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Abstract Research question What is the effect of C-type natriuretic peptide (CNP) on human sperm capacitation in vitro and what is the mechanism of this effect? Design CNP/NPR-B expression in the female rat genital tract was examined by immunohistochemistry and western blot assay, and then the role of CNP in human sperm capacitation was determined. The signal transduction pathway of CNP in the process was determined to elucidate the regulation mechanism of CNP by enzyme-linked immunosorbent assay and flow cytometry. Results Both CNP and NPR-B were expressed in the genital tract of female rats, especially in the mucosa epithelium cell of the oviduct; the CNP level in the rat oviduct was higher than that in the cervix. Both CNP and NPR-B level in the rat oviduct varied during the oestrus cycle, maximal expression being observed at proestrus. Furthermore, intracellular cGMP level in spermatozoa was significantly enhanced by CNP (P < 0.01). PKG activity was detected in the spermatozoa, and it can be activated by the CNP and 8-Br-cGMP (cGMP analogue). The PKG inhibitor KT5823 inhibited the effect of CNP on sperm hyperactivation and the acrosome reaction. Finally, Ca2+ and tyrosine phosphorylation levels in spermatozoa were markedly improved by CNP and 8-Br-cGMP but significantly inhibited by the addition of KT5823 (P < 0.05). Conclusions CNP secreted by the female genital tract might bind to NPR-B on the spermatozoa. It successively stimulated intracellular cGMP/PKG signalling, increased Ca2+ and tyrosine-phosphorylated proteins, promoted hyperactivation and induced the acrosome reaction, which ultimately facilitated sperm capacitation. [ABSTRACT FROM AUTHOR]

Abstract Increased protein tyrosine phosphorylation and the appearance of a phosphorylated protein of 32 kD (p32) are reported among the capacitation-like changes in cryopreserved boar sperm. Egg yolk freezing extenders are composed by two fractions: insoluble granules and soluble plasma, which contains the low density lipoproteins (LDL) proposed as responsible for the egg yolk cryoprotective action. The aim of this work was to analyze the effects of complete egg yolk and its insoluble, soluble and LDL fractions on boar sperm quality and protein tyrosine phosphorylation after the first stage of a standard cryopreservation protocol. Semen samples in Androstar® Plus diluent were centrifuged and resuspended in the different egg yolk extenders. Temperature was decreased from 17 °C to 5 °C and sperm quality, protein tyrosine phosphorylation and protein pattern were analyzed. Results showed that complete egg yolk as well as soluble and LDL egg yolk fractions maintained sperm quality after temperature decrease. Cooling without any lipid component or in the presence of the insoluble fraction, significantly reduced sperm motility. About sperm protein tyrosine phosphorylation analysis, the p32 band appeared before treatments or after cooling in Androstar® Plus diluent. Complete egg yolk and its insoluble fraction interfered with sperm tyrosine phosphorylation even after cells were extensively washed. Analysis of extenders revealed a high amount of tyrosine phosphorylated proteins in the insoluble fraction, which may have co-precipitate with sperm in experiments. Samples submitted to temperature decrease from 17 °C to 5 °C in the presence of soluble and LDL egg yolk fractions in Androstar® Plus diluent did not show any change in the p32 band associated with sperm capacitation. However, a tyrosine-phosphorylated protein of 33 kD present in clarified egg yolk was also observed in sperm treated with this extender. Protein transference from plasma and LDL egg yolk extenders was also observed in sperm protein profile. Results suggested that soluble and LDL fractions might have a protective action preventing sperm protein tyrosine phosphorylation during cooling from 17 °C to 5 °C. Further studies are needed to expand the knowledge of the LDL protection mechanism as well as to determine the possible benefits of clarified egg yolk in freezing protocols. Highlights • Egg yolk interfered with sperm tyrosine phosphorylation analysis even in washed cells. • Soluble and LDL egg yolk fractions in Androstar® plus protected sperm during cooling. • No change in p32 band associated to sperm capacitation took place in these conditions. • A soluble egg yolk tyrosine-phosphorylated protein of 33 kD was transferred to sperm. • Protein transference from soluble and LDL egg yolk to sperm was observed. [ABSTRACT FROM AUTHOR]

Valproic acid (VPA), an antiepileptic drug, has been demonstrated to damage histology and to change tyrosine phosphorylation patterns with increased oxidative stress in perirenal tissues. This study aimed to investigate the effect of VPA on microstructure, tyrosine phosphorylation, and lipid peroxidation of rat kidney. Adult male rats were divided into control and VPA-treated groups intraperitoneally injected with normal saline and VPA 500 mg/kgBW for 10 consecutive days, respectively (n = 7 each). The blood serum was examined for biochemical levels. The kidney tissues were routinely processed for histological observation. Total proteins from kidney were extracted to assay the malondialdehyde (MDA) levels and phosphorylation expression. The results showed that VPA significantly decreased blood glucose levels while tend to increase urea nitrogen and creatinine. MDA levels in VPA group were significantly higher that of control. Renal cortex of VPA-treated animals revealed vasodilatations. Although the ratio of a renal phosphorylated 72 kDa protein/ beta actin expression seemed to be not different in both groups, VPA significantly decreased the intensity of beta actin. In conclusion, VPA dilates renal microvasculature with increasing of MDA but suppresses the actin expression. [ABSTRACT FROM AUTHOR]

