Non-receptor Protein Tyrosine Kinase Research Articles

Effects of syndecan-4 silencing on the extracellular matrix remodeling in anoikis-resistant endothelial cells.

Anoikis is a process of programmed cell death induced by the loss of cell/matrix interactions. In previous work, we have shown that the acquisition of anoikis resistance upregulates syndecan-4 (SDC4) expression in endothelial cells. In addition, SDC4 gene silencing by microRNA interference reverses the transformed phenotype of anoikis-resistant endothelial cells. Due to this role of SDC4 in regulating the behavior of anoikis-resistant endothelial cells, we have evaluated that the functional consequences of SDC4 silencing in the extracellular matrix (ECM) remodeling in anoikis-resistant rabbit aortic endothelial cells submitted to SDC4 gene silencing (miR-Syn4-Adh-1-EC). For this, we evaluated the expression of adhesive proteins, ECM receptors, nonreceptor protein-tyrosine kinases, and ECM-degrading enzymes and their inhibitors. Altered cell behavior was monitored by adhesion, migration, and tube formation assays. We found that SDC4 silencing led to a decrease in migration and angiogenic capacity of anoikis-resistant endothelial cells; this was accompanied by an increase in adhesion to fibronectin. Furthermore, after SDC4 silencing, we observed an increase in the expression of fibronectin, collagen IV, and vitronectin, and a decrease in the expression of integrin α5β1 and αvβ3, besides that, silenced cells show an increase in Src and FAK expression. Quantitative polymerase chain reactionand Western blot analysis demonstrated that SDC4 silencing leads to altered gene and protein expression of MMP2, MMP9, and HSPE. Compared withparental cells, SDC4 silenced cells showed a decrease in nitric oxide production and eNOS expression. In conclusion, these data demonstrate that SDC4 plays an important role in ECM remodeling. In addition, our findings represent an important step towardunderstanding the mechanism by which SDC4 can reverse the transformed phenotype of anoikis-resistant endothelial cells.

Traditional Chinese Medicine Targeting Sarcoma Virus Oncogene-related Diseases.

The sarcoma virus oncogene (Src) tyrosine kinase, a nonreceptor protein-tyrosine kinase, plays a crucial role in cell survival, migration, differentiation and proliferation. The study of Src has developed considerably since it was first discovered as a proto-oncogene. Src has also been associated with inflammation and bone-related diseases. Src inhibitors (bosutinib, ponatinib, dasatinib, and vandetanib) have been put into clinical use. However, their side effects and cardiovascular toxicity may be a concern. There is an urgent need to explore new Src inhibitors. Traditional Chinese medicine (TCM), which has a vast history, can provide a broad resource base. Many natural compounds and TCM extracts have the potential for anti-Src treatment. This article describes the natural compounds and extracts from TCM.

Upregulation of the protein kinase Lyn is associated with renal injury in type 2 diabetes patients

Background The role of inflammation in the pathogenesis of type 2 diabetes mellitus (T2DM) is well established. Lyn, a member of the nonreceptor protein tyrosine kinase Src family, has been reported to modulate inflammatory signaling pathways. Methods Lyn expression was assessed in kidney biopsies of 11 patients with diabetic kidney disease (DKD) and in kidney tissues of streptozotocin (STZ)-induced DKD mice. 102 recruited T2DM patients were divided into three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. Twenty-one healthy volunteers were recruited as a control group. Clinical data, blood and urine samples of all individuals were collected for analysis. Results Lyn expression was augmented in the kidneys of DKD patients and STZ-induced diabetic mice. Compared with control and normoalbuminuria groups, both mRNA and protein expression of Lyn in peripheral blood mononuclear cells (PBMCs) in the macroalbuminuria group were significantly increased (p < .05). Elevated Lyn levels were independently related to urine albumin/urine creatinine ratio and were positively associated with key inflammatory factors, namely interleukin-1β, monocyte chemoattractant protein-1, and tumor necrosis factor-α. Additionally, Lyn exhibited a noteworthy connection with renal tubular injury indicators, specifically urinary neutrophil gelatinase-associated lipocalin and urinary retinol binding protein. ROC curve analysis showed that Lyn could predict albuminuria in diabetic patients with an area under the curve of 0.844 (95% CI: 0.764–0.924). Conclusion Lyn levels in PBMCs exhibited a positive correlation with the severity of albuminuria, renal tubular damage, and inflammatory responses. Hence, Lyn may be a compelling candidate for predicting albuminuria levels in diabetes.

