phenotypes of UHRF1P transgenic mice were obliterated by MAP4K3 knockout. Collectively, UHRF1P overexpression in T cells inhibits the E3 ligase function of its parental UHRF1 and induces autoimmune diseases.

Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck.

BACKGROUND: T cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation. METHODS: Whole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells. RESULTS: TRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation. CONCLUSIONS: TRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.

The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling.

DUSP22 is a dual-specificity phosphatase that inhibits T cell activation by inactivating the kinase Lck. Here we show that the E3 ubiquitin ligase UBR2 is a positive upstream regulator of Lck during T-cell activation. DUSP22 dephosphorylates UBR2 at specific Serine residues, leading to ubiquitin-mediated UBR2 degradation. UBR2 is also modified by the SCF E3 ubiquitin ligase complex via Lys48-linked ubiquitination at multiple Lysine residues. Single-cell RNA sequencing analysis and UBR2 loss of function experiments showed that UBR2 is a positive regulator of proinflammatory cytokine expression. Mechanistically, UBR2 induces Lys63-linked ubiquitination of Lck at Lys99 and Lys276 residues, followed by Lck Tyr394 phosphorylation and activation as part of TCR signalling. Inflammatory phenotypes induced by TCR-triggered Lck activation or knocking out DUSP22, are attenuated by genomic deletion of UBR2. UBR2-Lck interaction and Lck Lys63-linked ubiquitination are induced in the peripheral blood T cells of human SLE patients, which demonstrate the relevance of the UBR2-mediated regulation of inflammation to human pathology. In summary, we show here an important regulatory mechanism of T cell activation, which finetunes the balance between T cell response and aggravated inflammation.

Dual-Specificity Phosphatase 6 Deficiency Attenuates Arterial-Injury-Induced Intimal Hyperplasia in Mice.

In response to injury, vascular smooth muscle cells (VSMCs) of the arterial wall dedifferentiate into a proliferative and migratory phenotype, leading to intimal hyperplasia. The ERK1/2 pathway participates in cellular proliferation and migration, while dual-specificity phosphatase 6 (DUSP6, also named MKP3) can dephosphorylate activated ERK1/2. We showed that DUSP6 was expressed in low baseline levels in normal arteries; however, arterial injury significantly increased DUSP6 levels in the vessel wall. Compared with wild-type mice, Dusp6-deficient mice had smaller neointima. In vitro, IL-1ß induced DUSP6 expression and increased VSMC proliferation and migration. Lack of DUSP6 reduced IL-1ß-induced VSMC proliferation and migration. DUSP6 deficiency did not affect IL-1ß-stimulated ERK1/2 activation. Instead, ERK1/2 inhibitor U0126 prevented DUSP6 induction by IL-1ß, indicating that ERK1/2 functions upstream of DUSP6 to regulate DUSP6 expression in VSMCs rather than downstream as a DUSP6 substrate. IL-1ß decreased the levels of cell cycle inhibitor p27 and cell-cell adhesion molecule N-cadherin in VSMCs, whereas lack of DUSP6 maintained their high levels, revealing novel functions of DUSP6 in regulating these two molecules. Taken together, our results indicate that lack of DUSP6 attenuated neointima formation following arterial injury by reducing VSMC proliferation and migration, which were likely mediated via maintaining p27 and N-cadherin levels.

DUSP8 induces TGF-ß-stimulated IL-9 transcription and Th9-mediated allergic inflammation by promoting nuclear export of Pur-α.