Highlights • 6 μM DBP, 3 μM MBP and their mixture affect human sperm motility. • 6 μM DBP, 3 μM MBP and their mixture reduce human sperm penetration ability. • 6 μM DBP, 3 μM MBP and their mixture inhibit human sperm capacitation. • 6 μM DBP, 3 μM MBP and their mixture suppress sperm tyrosine phosphorylation. Abstract Di- n -butyl phthalate (DBP), a plastic-derived, endocrine-disrupting chemical, is regarded as a male reproductive toxicant. In this study, we investigated the in vitro actions of DBP and mono -n- butyl phthalate (MBP, the main metabolite of DBP) on human sperm functions. Human sperm were treated with DBP (2 nM-6 μM), MBP (1 nM-3 μM), and a mixture of DBP and MBP in vitro. The results showed that only DBP at 6 μM, a dose reported in semen of infertile men, MBP at 3 μM (three times of the reported maximum MBP concentration in semen), and their mixture, had obvious adverse effects on sperm motility, penetration ability and capacitation. In addition, these doses of phthalates suppressed human sperm tyrosine phosphorylation, a key signaling pathway that regulates sperm functions. Our findings indicate that DBP and MBP may compromise human sperm functions by inhibiting sperm tyrosine phosphorylation once they accumulate in semen at high levels. [ABSTRACT FROM AUTHOR]

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase- Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited. [ABSTRACT FROM AUTHOR]

Summary: Plants have evolved many receptor‐like cytoplasmic kinases (RLCKs) to modulate their growth, development, and innate immunity. Broad‐Spectrum Resistance 1 (BSR1) encodes a rice RLCK, whose overexpression confers resistance to multiple diseases, including fungal rice blast and bacterial leaf blight. However, the mechanisms underlying resistance remain largely unknown. In the present study, we report that BSR1 is a functional protein kinase that autophosphorylates and transphosphorylates an artificial substrate in vitro. Although BSR1 is classified as a serine/threonine kinase, it was shown to autophosphorylate on tyrosine as well as on serine/threonine residues when expressed in bacteria, demonstrating that it is a dual‐specificity kinase. Protein kinase activity was found to be indispensable for resistance to rice blast and leaf blight in BSR1‐overexpressing plants. Importantly, tyrosine phosphorylation of BSR1 was critical for proper localization of BSR1 in rice cells and played a crucial role in BSR1‐mediated resistance to multiple diseases, as evidenced by compromised disease resistance in transgenic plants overexpressing a mutant BSR1 in which Tyr‐63 was substituted with Ala. Overall, our data indicate that BSR1 is a non‐receptor dual‐specificity kinase and that both tyrosine and serine/threonine kinase activities are critical for the normal functioning of BSR1 in the resistance to multiple pathogens. Our results support the notion that tyrosine phosphorylation plays a major regulatory role in the transduction of defense signals from cell‐surface receptor complexes to downstream signaling components in plants. Significance Statement: BSR1, encoding a rice receptor‐like cytoplasmic kinase, confers resistance to multiple diseases when overexpressed in rice. However, the mechanisms underlying this resistance remain largely elusive. We show that BSR1 is a dual‐specificity kinase that phosphorylates both tyrosine and serine/threonine residues and that tyrosine phosphorylation of BSR1 plays a crucial role in BSR1‐mediated resistance to multiple diseases. [ABSTRACT FROM AUTHOR]

Abstract Alkylating reagent chemotherapy for human cancers is not curative, and relapse occurs due to the continued presence of tumor cells, referred to as minimal residual disease (MRD). The survival of MRD cells after chemotherapy, a phenomenon referred to as intrinsic resistance, depends on reactive oxygen species. Well-differentiated regions of the tumor are intrinsically resistant to chemotherapy. Receptor tyrosine kinase erythropoietin-producing human hepatocellular receptor A4 (EphA4) protein is highly expressed in the well-differentiated tumor-derived cervical cancer cell line Caski, but not in poorly differentiated tumor-derived cervical cancer cell lines such as HeLa or SiHa. Here, we report that reactive oxygen species produced by cisplatin exposure induce tyrosine phosphorylation of EphA4. After observing that EphA4 is activated by cisplatin, we rationalized a combination chemotherapy that induces well-differentiated cervical cancer death. Pharmacological inhibition of EphA4 increased cisplatin-induced cell death in Caski cells. Moreover, we observed increased expression levels of the senescence marker cyclin-dependent kinase inhibitor 2A (p16) in the absence of EphA4 kinase function after stimulation of Caski cells with cisplatin exposure. Mechanistically, cisplatin induces chemotherapy resistance of Caski cells by upregulating Lyn, a Src family kinase (SFK) that interacts with EphA4, through a pathway involving reactive oxygen species. Thus, the reactive oxygen species–SFK–EphA4 axis presents new potential drug targets for chemotherapy resistance. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Resveratrol upregulates the expression of TJ proteins. • Resveratrol can promote TJ barrier integrity in intestinal Caco-2 cells. • Resveratrol protects against IL-6- and H 2 O 2 -induced barrier disruption. • Resveratrol supplements may be used to protect the intestinal TJ barrier in humans. Abstract It has been shown that some polyphenols participate in barrier regulation. Here, we investigated the effects of resveratrol on Caco-2 cell monolayers, with a focus on the modulation of tight junction (TJ) structure. The effects of resveratrol on barrier impairment in the cells following treatment with H 2 O 2 and interleukin (IL)-6 were investigated. Measurement of transepithelial electrical resistance showed that resveratrol enhances TJ barrier integrity. Immunoblot and immunofluorescence analyses revealed that resveratrol upregulates the expression of TJ proteins and their distribution in the cytoskeletal fraction. Furthermore, resveratrol ameliorated H 2 O 2 -induced epithelial barrier disruption in the Caco-2 cell model of oxidative-stress-induced barrier impairment by inhibiting tyrosine phosphorylation of occludin. It was found that resveratrol mitigated IL-6-induced hyperpermeability in the inflamed cell model by downregulating claudin-2 expression. The results show that resveratrol enhances TJ barrier integrity and mediates TJ barrier protection against damage induced by oxidative stress and pro-inflammatory cytokines in Caco-2 cells. [ABSTRACT FROM AUTHOR]

