DUSP22 inhibits lung tumorigenesis by suppression of EGFR/c-Met signaling.

DUSP22, an atypical dual-specificity phosphatase enzyme, plays a significant role in regulating multiple kinase signaling pathways by dephosphorylation. Our study demonstrated that decreased DUSP22 expression is associated with shorter disease-free survival, advanced TNM (tumor, lymph nodes, and metastasis), cancer stage, and higher tumor grade in lung adenocarcinoma (LUAD) patients. Exogenous DUSP22 expression reduces the colony-forming capacity of lung cancer cells and inhibits xenograft tumor growth primarily by targeting EGFR and suppressing its activity through dephosphorylation. Knockdown of DUSP22 using shRNA enhances EGFR dependency in HCC827 lung cancer cells and increases sensitivity to gefitinib, an EGFR inhibitor. Consistently, genetic deletion of DUSP22 enhances EGFRdel (exon 19 deletion)-driven lung tumorigenesis and elevates EGFR activity. Pharmacological inhibition of DUSP22 activates EGFR, ERK1/2, and upregulates downstream PD-L1 expression. Additionally, lentiviral deletion of DUSP22 by shRNA enhances lung cancer cell migration through EGFR/c-Met and PD-L1-dependent pathways. Gefitinib, an EGFR inhibitor, mechanistically suppresses migration induced by DUSP22 deletion and inhibits c-Met activity. Furthermore, cabozantinib, a c-Met inhibitor, reduces migration and attenuates EGFR activation caused by DUSP22 deletion. Collectively, our findings support the hypothesis that loss of DUSP22 function in lung cancer cells confers a survival advantage by augmenting EGFR signaling, leading to increased activation of downstream c-Met, ERK1/2, and PD-L1 axis, ultimately contributing to the progression of advanced lung cancer.

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.

Hematopoietic progenitor kinase 1 inhibits the development and progression of pancreatic intraepithelial neoplasia.

Ras plays an essential role in the development of acinar-to-ductal metaplasia (ADM) and pancreatic ductal adenocarcinoma (PDAC). However, mutant Kras is an inefficient driver for PDAC development. The mechanisms of the switching from low Ras activity to high Ras activity that are required for development and progression of pancreatic intraepithelial neoplasias (PanINs) are unclear. In this study, we found that hematopoietic progenitor kinase 1 (HPK1) was upregulated during pancreatic injury and ADM. HPK1 interacted with the SH3 domain and phosphorylated Ras GTPase-activating protein (RasGAP) and upregulated RasGAP activity. Using transgenic mouse models of HPK1 or M46, a kinase-dead mutant of HPK1, we showed that HPK1 inhibited Ras activity and its downstream signaling and regulated acinar cell plasticity. M46 promoted the development of ADM and PanINs. Expression of M46 in KrasG12D Bac mice promoted the infiltration of myeloid-derived suppressor cells and macrophages, inhibited the infiltration of T cells, and accelerated the progression of PanINs to invasive and metastatic PDAC, while HPK1 attenuated mutant Kras-driven PanIN progression. Our results showed that HPK1 plays an important role in ADM and the progression of PanINs by regulating Ras signaling. Loss of HPK1 kinase activity promotes an immunosuppressive tumor microenvironment and accelerates the progression of PanINs to PDAC.

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.

DUSP3 regulates phosphorylation-mediated degradation of occludin and is required for maintaining epithelial tight junction.