Dual-specificity phosphatase 8 (DUSP8) is a MAPK phosphatase that dephosphorylates and inactivates the kinase JNK. DUSP8 is highly expressed in T cells; however, the in vivo role of DUSP8 in T cells remains unclear. Using T cell-specific Dusp8 conditional KO (T-Dusp8 cKO) mice, mass spectrometry analysis, ChIP-Seq, and immune analysis, we found that DUSP8 interacted with Pur-α, stimulated interleukin-9 (IL-9) gene expression, and promoted Th9 differentiation. Mechanistically, DUSP8 dephosphorylated the transcriptional repressor Pur-α upon TGF-ß signaling, leading to the nuclear export of Pur-α and subsequent IL-9 transcriptional activation. Furthermore, Il-9 mRNA levels were induced in Pur-α-deficient T cells. In addition, T-Dusp8-cKO mice displayed reduction of IL-9 and Th9-mediated immune responses in the allergic asthma model. Reduction of Il-9 mRNA levels in T cells and allergic responses of T-Dusp8-cKO mice was reversed by Pur-α knockout. Remarkably, DUSP8 protein levels and the DUSP8-Pur-α interaction were indeed increased in the cytoplasm of T cells from people with asthma and patients with atopic dermatitis. Collectively, DUSP8 induces TGF-ß-stimulated IL-9 transcription and Th9-induced allergic responses by inhibiting the nuclear translocation of the transcriptional repressor Pur-α. DUSP8 may be a T-cell biomarker and therapeutic target for asthma and atopic dermatitis.

ACE2 in chronic disease and COVID-19: gene regulation and post-translational modification.

Angiotensin-converting enzyme 2 (ACE2), a counter regulator of the renin-angiotensin system, provides protection against several chronic diseases. Besides chronic diseases, ACE2 is the host receptor for SARS-CoV or SARS-CoV-2 virus, mediating the first step of virus infection. ACE2 levels are regulated by transcriptional, post-transcriptional, and post-translational regulation or modification. ACE2 transcription is enhanced by transcription factors including Ikaros, HNFs, GATA6, STAT3 or SIRT1, whereas ACE2 transcription is reduced by the transcription factor Brg1-FoxM1 complex or ERRα. ACE2 levels are also regulated by histone modification or miRNA-induced destabilization. The protein kinase AMPK, CK1α, or MAP4K3 phosphorylates ACE2 protein and induces ACE2 protein levels by decreasing its ubiquitination. The ubiquitination of ACE2 is induced by the E3 ubiquitin ligase MDM2 or UBR4 and decreased by the deubiquitinase UCHL1 or USP50. ACE2 protein levels are also increased by the E3 ligase PIAS4-mediated SUMOylation or the methyltransferase PRMT5-mediated ACE2 methylation, whereas ACE2 protein levels are decreased by AP2-mediated lysosomal degradation. ACE2 is downregulated in several human chronic diseases like diabetes, hypertension, or lung injury. In contrast, SARS-CoV-2 upregulates ACE2 levels, enhancing host cell susceptibility to virus infection. Moreover, soluble ACE2 protein and exosomal ACE2 protein facilitate SARS-CoV-2 infection into host cells. In this review, we summarize the gene regulation and post-translational modification of ACE2 in chronic disease and COVID-19. Understanding the regulation and modification of ACE2 may help to develop prevention or treatment strategies for ACE2-mediated diseases.

DUSP6 Deficiency Attenuates Neurodegeneration after Global Cerebral Ischemia.

Transient global cerebral ischemia (tGCI) resulting from cardiac arrest causes selective neurodegeneration in hippocampal CA1 neurons. Although the effect is clear, the underlying mechanisms directing this process remain unclear. Previous studies have shown that phosphorylation of Erk1/2 promotes cell survival in response to tGCI. DUSP6 (also named MKP3) serves as a cytosolic phosphatase that dephosphorylates Erk1/2, but the role of DUSP6 in tGCI has not been characterized. We found that DUSP6 was specifically induced in the cytoplasm of hippocampal CA1 neurons 4 to 24 h after tGCI. DUSP6-deficient mice showed normal spatial memory acquisition and retention in the Barnes maze. Impairment of spatial memory acquisition and retention after tGCI was attenuated in DUSP6-deficient mice. Neurodegeneration after tGCI, revealed by Fluoro-Jade C and H&E staining, was reduced in the hippocampus of DUSP6-deficient mice and DUSP6 deficiency enhanced the phosphorylation and nuclear translocation of Erk1/2 in the hippocampal CA1 region. These data support the role of DUSP6 as a negative regulator of Erk1/2 signaling and indicate the potential of DUSP6 inhibition as a novel therapeutic strategy to treat neurodegeneration after tGCI.

Dual-specificity phosphatases 22-deficient T cells contribute to the pathogenesis of ankylosing spondylitis.