Serine/arginine‐rich splicing factor 1 (SRSF1) has been linked to various human cancers including pediatric acute lymphoblastic leukemia (ALL). Our previous study has shown that SRSF1 potentially contributes to leukemogenesis; however, its underlying mechanism remains unclear. In this study, leukemic cells were isolated from pediatric ALL bone marrow samples, followed by immunoprecipitation assays and mass spectrometry analysis specific to SRSF1. Subcellular localization of the SRSF1 protein and its mutants were analyzed by immunofluorescence staining. Cell growth, colony formation, cell apoptosis, and the cell cycle were investigated using stable leukemic cell lines generated with lentivirus‐mediated overexpressed WT or mutant plasmids. Cytotoxicity of the Tie2 kinase inhibitor was also evaluated. Our results showed the phosphorylation of SRSF1 at tyrosine 19 (Tyr‐19) was identified in newly diagnosed ALL samples, but not in complete remission or normal control samples. Compared to the SRSF1 WT cells, the missense mutants of the Tyr‐19 phosphorylation affected the subcellular localization of SRSF1. In addition, the Tyr‐19 phosphorylation of SRSF1 also led to increased cell proliferation and enhanced colony‐forming properties by promoting the cell cycle. Remarkably, we further identified the kinase Tie2 as a potential therapeutic target in leukemia cells. In conclusion, we identify for the first time that the phosphorylation state of SRSF1 is linked to different phases in pediatric ALL. The Tyr‐19 phosphorylation of SRSF1 disrupts its subcellular localization and promotes proliferation in leukemia cells by driving cell‐cycle progression. Inhibitors targeting Tie2 kinase that could catalyze Tyr‐19 phosphorylation of SRSF1 offer a promising therapeutic target for treatment of pediatric ALL. Our results underscore the phosphorylation of serine/arginine‐rich splicing factor 1 (SRSF1) at the tyrosine 19 (Tyr‐19) residue disrupts subcellular localization of SRSF1 and promotes cell proliferation in leukemic cells by accelerating cell‐cycle progression. This study undoubtedly adds a new layer in understanding of how the posttranslational mechanism supports leukemia progression. Inhibitors targeting Tie2 kinase that could catalyze Tyr‐19 phosphorylation of SRSF1 offer a promising therapeutic target for the treatment of pediatric ALL. [ABSTRACT FROM AUTHOR]

Stress affects the male reproductive system and can cause sub-fertility or infertility. Although Phyllanthus emblica L. (PE) extract has been shown to have high antioxidant capacity and protective properties in damaged tissue, the preventive effects of PE extract on testicular function from stress-related impairment have never been demonstrated. This study aimed to investigate the effects of PE aqueous leaf extract on testicular impairment and protein marker changes in rats suffering from chronic stress. Adult male rats were divided into four groups: a control group, a chronic stress (CS) group, and two groups with CS that received different doses of PE extract (50 or 100 mg/kg body weight (BW)). In the treatment groups, the animals were given PE extract daily before stress induction for 42 consecutive days. Stress was induced through immobilization (4 h/d) followed by forced cold swimming (15 min/d). Sperm quality and the histology of the testes and caudal epididymis were examined, as were levels of serum corticosterone, testosterone, and malondialdehyde (MDA). The expressions of testicular steroidogenic acute regulatory (StAR) and tyrosine-phosphorylated proteins were investigated using immuno-Western blot analysis, as these proteins are assumed to play important roles in spermatogenesis and androgen synthesis. The results showed that PE (50 mg/kg BW) significantly increased sperm concentration and testosterone levels, while decreasing corticosterone levels, MDA levels, sperm head abnormalities, and acrosome-reacted sperm in CS rats. In addition, PE at both doses was found to diminish testicular histopathology in the CS rats. We also found that 50 mg/kg BW of PE significantly improved StAR protein expression and altered the intensities of some tyrosine-phosphorylated proteins in testis. We conclude that PE leaf extract at 50 mg/kg BW can prevent testicular damage in rats with CS. [ABSTRACT FROM AUTHOR]