PTK2 promotes uveal melanoma metastasis by activating epithelial‐to‐mesenchymal transition

AbstractUveal melanoma (UM) is the most common primary intraocular malignant tumor in adults. More than half of UM cases develop distant metastasis to the liver, lung, bone, and other organs, which frequently results in patient death. However, the mechanisms underlying UM metastasis remain largely unknown. Here, we report that protein tyrosine kinase 2 (PTK2), a nonreceptor protein tyrosine kinase also known as focal adhesion kinase (FAK), was overexpressed in most examined UM specimens. Furthermore, we identified PTK2 expression as a novel independent risk factor that predicts poor prognosis of UM patients. Mechanistic studies revealed that PTK2 promotes the EMT phenotype, leading to metastasis of UM cells. We showed that PTK2, located on chromosome 8q, is a functional gene of chromosome 8q gain to UM metastasis, which may represent the molecular mechanism for the aberrant expression and activation of PTK2 in UM. Our data reveal a novel role and mechanism of PTK2 in the metastatic process of UM, suggesting PTK2 may be a potential prognostic biomarker for UM metastasis and a promising therapeutic target for UM treatment.

Exploring New Therapeutic Approaches for Rheumatoid Arthritis Based on Basic Signaling Pathways

RA is an incurable systemic inflammation the main features are infiltration of pro-inflammatory factors with joint swelling and pain as the main clinical manifestation. Studies have shown that there are many causes of RA, among which immune factors are the most important pathogenesis of rheumatoid arthritis. At present, emerging studies have found that PI3K / AKT / mTOR signaling axis is abnormal in patients with RA, which opens up new ideas for the treatment of RA through the normal expression of signal transduction by targeted drugs. Janus kinase family ( JAKs ), are up-regulated in non-receptor protein tyrosine kinase cytokine signaling ( SOCS ) inhibitors, suggesting that the existence of SOCS is most possibly to constitute the main machine-made of negative adjustment of JAK / STAT signaling. In RA patients, there is a correlation between the activation of mTOR signaling and the quantity of osteoclasts. The recently discovered cytokine IL-22 has been shown to significantly increase the number of fibroblasts isolated from the skin of the patients with psoriasis. The dual inhibitor of P1-3K / mTOR, NVP-BEZ235, effectively blocks the spread. This article mainly explores and discusses new targeted drugs such as tofacitinib and Ruxolitinib by discussing new pathological mechanisms and conventional diagnosis and treatment methods of RA. Since RA is one of the most representative diseases of skeletal immune diseases, the study of ra abnormal signaling pathways to discuss related treatment methods provides valuable insights into the disease and other inflammatory and autoimmune diseases.

TEC Genetic Polymorphism is Associated with TNF-α Inhibitor Treatment Response in Rheumatoid Arthritis Patients

Numerous genetic and clinical factors influence the progression of rheumatoid arthritis and treatment response. Single nucleotide polymorphisms (SNPs) have been associated with different treatment responses in TNF-α inhibitor users. This study aimed to determine if Tec protein tyrosine kinase (TEC), which belongs to the Tec family of non-receptor protein tyrosine kinases, is associated with treatment response in rheumatoid arthritis (RA) patients. RA patients from three university hospitals were recruited, and clinical data were collected from electronic medical records. One hundred fourteen patients treated with TNF-α inhibitor were included in the analysis. SNP-response associations were evaluated using logistic regression. Among the five SNPs analyzed, rs1472971 and rs11727166 were statistically significant genetic factors of TNF-α inhibitor treatment response. This study provides genetic evidence that TEC polymorphism can be a prognostic marker in treating RA patients.