BACKGROUND: Dual-specificity phosphatases (DUSPs) can dephosphorylate both tyrosine and serine/threonine residues of their substrates and regulate T cell-mediated immunity and autoimmunity. The aim of this study was to investigate the potential roles of DUSPs in ankylosing spondylitis (AS). METHODS: Sixty AS patients and 45 healthy controls were enrolled in this study. Associations of gene expression of 23 DUSPs in peripheral T cells with inflammatory cytokine gene expression and disease activity of AS were analyzed. Finally, we investigated whether the characteristics of AS are developed in DUSP-knockout mice. RESULTS: The mRNA levels of DUSP4, DUSP5, DUSP6, DUSP7, and DUSP14 in peripheral T cells were significantly higher in AS group than those of healthy controls (all p < 0.05), while DUSP22 (also named JKAP) mRNA levels were significantly lower in AS group than healthy controls (p < 0.001). The mRNA levels of DUSP4, DUSP5, DUSP6, DUSP7, and DUSP14 in T cells were positively correlated with mRNA levels of tumor necrosis factor-α (TNF-α), whereas DUSP22 was inversely correlated (all p < 0.05). In addition, inverse correlations of DUSP22 gene expression in peripheral T cells with C-reactive protein, erythrocyte sedimentation rate, and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) were observed (all p < 0.05). More importantly, aged DUSP22 knockout mice spontaneously developed syndesmophyte formation, which was accompanied by an increase of TNF-α+, interleukin-17A+, and interferon-γ+ CD3+ T cells. CONCLUSIONS: DUSP22 may play a crucial role in the pathogenesis and regulation of disease activity of AS.

MAP4K3/GLK inhibits Treg differentiation by direct phosphorylating IKKß and inducing IKKß-mediated FoxO1 nuclear export and Foxp3 downregulation.

Rationale: GLK (MAP4K3) activates PKCθ-IKKß axis in T-cell activation and induces IL-17A-mediated autoimmune diseases. Attenuation of Treg differentiation and function by GLK could also contribute to autoimmune diseases. Methods: We analyzed the roles of GLK and IKKß in Treg differentiation and function using T-cell-specific GLK transgenic mice and IKKß conditional knockout mice. The mechanism of GLK/IKKß-mediated attenuation of Treg differentiation/function was studied by chromatin-immunoprecipitation, reporter assays, in vitro kinase assays, protein-protein interaction assays, mass spectrometry, confocal microscopy, flow cytometry, and single-cell RNA sequencing (scRNA-seq) analysis. Results: We found that GLK signaling inhibited Foxp3 transcription by blocking the function of the transcription factor FoxO1. Mechanistically, GLK directly phosphorylated and activated IKKß at Ser733 in a PKCθ-independent manner. The phospho-IKKß Ser733 induced FoxO1 Ser319 phosphorylation and nuclear export, leading to Foxp3 downregulation. Consistently, scRNA-seq analyses showed that Foxp3 mRNA levels were inversely correlated with FoxO1 mRNA levels in GLK transgenic CD4+ T cells. Conclusions: GLK-IKKß-FoxO1 signaling axis inhibits Foxp3 transcription, leading to reduction of Treg differentiation and suppressive activity, as well as induction of autoimmune disease.

SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19.

ACE2 on epithelial cells is the SARS-CoV-2 entry receptor. Single-cell RNA-sequencing data derived from two COVID-19 cohorts revealed that MAP4K3/GLK-positive epithelial cells were increased in patients. SARS-CoV-2-induced GLK overexpression in epithelial cells was correlated with COVID-19 severity and vesicle secretion. GLK overexpression induced the epithelial cell-derived exosomes containing ACE2; the GLK-induced exosomes transported ACE2 proteins to recipient cells, facilitating pseudovirus infection. Consistently, ACE2 proteins were increased in the serum exosomes from another COVID-19 cohort. Remarkably, SARS-CoV-2 spike protein-stimulated GLK, and GLK stabilized ACE2 in epithelial cells. Mechanistically, GLK phosphorylated ACE2 at two serine residues (Ser776, Ser783), leading to the dissociation of ACE2 from its E3 ligase UBR4. Reduction in UBR4-induced Lys48-linked ubiquitination at three lysine residues (Lys26, Lys112, Lys114) of ACE2 prevented its degradation. Furthermore, SARS-CoV-2 pseudovirus or live virus infection in humanized ACE2 mice induced GLK and ACE2 protein levels, and ACE2-containing exosomes. Collectively, ACE2 stabilization by SARS-CoV-2-induced MAP4K3/GLK may contribute to the pathogenesis of COVID-19.