Peroxiredoxin-2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells. Prx2 belongs to a well-known family of antioxidants, the peroxiredoxins (Prxs), that are widely expressed in mammalian cells. Prx2 is a typical, homodimeric, 2-Cys Prx that uses two cysteine residues to accomplish the task of detoxifying a vast range of organic peroxides, H2O2, and peroxynitrite. Although progress has been made on functional characterization of Prx2, much still remains to be investigated on Prx2 post-translational changes. Here, we first show that Prx2 is Tyrosine (Tyr) phosphorylated by Syk in red cells exposed to oxidation induced by diamide. We identified Tyr-193 in both recombinant Prx2 and native Prx2 from red cells as a specific target of Syk. Bioinformatic analysis suggests that phosphorylation of Tyr-193 allows Prx2 conformational change that is more favorable for its peroxidase activity. Indeed, Syk-induced Tyr phosphorylation of Prx2 enhances in vitro Prx2 activity, but also contributes to Prx2 translocation to the membrane of red cells exposed to diamide. The biologic importance of Tyr-193 phospho-Prx2 is further supported by data on red cells from a mouse model of humanized sickle cell disease (SCD). SCD is globally distributed, hereditary red cell disorder, characterized by severe red cell oxidation due to the pathologic sickle hemoglobin. SCD red cells show Tyr-phosphorylated Prx2 bound to the membrane and increased Prx2 activity when compared to healthy erythrocytes. Collectively, our data highlight the novel link between redox related signaling and Prx2 function in normal and diseased red cells.

Vav1 works both as a catalytic Rho GTPase activator and an adaptor molecule. These functions, which are critical for T cell development and antigenic responses, are tyrosine phosphorylation-dependent. However, it is not known whether other posttranslational modifications can contribute to the regulation of the biological activity of this protein. Here, we show that Vav1 becomes acetylated on lysine residues in a stimulation- and SH2 domain-dependent manner. Using a collection of both acetylation- and deacetylation-mimicking mutants, we show that the acetylation of four lysine residues (Lys222, Lys252, Lys587, and Lys716) leads to the downmodulation of the adaptor function of Vav1 that triggers the stimulation of the nuclear factor of activated T cells (NFAT). These sites belong to two functional subclasses according to mechanistic criteria. We have also unveiled additional acetylation sites potentially involved in either the stimulation (Lys782) or the downmodulation (Lys335, Lys374) of specific Vav1-dependent downstream responses. Collectively, these results indicate that Nε-lysine acetylation can play variegated roles in the regulation of Vav1 signaling. Unlike the case of the tyrosine phosphorylation step, this new regulatory layer is not conserved in other Vav family paralogs.

ABSTRACT Enteric pathogens with low infectious doses rely on the ability to orchestrate the expression of virulence and metabolism-associated genes in response to environmental cues for successful infection. Accordingly, the human pathogen enterohemorrhagic Escherichia coli (EHEC) employs a complex multifaceted regulatory network to link the expression of type III secretion system (T3SS) components to nutrient availability. While phosphorylation of histidine and aspartate residues on two-component system response regulators is recognized as an integral part of bacterial signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of E. coli identified 342 phosphorylated proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. The present study demonstrates that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression under glycolytic conditions that are typical for the colonic lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating the expression of ler, which encodes the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA binding to fine-tune the expression of virulence-associated genes, including those of the locus of enterocyte effacement pathogenicity island that encode the T3SS, and thereby negatively affects the formation of attaching and effacing lesions. Our data indicate that tyrosine phosphorylation provides an additional mechanism to control the DNA binding of Cra and other LacI/GalR family regulators, including LacI and PurR. This study describes an initial effort to unravel the role of global phosphotyrosine signaling in the control of EHEC virulence potential. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) causes outbreaks of hemorrhagic colitis and the potentially fatal hemolytic-uremic syndrome. Successful host colonization by EHEC relies on the ability to coordinate the expression of virulence factors in response to environmental cues. A complex network that integrates environmental signals at multiple regulatory levels tightly controls virulence gene expression. We demonstrate that EHEC utilizes a previously uncharacterized phosphotyrosine signaling pathway through Cra to fine-tune the expression of virulence-associated genes to effectively control T3SS production. This study demonstrates that tyrosine phosphorylation negatively affects the DNA-binding capacity of Cra, which affects the expression of genes related to virulence and metabolism. We demonstrate for the first time that phosphotyrosine-mediated control affects global transcription in EHEC. Our data provide insight into a hitherto unexplored regulatory level of the global network controlling EHEC virulence gene expression.