LCK facilitates DNA damage repair by stabilizing RAD51 and BRCA1 in the nucleus of chemoresistant ovarian cancer

Poly-ADP Ribose Polymerase (PARP) targeted therapy is clinically approved for the treatment of homologous recombination (HR) repair deficient tumors. The remarkable success of this therapy in the treatment of HR repair deficient cancers has not translated to HR-proficient cancers. Our studies identify the novel role of non-receptor lymphocyte-specific protein tyrosine kinase (LCK) in the regulation of HR repair in endometrioid epithelial ovarian cancer (eEOC) model. We show that DNA damage leads to direct interaction of LCK with the HR repair proteins RAD51 and BRCA1 in a kinase dependent manner RAD51 and BRCA1 stabilization. LCK expression is induced and activated in the nucleus in response to DNA damage insult. Disruption of LCK expression attenuates RAD51, BRCA1, and BRCA2 protein expression by hampering there stability and results in inhibition of HR-mediated DNA repair including suppression of RAD51 foci formation, and augmentation of γH2AX foci formation. In contrast LCK overexpression leads to increased RAD51 and BRCA1 expression with a concomitant increase in HR DNA damage repair. Importantly, attenuation of LCK sensitizes HR-proficient eEOC cells to PARP inhibitor in cells and pre-clinical mouse studies. Collectively, our findings identify a novel therapeutic strategy to expand the utility of PARP targeted therapy in HR proficient ovarian cancer.Graphical

NMR approaches to study proteins integrating globular and disordered domains: the case of c-Src

Abstract Nuclear Magnetic Resonance is one of the most versatile structural biology tools. Its unique capacities remain unchallenged by the advances in other techniques, experimental, like cryo-electron microscopy, or computational, such as AlphaFold. In this perspective article we present the role played by various NMR techniques in the study of c-Src, a non-receptor protein tyrosine kinase that contains globular and intrinsically disordered domains. We show (i) how NMR helped chemical biology to discover the regulatory role of the Unique domain, (ii) its role in the characterization of the fuzzy intramolecular complex connecting the disordered region with the globular core through the SH3 domain, (iii) the identification of salt bridges connecting the main post-translational sites of the Unique domain with neighbor basic residues, and, (iv) the characterization of breathing motions and the independent dynamics of the two lobes of the kinase domain.

Proteomic and phosphoproteomic analysis reveal threonine deficiency increases hepatic lipid deposition in Pekin ducks via reducing STAT phosphorylation.

Dietary threonine (Thr) deficiency enhances triglyceride (TG) deposition in the liver of Pekin ducks, which injures hepatic function and impairs growth performance. However, the underlying molecular mechanisms remain unclear. In the present study, we investigated the effects of dietary Thr deficiency on the expressions of proteins and phosphoproteins in liver of Pekin ducks, to identify the underlying molecular changes. A total of 300 one-day-old ducklings were divided into 3 groups with 10 replicates of 10 birds. All ducks were fed corn-wheat-peanut meal diets containing 0.46%, 0.71%, and 0.96% Thr, respectively, from 1 to 21 days of age. Growth performance, serum parameters, hepatic TG content, and expression of genes involved in lipid metabolism of Pekin ducks were determined. A Thr deficiency group (Thr-D, 0.46% Thr) and a Thr sufficiency group (Thr-S, 0.71% Thr) were selected for subsequent proteomic and phosphoproteomic analysis. The results showed that Thr-D reduced the growth performance (P<0.001), and increased the plasma concentrations of cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and hepatic TG (P<0.05). Thr-D increased gene expression related to fatty acid and TG synthesis (P<0.05). A total of 176 proteins and 259 phosphosites (containing 198 phosphoproteins) were observed to be differentially expressed as a result of Thr-D. The upregulated proteins were enriched in the pathway related to amino acid metabolism, peroxisome. The downregulated proteins were enriched in linolenic and arachidonic acid metabolism, and the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. The upregulated phosphoproteins were enriched in the pathways related to fatty acid biosynthesis, fructose and mannose metabolism, and glycolysis/gluconeogenesis. Thr-D reduced the phosphorylation of STAT1 at S729 and STAT3 at S728, and expression of STAT5B. In contrast, Thr-D increased non-receptor tyrosine-protein kinase (TYK2) expression and STAT1 phosphorylation at S649. Taken together, dietary Thr-D increased hepatic TG accumulation by upregulating the expression of genes and proteins, and phosphoproteins related to fatty acid and triglyceride synthesis. Furthermore, these processes might be regulated by the JAK-STAT signaling pathway, especially the phosphorylation of STAT1 and STAT3.