Induction of Interferon-γ and Tissue Inflammation by Overexpression of Eosinophil Cationic Protein in T Cells and Exosomes.

OBJECTIVE: T cells play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Serum-derived exosomes are increased in SLE patients and are correlated with disease severity. This study was undertaken to investigate whether T cell-derived exosomal proteins play a role in SLE pathogenesis. METHODS: We characterized proteins in T cell-derived exosomes from SLE patients and healthy controls by MACSPlex exosome analysis and proteomics. To study the potential pathogenic functions of the exosomal protein identified, we generated and characterized T cell-specific transgenic mice that overexpressed that protein in T cells. RESULTS: We identified eosinophil cationic protein (ECP, also called human RNase III) as overexpressed in SLE T cell-derived exosomes. T cell-specific ECP-transgenic mice (n = 5 per group) displayed early induction of serum interferon-γ (IFNγ) levels (P = 0.062) and inflammation of multiple tissue types. Older T cell-specific ECP-transgenic mice (n = 3 per group) also displayed an increase in follicular helper T cell and plasma B cell numbers, and in autoantibody levels (P < 0.01). Single-cell RNA sequencing showed the induction of IFNγ messenger RNA (P = 2.2 × 10-13 ) and inflammatory pathways in ECP-transgenic mouse T cells. Notably, adoptively transferred ECP-containing exosomes stimulated serum autoantibody levels (P < 0.01) and tissue IFNγ levels in the recipient mice (n = 3 per group). The transferred exosomes infiltrated into multiple tissues of the recipient mice, resulting in hepatitis, nephritis, and arthritis. CONCLUSION: Our findings indicate that ECP overexpression in T cells or T cell-derived exosomes may be a biomarker and pathogenic factor for nephritis, hepatitis, and arthritis associated with SLE.

Genomic sequencing and functional analyses identify MAP4K3/GLK germline and somatic variants associated with systemic lupus erythematosus.

OBJECTIVES: MAP4K3 (GLK) overexpression in T cells induces interleukin (IL)-17A production and autoimmune responses. GLK overexpressing T-cell population is correlated with severity of human systemic lupus erythematosus (SLE); however, it is unclear how GLK is upregulated in patients with SLE. METHODS: We enrolled 181 patients with SLE and 250 individuals without SLE (93 healthy controls and 157 family members of patients with SLE) in two independent cohorts from different hospitals/cities. Genomic DNAs of peripheral blood mononuclear cells were subjected to next-generation sequencing to identify GLK gene variants. The functional consequences of the identified GLK germline or somatic variants were investigated using site-directed mutagenesis and cell transfection, followed by reporter assays, mass spectrometry, immunoblotting, coimmunoprecipitation, and in situ proximity ligation assays. RESULTS: We identified 58 patients with SLE from Cohort #1 and #2 with higher frequencies of a somatic variant (chr2:39 477 124 A>G) in GLK 3'-untranslated region (UTR); these patients with SLE showed increased serum anti-double-stranded DNA levels and decreased serum C3/C4 levels. This somatic variant in 3'-UTR enhanced GLK mRNA levels in T cells. In addition, we identified five patients with SLE with GLK (A410T) germline variant in Cohort #1 and #2, as well as two other patients with SLE with GLK (K650R) germline variant in Cohort #1. Another GLK germline variant, A579T, was also detected in one patient with SLE from Cohort #2. Both GLK (A410T) and GLK (K650R) mutants inhibited GLK ubiquitination induced by the novel E3 ligase makorin ring-finger protein 4 (MKRN4), leading to GLK protein stabilisation. CONCLUSIONS: Multiple GLK germline and somatic variants cause GLK induction by increasing mRNA or protein stability in patients with SLE.

BPI overexpression suppresses Treg differentiation and induces exosome-mediated inflammation in systemic lupus erythematosus.