Neurodegenerative diseases, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis, are chronic disorders of the CNS that are characterized by progressive neuronal dysfunction. These diseases have diverse clinical and pathological features and their pathogenetic mechanisms are not yet fully understood. Currently, widely accepted hypotheses include the accumulation of misfolded proteins, oxidative stress from reactive oxygen species, mitochondrial dysfunction, DNA damage, neurotrophin dysfunction, and neuroinflammatory processes. In the CNS of patients with neurodegenerative diseases, a variety of abnormally phosphorylated proteins play important roles in pathological processes such as neuroinflammation and intracellular accumulation of β-amyloid plaques and tau. In recent years, the roles of abnormal tyrosine phosphorylation of intracellular signaling molecules regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) in neurodegenerative diseases have attracted increasing attention. Here, we summarize the roles of signaling pathways related to protein tyrosine phosphorylation in the pathogenesis of neurodegenerative diseases and the progress of therapeutic studies targeting PTKs and PTPs that provide theoretical support for future studies on therapeutic strategies for these devastating and important neurodegenerative diseases., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Association of Neuropathologists, Inc. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Tyrosine phosphorylation, as a hallmark in cellular signal transduction, is important for a diverse array of cellular processes, such as proliferation, metabolism, motility, and survival. Aberrant tyrosine phosphorylation plays a causal role in many diseases, especially the cancer. Detecting protein phosphorylation status in the cancer cells or tissues is vital for assessing the pathological phase, discovering the cancer biomarkers, and identifying the drug targets. However, the common biochemical detection methods remain through anti-pTyr antibodies, which are known to have limited sensitivity, poor reproducibility and high cost. Recent studies have proved that superbinder SH2 domain is a good replacement of anti-pTyr antibodies for the specific enrichment of pTyr peptides in phosphoproteomics analysis. In this work, we exploited a series of affinity reagents based on superbinder SH2 derived from Src protein for detecting the pTyr-containing proteins to replace anti-pY antibodies in immunoblotting and immunofluorescence techniques. The excellent performance of HRP-sSH2 and EGFP-sSH2 was verified by the analysis of several different tumor cell samples and was compared with most commonly used commercial antibodies. EGFP-sSH2-(Arg) 9 might be applied as the probe for direct fluorescence imaging in live cells via efficiently penetrating cell membranes and specifically binding with pTyr proteins. In summary, we have developed three novel, convenient, sensitive, and cost-effective affinity reagents that would have wide applications in protein tyrosine phosphorylation analysis for the tumor research and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Highlights • Hepatic ischemia reperfusion leads to hippocampus injury in young mice. • Hepatic ischemia reperfusion leads to long-term cognitive impairment. • Hepatic ischemia reperfusion enhances tyrosine phosphorylation of Src and NR2A. • The hippocampus injury is associated with Src-PSD95-NR2A pathway. Abstract Hepatic ischemia reperfusion (HIR) has been found to induce hippocampus injury and cognitive dysfunction. The N-methyl- d -aspartate (NMDA) receptor subunit 2A (NR2A) is an important factor mediating excitotoxicity and neurons injury, and autophosphorylation of Src can up-regulate tyrosine phosphorylation of NR2A to improve its activity. However, the role of Src and NR2A in HIR-induced hippocampus injury in young mice remains unknown. In this study, we found that serum biomarkers of brain injury (S100β and NSE) increased significantly and reached highest after reperfusion of 3 days which had the same trend with the levels of p-Src and p-NR2A. Interactions between Src and NR2A or PSD95 were increased after HIR. Hippocampal neuron apoptosis was increased, and long-term cognitive impairment was found after reperfusion of 1 month. Inhibition of Src and NR2A with PP2 and NVP-AAM077 respectively not only down-regulated the levels of p-Src and p-NR2A, but also ameliorated hippocampal neurons apoptosis and long-term cognitive impairment after HIR. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ) and interleukin (IL)-6 were increased after reperfusion of 3 days, while PP2 and NVP-AAM077 treatment didn't attenuate the changes. And no difference was found in serum TNF-α, IFN-γ, IL-6 concentrations as well as the levels of Src, p-Src, NR2A, p-NR2A, PSD95 among the four groups after reperfusion of 1 month. In summary, HIR can lead to hippocampus injury and long-term cognitive dysfunction, and Src-PSD95-NR2A pathway plays an important role in the process. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor receptor 2 (FGFR2) was demonstrated to correlate to the progression and prognosis of intrahepatic cholangiocarcinoma (ICC) by numerous evidences. However, as a well‐recognized suppressor of FGFR2 signalling, the clinical significance of Sprouty (SPRY) family of ICC has not been investigated. In our study, the expressions of SPRY1‐4 in 20 pairs of fresh tumour tissues were detected with qPCR, and in 108 cases of paraffin‐embedded tissues with immunohistochemistry. The prognostic value of SPRY family in ICC was estimated with univariate analysis and multivariate analysis. As a result, SPRY2 was identified as an independent prognostic biomarker predicting favourable prognosis of ICC. High SPRY2 expression was correlated with good differentiation of ICC. With silencing SPRY2 expression, we demonstrated that SPRY2 could suppress FGFR2‐induced ERK phosphorylation, migration, invasion and epithelial‐mesenchymal transition (EMT) under FGF1 stimulation. By overexpressing SPRY2‐wide type or SPRY2‐Y55F, the tyrosine‐55 of SPRY2 was demonstrated to be essential in suppressing ERK phosphorylation, tumour invasion and EMT of ICC cells. In conclusion, SPRY2 was correlated with favourable prognosis of ICC via suppressing FGFR2‐induced ERK phosphorylation, invasion and EMT. The phosphorylation of SPRY2‐Y55 was required in this tumour‐suppressing function of SPRY2. [ABSTRACT FROM AUTHOR]