Focal adhesion kinase induces cardiac remodeling through NF-κB-mediated inflammatory responses in diabetic cardiomyopathy

BackgroundHyperglycemia-induced chronic inflammation is a crucial risk factor that causes undesirable cardiac alternations in diabetic cardiomyopathy (DCM). Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that primarily regulates cell adhesion and migration. Based on recent studies, FAK is involved in inflammatory signaling pathway activation in cardiovascular diseases. Here, we evaluated the possibility of FAK as a therapeutic target for DCM. MethodsA small molecular selective FAKinhibitor, PND-1186 (PND), was used to evaluate the effect of FAK on DCM in both high glucose-stimulated cardiomyocytes and streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) mice. ResultsIncreased FAK phosphorylation was found in the hearts of STZ-induced T1DM mice. PND treatment significantly decreased the expression of inflammatory cytokines and fibrogenic markers in cardiac specimens of diabetic mice. Notably, these reductions were correlated with improved cardiac systolic function. Furthermore, PND suppressed transforming growth factor-β-activated kinase 1 (TAK1) phosphorylation and NF-κB activation in the hearts of diabetic mice. Cardiomyocytes were identified as the main contributor to FAK-mediated cardiac inflammation and the involvement of FAK in cultured primary mouse cardiomyocytes and H9c2 cells was identified. Both FAK inhibition or FAK deficiency prevented hyperglycemia-induced inflammatory and fibrotic responses in cardiomyocytes owing to the inhibition of NF-κB. Herein, FAK activation was revealed to FAK directly binding to TAK1, leading to activation of TAK1 and downstream NF-κB signaling pathway. ConclusionsFAK is a key regulator of diabetes-associated myocardial inflammatory injury by directly targeting to TAK1.

Targeting the Src N-terminal regulatory element in cancer.

The signaling pathways displayed by cancer cells are often composed by the same components than the physiological ones, yet the overall result is a pathological deregulation. The non-receptor protein tyrosine kinase Src is a good example. Src is the first described proto-oncogene and a demonstrated player in cancer progression, as it affects proliferation, invasion, survival, cancer stemness, and drug resistance. Src activation is linked to poor prognosis in many cancer types, yet mutations in this protein are rarely observed. In addition, being a demonstrated cancer target, unspecific inhibition of the kinase activity has proven inefficient in clinics since the inhibition of Src in non-cancerous cells results in unacceptable toxicity. Thus, there is a need for new target regions in Src that could inhibit Src activity only in certain cell types, e.g., cancer cells, while maintaining the normal physiological activity in healthy cells. The Src N-terminal regulatory element (SNRE) includes the poorly studied intrinsically disordered region with unique sequences for each of the members of the Src family. In this perspective, we discuss the non-canonical regulatory mechanisms involving the SNRE and their potential use as oncotargets.

Comprehensive Analysis of Prognostic Value and Immune Infiltration of Src Family Kinases in Hepatocellular Carcinoma.