BACKGROUND: Tight junctions (TJ) are multi-protein complexes that hold epithelial cells together and form structural and functional barriers for maintaining proper biological activities. Dual specificity phosphatase 3 (DUSP3), a suppressor of multiple protein tyrosine (Tyr) kinases, is decreased in lung cancer tissues. Here we demonstrated the role of DUSP3 in regulation of epithelial TJ. METHODS: Barrier functions of TJ were examined in wild-type or DUSP3-deficient lung epithelial cells. Animal and clinical data were analyzed for the association between DUSP3 deficiency and lung cancer progression. Proximity ligation assay, immunoblotting, and phosphatase assay were performed to study the effect of DUSP3 on the TJ protein occludin (OCLN). Mutations of Tyr residues on OCLN showed the role of Tyr phosphorylation in regulating OCLN. RESULTS: Compared to those of the DUSP3-expressing cells, we found the expression and distribution of ZO-1, a TJ-anchoring molecule, were abnormal in DUSP3-deficient cells. OCLN had an increased phosphorylation level in DUSP3-deficient cells. We identified that OCLN is a direct substrate of DUSP3. DUSP3 regulated OCLN ubiquitination and degradation through decreasing OCLN tyrosine phosphorylation directly or through suppressing focal adhesion kinase, the OCLN kinase. CONCLUSION: Our study revealed that DUSP3 is an important TJ regulatory protein and its decrease may be involved in progression of epithelial cancers.

Identification of a gut microbiota member that ameliorates DSS-induced colitis in intestinal barrier enhanced Dusp6-deficient mice.

Strengthening the gut epithelial barrier is a potential strategy for management of gut microbiota-associated illnesses. Here, we demonstrate that dual-specificity phosphatase 6 (Dusp6) knockout enhances baseline colon barrier integrity and ameliorates dextran sulfate sodium (DSS)-induced colonic injury. DUSP6 mutation in Caco-2 cells enhances the epithelial feature and increases mitochondrial oxygen consumption, accompanied by altered glucose metabolism and decreased glycolysis. We find that Dusp6-knockout mice are more resistant to DSS-induced dysbiosis, and the cohousing and fecal microbiota transplantation experiments show that the gut/fecal microbiota derived from Dusp6-knockout mice also confers protection against colitis. Further culturomics and mono-colonialization experiments show that one gut microbiota member in the genus Duncaniella confers host protection from DSS-induced injury. We identify Dusp6 deficiency as beneficial for shaping the gut microbiota eubiosis necessary to protect against gut barrier-related diseases.

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.

DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis.

Dual-specificity phosphatases (DUSPs) regulate the activity of various downstream kinases through serine or threonine or tyrosine dephosphorylation. Loss of function and aberrant expression of DUSPs has been implicated in cancer progression and poor survival, yet the function of DUSP22 in prostate cancer (PCa) cells is not clear. Gene Expression Omnibus and cBioPortal microarray database analyses showed that DUSP22 expression was lower in PCa tissues than normal prostate tissues, and altered DUSP22 expression was associated with shorter progression-free and disease-free survival of patients with PCa. Exogenous DUSP22 expression in LNCaP, PC3, and C4-2B PCa cells inhibited cellular proliferation and colony formation, supporting a growth inhibitory role for DUSP22 in PCa cells. DUSP22 expression significantly attenuated epidermal growth factor (EGF) receptor (EGFR) and its downstream ERK1/2 signaling by dephosphorylation. However, DUSP22 failed to suppress the growth of CWR22Rv1 and DU145 cells with elevated phosphorylated (p-)ERK1/2 levels. A serine-to-alanine mutation at position 58, a potential ERK1/2-targeted phosphorylation site in DUSP22, was sufficient to suppress growth of CWR22Rv1 cells with elevated p-ERK1/2 levels, suggesting a mutually antagonistic relationship between DUSP22 and ERK1/2 dependent on phosphorylation status. We showed that DUSP22 can suppress prostate-specific antigen gene expression through phosphatase-dependent pathways, suggesting that DUSP22 is an important regulator of the androgen receptor (AR) in PCa cells. Mechanistically, DUSP22 can interact with AR as a regulatory partner and interfere with EGF-induced AR phosphorylation at Tyr534, suggesting that DUSP22 serves as a crucial suppressor of both EGFR and AR-dependent signaling in PCa cells via dephosphorylation. Our findings indicate that loss of function of DUSP22 in PCa cells leads to aberrant activation of both EGFR-ERKs and AR signaling and ultimately progression of PCa, supporting the potential for novel therapeutic design of harnessing DUSP22 in the treatment of PCa.-Lin, H.-P., Ho, H.-M., Chang, C.-W., Yeh, S.-D., Su, Y.-W., Tan, T.-H., Lin, W.-J. DUSP22 suppresses prostate cancer proliferation by targeting the EGFR-AR axis.