Abstract SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) binds with the Y1356-phosphorylated hepatocyte growth factor (HGF) receptor, c-MET, through its SH2 domain, which is essential for the role of SHIP2 in HGF-induced cell scattering and cell spreading. Previously, the experimental structure of the SH2 domain from SHIP2 (SHIP2-SH2) had not been reported, and its interaction with the Y1356-phosphorylated c-MET had not been investigated from a structural point of view. In this study, the solution structure of SHIP2-SH2 was determined by NMR spectroscopy, where it was found to adopt a typical SH2-domain fold that contains a positively-charged pocket for binding to phosphotyrosine (pY). The interaction between SHIP2-SH2 and a pY-containing peptide from c-MET (Y1356 phosphorylated) was investigated through NMR titrations. The results showed that the binding affinity of SHIP2-SH2 with the phosphopeptide is at low micromolar level, and the binding interface consists of the positively-charged pocket and its surrounding regions. Furthermore, R28, S49 and R70 were identified as key residues for the binding and may directly interact with the pY. Taken together, these findings provide structural insights into the binding of SHIP2-SH2 with the Y1356-phosphorylated c-MET, and lay a foundation for further studies of the interactions between SHIP2-SH2 and its various binding partners. Highlights • SHIP2-SH2 consists of two α-helices and six β-strands adopting a typical SH2 fold. • SHIP2-SH2 surface contains a positive-charged pocket for binding phosphotyrosine. • The pocket and its surrounding regions are for binding Y1356-phosphorylated c-MET. • The SHIP2-SH2 affinity for Y1356-phosphorylated c-MET is at low micromolar level. • R28, S49 and R70 of SHIP2-SH2 are crucial for binding Y1356-phosphorylated c-MET. [ABSTRACT FROM AUTHOR]

Triple negative breast cancer (TNBC) patients cannot benefit from EGFR-targeted therapy even though the EGFR is highly expressed, because patients exhibit resistance to these drugs. Unfortunately, the molecular mechanisms remain relatively unknown. ANXA2, highly expressed in invasive breast cancer cells, is closely related with poor prognosis, and acts as a molecular switch to EGFR activation. In this study, MDA-MB-231 cells and MCF7 cells were used. Our results showed that ANXA2 expression is inversely correlated with cell sensitivity to gefitinib. Knockdown of ANXA2 expression in MDA-MB-231 cells increased the gefitinib induced cell death. When ANXA2 was overexpressed in MCF7 cells, the gefitinib induced cell death was decreased. Furthermore, we demonstrated that phosphorylation of ANXA2 at Tyr23 is negatively correlated with the sensitivity of TNBC to gefitinib. Altogether, our results suggest a new role of ANXA2 in regulating sensitivity of TNBC MDA-MB-231 cells to the EGFR inhibitor gefitinib. [ABSTRACT FROM AUTHOR]

Transient receptor-potential, cation channel, subfamily M, member 4 (TRPM4) channels regulate a variety of physiological and pathological processes; however, their roles as functional channels under diverse conditions remain unclear. In this study, cytosolic protein tyrosine phosphatase non-receptor type 6 (PTPN6) interacted with TRPM4 channels. We confirmed their interaction by performing co-immunoprecipitation (Co-IP) assays following heterologous PTPN6 and TRPM4 channel expression in HEK293 cells. Furthermore, biomolecular fluorescence complementation (BiFC) image analysis confirmed TRPM4-PTPN6 binding. In addition, immunoblotting and Co-IP analyses revealed that TRPM4 expression significantly decreased in the membrane fraction of cells after PTPN6 was silenced with a specific short-hairpin RNA (shRNA-PTPN6). In agreement, TRPM4-induced changes in whole-cell currents were not detected in PTPN6-silenced HEK cells, in contrast to cells transfected with a scrambled RNA (scRNA) or in naïve HEK cells. These data suggest that PTPN6 inhibits TRPM4 channel activity by disrupting TRPM4 expression. Furthermore, TRPM4 channels were expressed in the membrane of naïve cells and scRNA transfectants, but not in those of PTPN6-silenced cells. These results indicated that PTPN6 is critically associated with TRPM4 trafficking. This role of PTPN6 in TRPM4 membrane localization was also demonstrated in HeLa cells. TRPM4 overexpression significantly enhanced cell proliferation in untreated HeLa cells, but not in HeLa cells with silenced PTPN6 expression. These findings indicate that PTPN6-dependent TRPM4 expression and trafficking to the plasma membrane is critical for cell proliferation in both HEK293 and HeLa cells. Therefore, PTPN6 is a novel therapeutic target for treating pathologic diseases involving TRPM4. [ABSTRACT FROM AUTHOR]

Highlights • TRIM25 undergoes tyrosine phosphorylation on Y278 residue upon viral infection. • c-Src interacts with TRIM25 and induces tyrosine phosphorylation. • Y278 phosphorylation is required for efficient RIG-I signaling. • Y278 phosphorylation facilitates TRIM25-mediated RIG-I ubiquitination. Abstract Retinoic acid-inducible gene I (RIG-I) detects viral RNAs and induces antiviral responses. During viral RNA recognition by RIG-I, tripartite motif protein 25 (TRIM25) plays a critical regulatory role by inducing K63-linked RIG-I polyubiquitination. Previous proteomics analysis revealed several phosphorylation sites on TRIM25, including tyrosine 278 (Y278), yet the roles of these modifications remain elusive. Here, we demonstrated that TRIM25 interacted with c-Src and underwent tyrosine phosphorylation by c-Src kinase upon viral infection and the phosphorylation is required for the complete activation of RIG-I signaling. Analysis using a c-Src inhibitor and TRIM25 mutant, in which tyrosine 278 is substituted by phenylalanine (Y278F), suggested that the phosphorylation positively regulates K63-linked polyubiquitination of RIG-I and subsequent antiviral signaling. The TRIM25 Y278F mutant displayed decreased E3-ubiquitin ligase activity in vitro , suggesting that this phosphorylation event affects the E3-ligase activity of TRIM25. Thus, we provide a molecular mechanism of c-Src-mediated positive regulation of RIG-I signaling. [ABSTRACT FROM AUTHOR]