Background: Serum-derived exosomes are correlated with disease severity of human systemic lupus erythematosus (SLE). The proteins in the T-cell-derived exosomes from SLE patients could contribute to inflammation. Methods: We characterized proteins of T cell-derived exosomes from SLE patients and healthy controls by proteomics. To study the potential pathogenic role of the identified exosomal protein, we generated and characterized T-cell-specific transgenic mice that overexpressed the identified protein in T cells using immunohistochemistry, immunoblotting, and single-cell RNA sequencing. Results: We identified an overexpressed protein, bactericidal/permeability-increasing protein (BPI), in SLE T cells and T-cell-derived exosomes. T-cell-specific BPI transgenic (Lck-BPI Tg) mice showed multi-tissue inflammation with early induction of serum IL-1ß levels, as well as serum triglyceride and creatinine levels. Interestingly, exosomes of Lck-BPI Tg T cells stimulated IL-1ß expression of wild-type recipient macrophages. Remarkably, adoptive transfer of BPI-containing exosomes increased serum IL-1ß and autoantibody levels in recipient mice. The transferred exosomes infiltrated into multiple tissues of recipient mice, resulting in hepatitis, nephritis, and arthritis. ScRNA-seq showed that Lck-BPI Tg T cells displayed a decrease of Treg population, which was concomitant with ZFP36L2 upregulation and Helios downregulation. Furthermore, in vitro Treg differentiation was reduced by BPI transgene and enhanced by BPI knockout. Conclusions: BPI is a negative regulator of Treg differentiation. BPI overexpression in T-cell-derived exosomes or peripheral blood T cells may be a biomarker and pathogenic factor for human SLE nephritis, hepatitis, and arthritis.

Structural Insights into the Active Site Formation of DUSP22 in N-loop-containing Protein Tyrosine Phosphatases.

Cysteine-based protein tyrosine phosphatases (Cys-based PTPs) perform dephosphorylation to regulate signaling pathways in cellular responses. The hydrogen bonding network in their active site plays an important conformational role and supports the phosphatase activity. Nearly half of dual-specificity phosphatases (DUSPs) use three conserved residues, including aspartate in the D-loop, serine in the P-loop, and asparagine in the N-loop, to form the hydrogen bonding network, the D-, P-, N-triloop interaction (DPN-triloop interaction). In this study, DUSP22 is used to investigate the importance of the DPN-triloop interaction in active site formation. Alanine mutations and somatic mutations of the conserved residues, D57, S93, and N128 substantially decrease catalytic efficiency (kcat/KM) by more than 102-fold. Structural studies by NMR and crystallography reveal that each residue can perturb the three loops and induce conformational changes, indicating that the hydrogen bonding network aligns the residues in the correct positions for substrate interaction and catalysis. Studying the DPN-triloop interaction reveals the mechanism maintaining phosphatase activity in N-loop-containing PTPs and provides a foundation for further investigation of active site formation in different members of this protein class.

DUSP11 Attenuates Lipopolysaccharide-Induced Macrophage Activation by Targeting TAK1.

Dual-specificity phosphatase 11 (DUSP11, also named as PIR1) is a member of the atypical DUSP protein tyrosine phosphatase family. DUSP11 is only known to be an RNA phosphatase that regulates noncoding RNA stability. To date, the role of DUSP11 in immune cell signaling and immune responses remains unknown. In this study, we generated and characterized the immune cell functions of DUSP11-deficient mice. We identified TGF-ß-activated kinase 1 (TAK1) as a DUSP11-targeted protein. DUSP11 interacted directly with TAK1, and the DUSP11-TAK1 interaction was enhanced by LPS stimulation in bone marrow-derived macrophages. DUSP11 deficiency enhanced the LPS-induced TAK1 phosphorylation and cytokine production in bone marrow-derived macrophages. Furthermore, DUSP11-deficient mice were more susceptible to LPS-induced endotoxic shock. The LPS-induced serum levels of IL-1ß, TNF-α, and IL-6 were significantly elevated in DUSP11-deficient mice compared with those of wild-type mice. The data indicate that DUSP11 inhibits LPS-induced macrophage activation by targeting TAK1.

MAP4K Family Kinases and DUSP Family Phosphatases in T-Cell Signaling and Systemic Lupus Erythematosus.