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.

RBM4-SRSF3-MAP4K4 splicing cascade modulates the metastatic signature of colorectal cancer cell.

Alternative splicing (AS) of pre-messenger (m)RNA is a pivotal mechanism in expanding proteomic diversity, which determines the functions of mammalian cells. By conducting transcriptome analyses to profile splicing events in human colorectal cancer (CRC) tissues compared to adjacent normal counterparts, we noted differential splicing profiles of serine/arginine-rich splicing factor 3 (SRSF3) and mitogen-activated protein 4 kinase 4 (MAP4K4) in cancerous tissues of CRC compared to adjacent normal tissues. In addition to SRSF3-mediated autoregulation, RNA-binding motif protein 4 (RBM4) constituted another mechanism in reprogramming the splicing profile of SRSF3. Upregulated expressions of SRSF3 in CRC cells modulated utilization of MAP4K4 exon 16 in a sequence-dependent manner. Alternatively spliced MAP4K4 variants exhibited differential effects on the phosphorylation of c-Jun N-terminal protein kinase 1 (JNK1) which subsequently modulated expression profiles of E-cadherin, N-cadherin, and vimentin, all of which are involved in the migration and invasion of CRC cells. Collectively, RBM4-SRSF3-MAP4K4 constitutes a novel mechanism for manipulating the metastasis of CRC cells through the JNK1 signaling pathway.

MAP4K4 and IL-6+ Th17 cells play important roles in non-obese type 2 diabetes.

Obesity is a causal factor of type 2 diabetes (T2D); however, people without obesity (including lean, normal weight, or overweight) may still develop T2D. Non-obese T2D is prevalent in Asia and also frequently occurs in Europe. Recently, multiple evidences oppose the notion that either obesity or central obesity (visceral fat accumulation) promotes non-obese T2D. Several factors such as inflammation and environmental factors contribute to non-obese T2D. According to the data derived from gene knockout mice and T2D clinical samples in Asia and Europe, the pathogenesis of non-obese T2D has been unveiled recently. MAP4K4 downregulation in T cells results in enhancement of the IL-6+ Th17 cell population, leading to insulin resistance and T2D in both human and mice. Moreover, MAP4K4 single nucleotide polymorphisms and epigenetic changes are associated with T2D patients. Interactions between MAP4K4 gene variants and environmental factors may contribute to MAP4K4 attenuation in T cells, leading to non-obese T2D. Future investigations of the pathogenesis of non-obese T2D shall lead to development of precision medicine for non-obese T2D.

Prediction of early hepatocellular carcinoma recurrence using germinal center kinase-like kinase.

Germinal center kinase-like kinase (GLK) is a key controller of autoimmunity. In this study, we assessed the clinical relevance and tumorigenic effects of GLK in hepatocellular carcinoma (HCC). Using immunohistochemistry, we showed that the GLK proportion score increased in both cancerous and adjacent non-cancerous liver tissue from patients with HCC recurrence. A Kaplan-Meier analysis revealed that patients with a wide distribution of GLK in non-cancerous liver tissue had a higher rate of HCC recurrence than those with very low or no GLK expression. Multivariate Cox regression analyses indicated that a high GLK proportion score in non-cancerous liver tissue was an independent predictor of early HCC recurrence after resection. Lentiviral vector-mediated overexpression of GLK activated the nuclear factor kappa B (NFκB) signaling cascade and accelerated cell cycle progression in primary human hepatocytes, thereby promoting proliferation. An increase in GLK expression coincided with NFκB activation and enhanced expression of proliferating cell nuclear antigen in HCC tissue. Our findings demonstrate a potential hepatocarcinogenic effect of GLK and the feasibility of using GLK to predict early HCC recurrence.

GLK/MAP4K3 overexpression associates with recurrence risk for non-small cell lung cancer.