Src family kinases (SFKs) belong to the non-receptor protein tyrosine kinase family and are generally dysregulated in a variety of tumors. This study aimed to thoroughly investigate the mutation status, expression level, prognostic value and relationship with immune infiltration of SFKs in hepatocellular carcinoma (HCC). TIMER2.0, UALCAN, cBioPortal, Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan-Meier Plotter were used to analyze the differential expression, genetic alteration, prognostic value and immune cell infiltration of SFKs in HCC patients. Furthermore, we used quantitative real-time PCR (qPCR) and western blot (WB) analysis to measure SFKs mRNA and protein expression in matching specimens of normal tissue and HCC. We analyzed the biological effects of FYN in Huh7 cells and subcutaneous xenograft tumor model. We also studied the biological effects of SRC on Huh7 cells. The mRNA expression levels of LYN, SRC and SRM were elevated in HCC tissues, whereas FYN was reduced. Approximately 10% genetic alterations rate of SFKs was observed in HCC. The mRNA levels of BLK, BRK, FRK, FYN, LCK, LYN, SRC, SRM and YES were correlated with clinical cancer stage. Elevated FYN mRNA levels in HCC were positively correlated with overall survival (OS), whereas SRC was negatively correlated with OS. All SFKs members in HCC were significantly associated with at least half of the six immune-infiltrating cells, including B cells, macrophages, dendritic cells, neutrophils, CD4+ T cells and CD8+ T cells. Furthermore, we confirmed that the protein expression level of FYN was decreased in patients with HCC and in a human hepatoma cell line. Overexpression of FYN suppressed Huh7 cell proliferation, migration, invasion, and tumorigenesis in xenograft nude mice. Knockdown of SRC inhibited Huh7 cell proliferation, migration and invasion. Dysregulated FYN and SRC expression in HCC is associated with poor prognosis and may be used as novel prognostic biomarkers in patients with HCC.

Pan-cancer Analysis Reveals SRC May Link Lipid Metabolism and Macrophages.

SRC is a member of the membrane-associated non-receptor protein tyrosine kinase superfamily. It has been reported to mediate inflammation and cancer. However, the exact molecular mechanism involved is still not clear. The current study was designed to explore the prognostic landscape of SRC and further investigate the relationship between SRC and immune infiltration in pan-cancer. Kaplan-Meier Plotter was used to detect the prognostic value of SRC in pan-cancer. Then using TIMER2.0 and CIBERSORT, the relationship between SRC and immune infiltration in pan-cancer was evaluated. Furthermore, the LinkedOmics database was used to screen SRC co-expressed genes, followed by functional enrichment of SRC co-expressed genes by Metascape online tool. STRING database and Cytoscape software were applied to construct and visualise the protein-protein interaction network of SRC co-expressed genes. MCODE plug-in was used to screen hub modules in the PPI network. The SRC co-expressed genes in hub modules were extracted, and the correlation analysis between interested SRC co-expressed genes and immune infiltration was conducted via TIMER2.0 and CIBERSORT. Our study demonstrated that SRC expression was significantly associated with overall survival and relapse-free survival in multiple cancer types. In addition, SRC expression was significantly correlated with the immune infiltration of B cells, dendritic cells, CD4+ T cells, macrophages, and neutrophils in pan-cancer. The expression of SRC had shown to have close correlations with M1 macrophage polarisation in LIHC, TGCT, THCA, and THYM. Moreover, the genes that co-expressed with SRC in LIHC, TGCT, THCA, and THYM were mainly enriched in lipid metabolism. Besides, correlation analysis showed that SRC co-expressed genes associated with lipid metabolism were also significantly correlated with the infiltration and polarisation of macrophages. These results indicate that SRC can serve as a prognostic biomarker in pan-cancer and is related to macrophages infiltration and interacts with genes involved in lipid metabolism.

FAK/IL-8 axis promotes the proliferation and migration of gastric cancer cells.