T cells play a critical role in the pathogenesis of systemic lupus erythematosus (SLE), which is a severe autoimmune disease. In the past 60 years, only one new therapeutic agent with limited efficacy has been approved for SLE treatment; therefore, the development of early diagnostic biomarkers and therapeutic targets for SLE is desirable. Mitogen-activated protein kinase kinase kinase kinases (MAP4Ks) and dual-specificity phosphatases (DUSPs) are regulators of MAP kinases. Several MAP4Ks and DUSPs are involved in T-cell signaling and autoimmune responses. HPK1 (MAP4K1), DUSP22 (JKAP), and DUSP14 are negative regulators of T-cell activation. Consistently, HPK1 and DUSP22 are downregulated in the T cells of human SLE patients. In contrast, MAP4K3 (GLK) is a positive regulator of T-cell signaling and T-cell-mediated immune responses. MAP4K3 overexpression-induced RORγt-AhR complex specifically controls interleukin 17A (IL-17A) production in T cells, leading to autoimmune responses. Consistently, MAP4K3 and the RORγt-AhR complex are overexpressed in the T cells of human SLE patients, as are DUSP4 and DUSP23. In addition, DUSPs are also involved in either human autoimmune diseases (DUSP2, DUSP7, DUSP10, and DUSP12) or T-cell activation (DUSP1, DUSP5, and DUSP14). In this review, we summarize the MAP4Ks and DUSPs that are potential biomarkers and/or therapeutic targets for SLE.

MAP4K3/GLK in autoimmune disease, cancer and aging.

MAP4K3 (also named GLK) is a serine/threonine kinase, which belongs to the mammalian Ste20-like kinase family. At 22 years of age, GLK was initially cloned and identified as an upstream activator of the MAPK JNK under an environmental stress and proinflammatory cytokines. The data derived from GLK-overexpressing or shRNA-knockdown cell lines suggest that GLK may be involved in cell proliferation through mTOR signaling. GLK phosphorylates the transcription factor TFEB and retains TFEB in the cytoplasm, leading to inhibition of cell autophagy. After generating and characterizing GLK-deficient mice, the important in vivo roles of GLK in T-cell activation were revealed. In T cells, GLK directly interacts with and activates PKCθ through phosphorylating PKCθ at Ser-538 residue, leading to activation of IKK/NF-κB. Thus, GLK-deficient mice display impaired T-cell-mediated immune responses and decreased inflammatory phenotypes in autoimmune disease models. Consistently, the percentage of GLK-overexpressing T cells is increased in the peripheral blood from autoimmune disease patients; the GLK-overexpressing T cell population is correlated with disease severity of patients. The pathogenic mechanism of autoimmune disease by GLK overexpression was unraveled by characterizing T-cell-specific GLK transgenic mice and using biochemical analyses. GLK overexpression selectively promotes IL-17A transcription by inducing the AhR-RORγt complex in T cells. In addition, GLK overexpression in cancer tissues is correlated with cancer recurrence of human lung cancer and liver cancer; the predictive power of GLK overexpression for cancer recurrence is higher than that of pathologic stage. GLK directly phosphorylates and activates IQGAP1, resulting in induction of Cdc42-mediated cell migration and cancer metastasis. Furthermore, treatment of GLK inhibitor reduces disease severity of mouse autoimmune disease models and decreases IL-17A production of human autoimmune T cells. Due to the inhibitory function of HPK1/MAP4K1 in T-cell activation and the promoting effects of GLK on tumorigenesis, HPK1 and GLK dual inhibitors could be useful therapeutic drugs for cancer immunotherapy. In addition, GLK deficiency results in extension of lifespan in Caenorhabditis elegans and mice. Taken together, targeting MAP4K3 (GLK) may be useful for treating/preventing autoimmune disease, cancer metastasis/recurrence, and aging.

MAP4K3/GLK Promotes Lung Cancer Metastasis by Phosphorylating and Activating IQGAP1.