Abstract Caspase-8 is involved in a number of cellular functions, with the most well established being the control of cell death. Yet caspase-8 is unique among the caspases in that it acts as an environmental sensor, transducing a range of signals to cells, modulating responses that extend far beyond simple survival. Ranging from the control of apoptosis and necroptosis and gene regulation to cell adhesion and migration, caspase-8 uses proteolytic and non-proteolytic functions to alter cell behavior. Novel interacting partners provide mechanisms for caspase-8 to position itself at signaling nodes that affect a variety of signaling pathways. Here, we examine the catalytic and noncatalytic modes of action by which caspase-8 influences cell adhesion and migration. The mechanisms vary from post-cleavage remodeling of the cytoskeleton to signaling elements that control focal adhesion turnover. This is facilitated by caspase-8 interaction with a host of cell proteins ranging from the proteases caspase-3 and calpain-2 to adaptor proteins such as p85 and Crk, to the Src family of tyrosine kinases. [ABSTRACT FROM AUTHOR]

Abstract: Aims: The Na,K‐ATPase is involved in a large number of regulatory activities including cSrc‐dependent signalling. Upon inhibition of the Na,K‐ATPase with ouabain, cSrc activation is shown to occur in many cell types. This study tests the hypothesis that acute potentiation of agonist‐induced contraction by ouabain is mediated through Na,K‐ATPase‐cSrc signalling‐dependent sensitization of vascular smooth muscle cells to Ca2+. Methods: Agonist‐induced rat mesenteric small artery contraction was examined in vitro under isometric conditions and in vivo in anaesthetized rats. Arterial wall tension and [Ca2+]i in vascular smooth muscle cells were measured simultaneously. Changes in cSrc and myosin phosphatase targeting protein 1 (MYPT1) phosphorylation were analysed by Western blot. Protein expression was examined with immunohistochemistry. The α1 and α2 isoforms of the Na,K‐ATPase were transiently downregulated by siRNA transfection in vivo. Results: Ten micromolar ouabain, but not digoxin, potentiated contraction to noradrenaline. This effect was not endothelium‐dependent. Ouabain sensitized smooth muscle cells to Ca2+, and this was associated with increased phosphorylation of cSrc and MYPT1. Inhibition of tyrosine kinase by genistein, PP2 or pNaKtide abolished the potentiating effect of ouabain on arterial contraction and Ca2+ sensitization. Downregulation of the Na,K‐ATPase α2 isoform made arterial contraction insensitive to ouabain and tyrosine kinase inhibition. Conclusion: Data suggest that micromolar ouabain potentiates agonist‐induced contraction of rat mesenteric small artery via Na,K‐ATPase‐dependent cSrc activation, which increases Ca2+ sensitization of vascular smooth muscle cells by MYPT1 phosphorylation. This mechanism may be critical for acute control of vascular tone. [ABSTRACT FROM AUTHOR]

Summary Hyperactivating mutations in the non-receptor tyrosine phosphatase SHP2 cause Noonan syndrome (NS). NS is associated with cognitive deficits, but how hyperactivation of SHP2 in NS changes neuron function is not well understood. We find that mice bearing an NS-associated SHP2 allele (NS mice) have selectively impaired Schaffer collateral-CA1 NMDA (N-methyl-D-aspartate) receptor (NMDAR)-mediated neurotransmission and that residual NMDAR-mediated currents decay faster in NS mice because of reduced contribution of GluN1:GluN2B diheteromers. Consistent with altered GluN2B function, we identify GluN2B Y1252 as an NS-associated SHP2 substrate both in vitro and in vivo . Mutation of Y1252 does not alter recombinant GluN1:GluN2B receptor kinetics. Instead, phospho-Y1252 binds the actin-regulatory adaptor protein Nck2, and this interaction is required for proper NMDAR function. These results establish SHP2 and Nck2 as NMDAR regulatory proteins and strongly suggest that NMDAR dysfunction contributes to NS cognitive deficits. [ABSTRACT FROM AUTHOR]