Gastric cancer (GC) is one of the most common malignancies in China and is associated with high mortality. The occurrence and development of gastric cancer are related to genetic and environmental factors. Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor protein tyrosine kinase that is activated by the extracellular matrix and growth factors. FAK is highly expressed in cancer and promotes its development by regulating cancer cell proliferation, migration, and angiogenesis. The expression of IL-8 is increased in many types of malignant tumor cells and is linked to their proliferation, migration, invasion, angiogenesis, and EMT. In this study, we found FAK to be essential for the proliferation, migration, and peritoneal metastasis of gastric cancer cells. To examine the molecular regulatory mechanisms of FAK in the peritoneal dissemination of gastric cancer, we performed RNA-seq analysis of MKN-45-FAK-/- and MKN45 cells and demonstrated that IL-8 was downregulated in FAK-deficient cells. Conversely, we confirmed that IL-8 activates FAK activity. We established that IL-8 promotes the proliferation, colony formation, andmigration of gastric cancer cells that are partially mediated by FAK. Thus, we propose that an IL-8-FAK-IL-8 positive feedback loop effects the proliferation and migration of gastric cancer cells.

A FRET-based respirasome assembly screen identifies spleen tyrosine kinase as a target to improve muscle mitochondrial respiration and exercise performance in mice

Aerobic muscle activities predominantly depend on fuel energy supply by mitochondrial respiration, thus, mitochondrial activity enhancement may become a therapeutic intervention for muscle disturbances. The assembly of mitochondrial respiratory complexes into higher-order “supercomplex” structures has been proposed to be an efficient biological process for energy synthesis, although there is controversy in its physiological relevance. We here established Förster resonance energy transfer (FRET) phenomenon-based live imaging of mitochondrial respiratory complexes I and IV interactions using murine myoblastic cells, whose signals represent in vivo supercomplex assembly of complexes I, III, and IV, or respirasomes. The live FRET signals were well correlated with supercomplex assembly observed by blue native polyacrylamide gel electrophoresis (BN-PAGE) and oxygen consumption rates. FRET-based live cell screen defined that the inhibition of spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase that belongs to the SYK/ zeta-chain-associated protein kinase 70 (ZAP-70) family, leads to an increase in supercomplex assembly in murine myoblastic cells. In parallel, SYK inhibition enhanced mitochondrial respiration in the cells. Notably, SYK inhibitor administration enhances exercise performance in mice. Overall, this study proves the feasibility of FRET-based respirasome assembly assay, which recapitulates in vivo mitochondrial respiration activities.

El papel del mentor en la cardiología

The maintenance of sarcoplasmic reticulum Ca2 + ATPase (SERCA2) activity is crucial for cardiac function and SERCA2 is dramatically reduced in the heart exposed to hypoxic/ischemic conditions. Previous work from our group showed that hypoxia upregulates the phosphorylated form of the Ca2 +-dependent nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (pPyk2) protein levels in a low-density lipoprotein receptor-related protein (LRP1)-dependent manner. Pyk2 in turn may modulate SERCA2 in cardiomyocytes although this remains controversial. We therefore aimed to investigate the role of LRP1 on hypoxia-induced SERCA2 depletion in cardiomyocytes and to establish LRP1 signalling mechanisms involved. Western blot analysis showed that hypoxia reduced SERCA2 concomitantly with a sustained increase in LRP1 and pPyk2 protein levels in HL-1 cardiomyocytes. By impairing hypoxia-induced Pyk2 phosphorylation and HIF-1α accumulation, LRP1 deficiency prevented SERCA2 depletion and reduction of the sarcoplasmic reticulum calcium content in cardiomyocytes. Moreover, the inhibition of Pyk2 phosphorylation (with the Src-family inhibitor PP2) or the specific silencing of Pyk2 (with siRNA-anti Pyk2) preserved low HIF-1α and high SERCA2 levels in HL-1 cardiomyocytes exposed to hypoxia. We determined that the LRP1/Pyk2 axis represses SERCA2 mRNA expression via HIF-1α since HIF-1α overexpression abolished the protective effect of LRP1 deficiency on SERCA2 depletion. Our findings show a crucial role of LRP1/Pyk2/HIF-1α in hypoxia-induced cardiomyocyte SERCA2 downregulation, a pathophysiological process closely associated with heart failure.

Design, synthesis, and biological evaluation of diaminopyrimidine derivatives as novel focal adhesion kinase inhibitors.