Lung cancer is the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of total lung cancers; 40% to 60% of NSCLC patients die of cancer recurrence after cancer resection. Since GLK (also named MAP4K3) induces activation of NF-κB, which contributes to tumor progression, we investigated the role of GLK in NSCLC. GLK protein levels of 190 samples from pulmonary tissue arrays and 58 pulmonary resection samples from stage I to stage III NSCLC patients were studied using immunohistochemistry or immunoblotting. High levels of GLK proteins were detected in pulmonary tissues from NSCLC patients. Elevated GLK protein levels were correlated with increased recurrence risks and poor recurrence-free survival rates in NSCLC patients after adjusting for pathologic stage, smoking status, alcohol status, and EGFR levels. Thus, GLK is a novel prognostic biomarker for NSCLC recurrence.

Epigenetic regulation of HGK/MAP4K4 in T cells of type 2 diabetes patients.

Type 2 diabetes (T2D) is a complex and heterogeneous disease. Obesity increases the risk of obese T2D; but in Asia non-obese T2D is prevalent. The cause of non-obese T2D has remained elusive. We studied the potential involvement of HGK/MAP4K4 in T2D using clinical samples from newly diagnosed, drug-naïve patients and healthy controls. HGK levels fell and IL-6 levels increased in T cells from T2D patients. Frequencies of IL-6-producing T cells were correlated with glucose levels after glucose-tolerance tests (but not body mass index and waist circumference) and inversely correlated with HGK expression levels. Moreover, methylation frequencies of the HGK promoter were increased in T2D patients and correlated with glucose levels after glucose-tolerance tests. The correlation was independent of body mass index. Demethylation treatment increased HGK expression levels and reduced IL-6 production in T2D T cells. This report identifies HGK methylation/downregulation in T cells as a potential biomarker for non-obese T2D.

MAP4K Family Kinases in Immunity and Inflammation.

MAP kinase kinase kinase kinases (MAP4Ks) belong to the mammalian Ste20-like family of serine/threonine kinases. MAP4Ks including MAP4K1/HPK1, MAP4K2/GCK, MAP4K3/GLK, MAP4K4/HGK, MAP4K5/KHS, and MAP4K6/MINK have been reported to induce JNK activation through activating the MAP3K-MAP2K cascade. The physiological roles of MAP4Ks in immunity and inflammation are largely unknown until recent studies using biochemical approaches and knockout mice. Surprisingly, JNK is not the major target of MAP4Ks in immune cells; MAP4Ks regulate immune responses through novel targets. HPK1 inhibits T-cell receptor (TCR) signaling and B-cell receptor signaling via inducing phosphorylation/ubiquitination of SLP-76 and BLNK, respectively. GLK activates TCR signaling through phosphorylating/activating PKCθ. T-cell-mediated immune responses and Th17-mediated experimental autoimmune diseases are enhanced in HPK1 knockout mice but ameliorated in GLK knockout mice. Consistently, HPK1 levels are decreased in peripheral blood mononuclear cells and T cells from patients with psoriatic arthritis and systemic lupus erythematosus (SLE), respectively. Moreover, GLK levels are increased in T cells from patients with SLE, rheumatoid arthritis, or adult-onset Still's disease; the percentages of GLK-overexpression T cells are correlated with the disease activity. In addition, HGK phosphorylates and induces TRAF2 protein degradation, leading to negative regulation of IL-6 production in resting T cells. Loss of HGK in T cells results in spontaneous systemic inflammation and type 2 diabetes in mice. HGK is also involved in cancer cell migration. To date, the phenotypes of knockout mice for GCK, KHS, and MINK have not been reported; the roles of these three MAP4Ks in immune cell signaling are discussed in this review. Taken together, MAP4K family kinases play diverse roles in immune cell signaling, immune responses, and inflammation.

PP4 is essential for germinal center formation and class switch recombination in mice.

PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig) VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23(cre)PP4(F/F) mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab) or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23(cre)PP4(F/F) mice, and their B cells showed a reduced efficiency of class switch recombination (CSR) in vitro upon stimulation by LPS or LPS plus IL-4. When CD23(cre)PP4(F/F) mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs) in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.