Spermatids are haploid differentiating cells that, in the meantime they differentiate, translocate along the seminiferous epithelium towards the tubule lumen to be just released as spermatozoa. The success of such a migration depends on dynamic of spermatid-Sertoli cell contacts, the molecular nature of which has not been well defined yet. It was demonstrated that the vascular endothelial cadherin (VEC) is expressed transitorily in the mouse seminiferous epithelium. Here, we evaluated the pattern of VEC expression by immunohistochemistry first in seminiferous tubules at different stages of the epithelial cycle when only unique types of germ cell associations are present. Changes in the pattern of VEC localization according to the step of spermatid differentiation were analysed in detail using testis fragments and spontaneously released germ cells. Utilizing the first wave of spermatogenesis as an in vivo model to have at disposal spermatids at progressive steps of differentiation, we checked for level of looser VEC association with the membrane by performing protein solubilisation under mild detergent conditions and assays through VEC-immunoblotting. Being changes in VEC solubilisation paralleled in changes in phosphotyrosine (pY) content, we evaluated if spermatid VEC undergoes Y658 phosphorylation and if this correlates with VEC solubilisation and spermatid progression in differentiation. Altogether, our study shows a temporally restricted pattern of VEC expression that culminates with the presence of round spermatids to progressively decrease starting from spermatid elongation. Conversely, pY658-VEC signs elongating spermatids; its intracellular polarized compartmentalization suggests a possible involvement of pY658-VEC in the acquisition of spermatid cell polarity. [ABSTRACT FROM AUTHOR]

IL-7 is required for T cell differentiation and mature T cell homeostasis and promotes pro-B cell proliferation and survival. Tyrosine phosphorylation plays a central role in IL-7 signaling. We identified by two-dimensional electrophoresis followed by anti-phosphotyrosine immunoblotting and mass spectrometry sixteen tyrosine phosphorylated proteins from the IL-7-dependent cell line D1. IL-7 stimulation induced the phosphorylation of the proteins STI1, ATIC and hnRNPH, involved in pathways related to survival, proliferation and gene expression, respectively, and increased the phosphorylation of CrkL, a member of a family of adaptors including the highly homologous Crk isoforms CrkII and CrkI, important in multiple signaling pathways. We observed an increased phosphorylation of CrkL in murine pro-B cells and in murine and human T cells. In addition, IL-7 increased the association of CrkL with the transcription factor Stat5, essential for IL-7 pro-survival activity. The selective tyrosine kinase inhibitor Imatinib. counteracted the IL-7 pro-survival effect in D1 cells and decreased CrkL phosphorylation. These data suggested that CrkL could play a pro-survival role in IL-7-mediated signaling. We observed that pro-B cells also expressed, in addition to CrkL, the Crk isoforms CrkII and CrkI and therefore utilized pro-B cells conditionally deficient in all three to evaluate the role of these proteins. The observation that the IL-7 pro-survival effect was reduced in Crk/CrkL conditionally-deficient pro-B cells further pointed to a pro-survival role of these adaptors. To further evaluate the role of these proteins, gene expression studies were performed in Crk/CrkL conditionally-deficient pro-B cells. IL-7 decreased the transcription of the receptor LAIR1, which inhibits B cell proliferation, in a Crk/CrkL-dependent manner, suggesting that the Crk family of proteins may promote pro-B cell proliferation. Our data contribute to the understanding of IL-7 signaling and suggest the involvement of Crk family proteins in pathways promoting survival and proliferation. [ABSTRACT FROM AUTHOR]

Abstract: The brief lifespan of the polymorphonuclear neutrophil (PMN) is regulated through its capacity to undergo apoptosis, a constitutive process that is actively inhibited during sepsis. We sought to define the cellular mechanisms through which Heat Shock Protein 90 (Hsp90) prolongs the survival of inflammatory PMN. We evaluated Hsp90 expression and interaction with client proteins in PMNs from patients with sepsis and in healthy control PMNs treated with LPS (1 μg/mL). Hsp90 activity was inhibited pharmacologically using radicicol (Rad; 1 μM), and Hsp90 transcription was silenced in septic PMN using siRNA. PMN apoptosis was evaluated by flow cytometry and expression of cleaved caspase‐8 and ‐3. Septic PMNs showed reduced rates of apoptosis compared with control PMNs 21 h after isolation, and Hsp90‐α mRNA was significantly more abundant in septic PMN. Caspase‐8 coimmunoprecipitated with Hsp90, c‐Src, and the p85 inhibitory subunit of PI3K in both septic and LPS‐treated PMN. Inhibition of Hsp90 activity with Rad or its translation using siRNA restored basal rates of apoptosis in both septic and LPS‐treated PMN. Radicicol further reduced c‐Src protein abundance, increased the ubiquitination of caspase‐8 and c‐Src, and enhanced the cleavage of caspase‐8 and ‐3. We conclude that Hsp90 prolongs the survival of activated neutrophils by stabilizing a molecular complex of c‐Src kinase and caspase‐8, preventing their ubiquitination, and resulting in inhibition of the catalytic activity of caspase‐8 and ‐3. [ABSTRACT FROM AUTHOR]

T cells possess an array of functional capabilities important for host defense against pathogens and tumors. T cell effector functions require the T cell antigen receptor (TCR). The TCR has no intrinsic enzymatic activity, and thus signal transduction from the receptor relies on additional signaling molecules. One such molecule is the cytoplasmic tyrosine kinase ZAP-70, which associates with the TCR complex and is required for initiating the canonical biochemical signal pathways downstream of the TCR. In this article, we describe recent structure-based insights into the regulation and substrate specificity of ZAP-70, and then we review novel methods for determining the role of ZAP-70 catalytic activity-dependent and -independent signals in developing and mature T cells. Lastly, we discuss the disease states in mouse models and humans, which range from immunodeficiency to autoimmunity, that are caused by mutations in ZAP-70. [ABSTRACT FROM AUTHOR]