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase that belongs to the family of focal adhesion complexes and is responsible for the development of various tumors. Herein, 24 diaminopyrimidine derivatives were designed and synthesized based on TAE-226. Several compounds with good activity were further evaluated regarding their antiproliferative activities against two cancer cells with high FAK expression. Compound A12 showed potent anticancer activity against A549 and MDA-MB-231 cell lines with IC50 values of 130 nM and 94 nM, respectively. In vitro metabolic stability and cytochrome P450 (CYP) inhibition assays showed that A12 exhibited favorable stability and weak inhibitory activity on CYP isoforms. Preliminary evaluation of kinase selectivity showed that A12 was a multi-kinase inhibitor. The acute toxicity in vivo indicated that A12 possessed acceptable safety. Compound A12 was also selected for molecular docking studies and the prediction of molecular properties and drug-like properties. These results indicated that compound A12 could be used as a potential lead compound targeting FAK for further development.

Structural Elements of the BCR Determine Permissiveness to Oncogenic RAS-Driven B-Cell Transformation

Background: RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) invariably promote constitutive ERK-activation and are oncogenic drivers in B-cell acute lymphoblastic leukemia (B-ALL; Irving et al., 2014). Conversely, persistent ERK-activation can induce plasma cell differentiation (Yasuda et al., 2011), negative B-cell selection and cell death (Limnander et al., 2011) during B-cell development. In RAS-associated autoimmune leukoproliferative disorder (RALD), RAS-activating germline mutations cause lymphoproliferative disorder but not leukemia. The molecular determinants driving these divergent outcomes of RAS-activation have yet to be characterized. For these reasons, we here examined the mechanisms that enable permissiveness to RAS-driven transformation of B-cells. Results: Studying RAS-pathway activating mutations, we found that these genetic lesions are very common in B-cell malignancies derived from the earliest (e.g. B-ALL) and the latest (e.g. multiple myeloma) stages of B-cell development. In contrast, RAS-pathway lesions are exceedingly rare in B-cell receptor (BCR)-expressing B-cell malignancies, including mantle cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma and diffuse large B-cell lymphoma (Figure 1). We also studied the consequence of oncogenic RAS-activation in mature B-cells, which express BCR. Consistent with previous findings that ERK-activation results in negative B-cell selection and cell death of B-cells (Limnander al., 2011), we found that expression of NRASG12D in mature B-cells resulted in upregulation of Prdm1, a tumor suppressor and mediator of negative B-cell selection and cell death (Hug et al., 2014; Setz et al., 2018). Altogether, our results suggest that mature B-cells are not permissive to RAS-driven transformation and prompted us to investigate whether expression of BCR counteracts constitutive ERK-activation resulting from oncogenic RAS signaling in B-cells. Reflecting incompatibility between oncogenic RAS-ERK activation and BCR-expressing malignant B-cells, expression of NRASG12D in BCR-positive mantle cell lymphoma cells transiently decreased surface expression of BCR but eventually led to depletion of cells from culture in growth competition assays. The non-receptor protein tyrosine kinase SYK is a key mediator of BCR signaling. Notably, treatment with small molecule inhibitor of SYK (entospletinib) largely reversed cell death caused by oncogenic RAS-signaling. Taken together, our results suggest that expression of an active BCR prevents permissiveness to constitutive ERK-activation. BCR consists of µ heavy chain (µHC) and conventional light chains (LCs; either kappa LC or lambda LC). Given that expression of a functional BCR opposes constitutive ERK-activation, this raised the question of whether structural elements of BCR are involved in determining outcome of RAS-activation. To this end, we performed CRISPR/Cas9-based gene editing to disrupt the constant region of immunoglobulin heavy chain (IGHM) or the constant region of the immunoglobulin kappa LC (IGKC) in kappa LC-expressing mantle cell lymphoma cells. While CRISPR-mediated deletion of IGHM reduced surface expression of IgM (μHC), this did not rescue cells from call death caused by oncogenic RAS-ERK activation. On the contrary, genetic ablation of IGKC enabled permissiveness to oncogenic RAS-signaling and prevented cell death resulting from constitutive ERK-activation. Hence, these findings showed that expression of kappa LC opposes constitutive RAS-ERK signaling in B-cell lymphoma cells. Conclusions: Surface expression of BCR is conserved in most mature B-cell lymphomas. Recent studies suggest that constitutive RAS-ERK signaling can override BCR requirement in B-cell lymphoma and that B-cell lymphoma cells acquiring RAS mutations may lose selective pressure to express a functional BCR (Varano et al., 2017). Here, our results demonstrated that conventional LCs of the BCR determine permissiveness to RAS-driven malignant transformation of B-cells. Conventional LCs were previously shown to inhibit the autonomous signaling capacity of BCR (Meixlsperger et al., 2007). We here showed that conventional LCs of BCR counteract oncogenic RAS signaling, providing a mechanistic basis of the low frequency of RAS-pathway activating mutations observed in BCR-expressing B-cell malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Membrane lipid rafts are required for AMPA receptor tyrosine phosphorylation.