Overexpression of the serine/threonine kinase GLK/MAP4K3 in human lung cancer is associated with poor prognosis and recurrence, however, the role of GLK in cancer recurrence remains unclear. Here, we report that transgenic GLK promotes tumor metastasis and cell migration through the scaffold protein IQ motif-containing GTPase-activating protein 1(IQGAP1). GLK transgenic mice displayed enhanced distant metastasis. IQGAP1 was identified as a GLK-interacting protein; two proline-rich regions of GLK and the WW domain of IQGAP1 mediated this interaction. GLK and IQGAP1 colocalized at the leading edge including filopodia and lamellipodia of migrating cells. GLK directly phosphorylated IQGAP1 at Ser-480 enhancing Cdc42 activation and subsequent cell migration. GLK-induced cell migration and lung cancer metastasis were abolished by IQGAP1 depletion. Consistently, human NSCLC patient tissues displayed increased phospho-IQGAP1, which correlated with poor survival. Collectively, GLK promotes lung cancer metastasis by binding to, phosphorylating, and activating IQGAP1. SIGNIFICANCE: These findings show the critical role of the GLK-IQGAP cascade in cell migration and tumor metastasis, suggesting it as a potential biomarker and therapeutic target for lung cancer recurrence.

AhR-ROR-γt complex is a therapeutic target for MAP4K3/GLKhighIL-17Ahigh subpopulation of systemic lupus erythematosus.

The cytokine IL-17A plays critical roles in the pathogenesis of autoimmune diseases. The frequencies of MAP kinase kinase kinase kinase 3 [also named germinal center kinase-like kinase (GLK)]-overexpressing T cells are correlated with disease severity of systemic lupus erythematosus (SLE). T-cell-specific GLK-transgenic mice develop spontaneous autoimmune responses through IL-17A. GLK signaling selectively stimulates IL-17A production in murine T cells through inducing aryl hydrocarbon receptor (AhR)-retinoic acid receptor-related orphan nuclear receptor-γt (ROR-γt) complex formation. Here, we investigated whether GLK-induced AhR-ROR-γt complex in T cells is a therapeutic target for human SLE. The population of GLK+IL-17A+ T cells was enhanced in the peripheral blood from patients with SLE compared with that of healthy controls using flow cytometry. The receiver operating characteristic curve analysis showed that increased GLK+IL-17A+ T-cell population in peripheral blood reflected an active stage of SLE. In addition, peripheral blood T cells from patients with SLE displayed induction of ROR-γt phosphorylation and the AhR-ROR-γt (and AhR-phosphorylated ROR-γt) complex. Moreover, we identified a small-molecule inhibitor, verteporfin, that inhibited GLK kinase activity and AhR-ROR-γt interaction. The small-molecule inhibitor verteporfin suppressed the disease severity in autoimmune mouse models and IL-17A production in T cells from patients with SLE. Collectively, the GLK-induced AhR-ROR-γt (and AhR-phosphorylated ROR-γt) complex is a therapeutic target for the GLKhighIL-17Ahigh subpopulation of human patients with SLE.-Chuang, H.-C., Chen, Y.-M., Chen, M.-H., Hung, W.-T., Yang, H.-Y., Tseng, Y.-H., Tan, T.-H. AhR-ROR-γt complex is a therapeutic target for MAP4K3/GLKhighIL-17Ahigh subpopulation of systemic lupus erythematosus.

Regulation of Dual-Specificity Phosphatase (DUSP) Ubiquitination and Protein Stability.

Mitogen-activated protein kinases (MAPKs) are key regulators of signal transduction and cell responses. Abnormalities in MAPKs are associated with multiple diseases. Dual-specificity phosphatases (DUSPs) dephosphorylate many key signaling molecules, including MAPKs, leading to the regulation of duration, magnitude, or spatiotemporal profiles of MAPK activities. Hence, DUSPs need to be properly controlled. Protein post-translational modifications, such as ubiquitination, phosphorylation, methylation, and acetylation, play important roles in the regulation of protein stability and activity. Ubiquitination is critical for controlling protein degradation, activation, and interaction. For DUSPs, ubiquitination induces degradation of eight DUSPs, namely, DUSP1, DUSP4, DUSP5, DUSP6, DUSP7, DUSP8, DUSP9, and DUSP16. In addition, protein stability of DUSP2 and DUSP10 is enhanced by phosphorylation. Methylation-induced ubiquitination of DUSP14 stimulates its phosphatase activity. In this review, we summarize the knowledge of the regulation of DUSP stability and ubiquitination through post-translational modifications.