Membrane lipid rafts are sphingolipids and cholesterol-enriched membrane microdomains, which form a center for the interaction or assembly of palmitoylated signaling molecules, including Src family non-receptor type protein tyrosine kinases. Lipid rafts abundantly exist in neurons and function in the maintenance of synapses. Excitatory synaptic strength is largely controlled by the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in the mammalian brain. AMPA receptor endocytosis from the synaptic surface is regulated by phosphorylation of the GluA2 subunit at tyrosine 876 by Src family kinases. Here, I revealed that tyrosine phosphorylated GluA2 is concentrated in the lipid rafts fraction. Furthermore, stimulation-induced upregulation of GluA2 tyrosine phosphorylation is disrupted by the treatment of neurons with a cholesterol-depleting compound, filipin III. These results indicate the importance of lipid rafts as enzymatic reactive sites for AMPA receptor tyrosine phosphorylation and subsequent AMPA receptor internalization from the synaptic surface.

The role of Syk phosphorylation in Fc receptor mediated mIgM+ B lymphocyte phagocytosis in flounder (Paralichthys olivaceus)

Spleen tyrosine kinase (Syk) is a non-receptor protein tyrosine kinase, and it mediates downstream signaling of FcR-mediated immune responses. Our previous work revealed that the expression of Syk was significantly up-regulated in flounder mIgM+ B lymphocytes after phagocytosis of antiserum-opsonized Edwardsiella tarda, which suggested Syk might be involved in Ig-opsonized phagocytosis. In this paper, phospho-Syk (pSyk) inhibitor was used to investigate the potential role of phosphorylated Syk in FcR-mediated phagocytosis of IgM+ B cells. Indirect immunofluorescence assay (IFA) and Western blotting showed that the level of phosphorylated Syk in the mIgM+ B lymphocytes treated with pSyk inhibitor was significantly lower compared to the control group after stimulation with flounder antiserum. Flow cytometry analysis showed that after 3 h incubation with antiserum-opsonized E. tarda, the phagocytosis rates of mIgM+ B lymphocytes from peripheral blood, spleen and head kidney pre-treated with pSyk inhibitor were 48.1%, 40.1% and 43.6% respectively, which were significantly lower than that of the control groups with 58.7%, 53.2% and 57.4%, respectively. And likewise, after pSyk inhibitor treatment, the proportions of mIgM+ B lymphocytes with higher intracellular reactive oxygen species (ROS) levels in peripheral blood, spleen and head kidney decreased to 15.2%, 12.0% and 12.1% from the control level of 26.5%, 25.9% and 26.3%, respectively. Moreover, the expression of three genes affected by pSyk, including phospholipase Cγ1 (PLCγ1), phospholipase Cγ2 (PLCγ2) and phosphatidylinositol 3 kinase (PI3K) were found to be significantly down-regulated in pSyk inhibitor-treated mIgM+ B lymphocytes post phagocytosis. These results suggest that pSyk plays a key role in FcR-mediated phagocytosis and bactericidal activity of mIgM+ B lymphocytes, which promotes further understanding of the regulatory role of pSyk in teleost B cells phagocytosis.