Increased Tyrosine Phosphorylation Research Articles

Csk controls leukocyte extravasation via local regulation of Src family kinases and cortactin signaling.

C-terminal Src kinase (Csk) targets Src family kinases (SFKs) and thereby inactivates them. We have previously shown that Csk binds to phosphorylated tyrosine 685 of VE-cadherin, an adhesion molecule of major importance for the regulation of endothelial junctions. This tyrosine residue is an SFK target, and its mutation (VE-cadherin-Y685F) inhibits the induction of vascular permeability in various inflammation models. Nevertheless, surprisingly, it increases leukocyte extravasation. Here, we investigated whether endothelial Csk is involved in these effects. We found that the deficiency of Csk in endothelial cells augments SFK activation and the phosphorylation of VE-cadherin-Y685 but had no net effect on vascular leak formation. In contrast, the lack of endothelial Csk enhanced leukocyte adhesion and transmigration in vitro and in vivo. Furthermore, the silencing of Csk increased tyrosine phosphorylation of the SFK substrate cortactin. Importantly, the effects of Csk silencing on the increase in SFK activation, cortactin phosphorylation, and neutrophil diapedesis were all dependent on Y685 of VE-cadherin. Deletion of cortactin, in turn, erased the supporting effect of Csk silencing on leukocyte transmigration. We have previously shown that leukocyte transmigration is regulated by endothelial cortactin in an ICAM-1-dependent manner. In line with this, blocking of ICAM-1 erased the supporting effect of Csk silencing on leukocyte transmigration. Collectively, our results establish a negative feedback loop that depends on the phosphorylation of VE-cadherin-Y685, which recruits Csk, which in turn dampens the activation of SFK and cortactin and thereby the clustering of ICAM-1 and the extravasation of neutrophils.

Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences

BackgroundExposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 μg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h.ResultsLead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria.ConclusionsPb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.Graphical

Effect of exogenous sperm capacitation inducers on stallion sperm

Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse.

Ritonavir Has Reproductive Toxicity Depending on Disrupting PI3K/PDK1/AKT Signaling Pathway.

Ritonavir (RTV) is an antiviral and a component of COVID-19 treatments. Moreover, RTV demonstrates anti-cancer effects by suppressing AKT. However, RTV has cytotoxicity and suppresses sperm functions by altering AKT activity. Although abnormal AKT activity is known for causing detrimental effects on sperm functions, how RTV alters AKT signaling in spermatozoa remains unknown. Therefore, this study aimed to investigate reproductive toxicity of RTV in spermatozoa through phosphoinositide 3-kinase/phosphoinositide-dependent protein kinase-1/protein kinase B (PI3K/PDK1/AKT) signaling. Duroc spermatozoa were treated with various concentrations of RTV, and capacitation was induced. Sperm functions (sperm motility, motion kinematics, capacitation status, and cell viability) and expression levels of tyrosine-phosphorylated proteins and PI3K/PDK1/AKT pathway-related proteins were evaluated. In the results, RTV significantly suppressed sperm motility, motion kinematics, capacitation, acrosome reactions, and cell viability. Additionally, RTV significantly increased levels of phospho-tyrosine proteins and PI3K/PDK1/AKT pathway-related proteins except for AKT and PI3K. The expression level of AKT was not significantly altered and that of PI3K was significantly decreased. These results suggest RTV may suppress sperm functions by induced alterations of PI3K/PDK1/AKT pathway through abnormally increased tyrosine phosphorylation. Therefore, we suggest people who use or prescribe RTV need to consider its male reproductive toxicity.

Betaine for the prevention and treatment of insulin resistance and fatty liver in a high-fat dietary model of insulin resistance in C57BL mice.

The aim was to investigate mechanisms by which betaine improves hepatic insulin signaling in a dietary mouse model of insulin resistance and fatty liver. C57BL 6J mice were fed a standard diet (SF), a standard diet with betaine (SFB), a nutritionally complete high fat (HF) diet, or a high fat diet with betaine (HFB) for 14 weeks. In a separate experiment, mice were fed high fat diet for 18 weeks, half of whom received betaine for the final 4 weeks. Activation of insulin signaling in the liver was assessed by western blot. Insulin signaling was also assessed in insulin resistant primary human hepatocytes treated with betaine. As compared with SF, mice receiving HF diet were heavier, had more hepatic steatosis, and abnormal glucose tolerance test (GTT). Betaine content in liver and serum was 50% lower in HF than in SF; betaine supplementation restored serum and liver betaine content. Betaine treatment of HF reduced whole body insulin resistance as measured by GTT. Betaine treatment of HF increased tyrosine phosphorylation of insulin receptor substrate-1 and phosphorylation (activation) of Akt, and increased hepatic glycogen content. In vitro, betaine reversed insulin resistance in primary human hepatocytes by increasing insulin-stimulated tyrosine phosphorylation of IRS1 and of Akt. Betaine supplementation reduced whole body insulin resistance and increased activation of insulin signaling pathways in the liver in a mouse model of insulin resistance and fatty liver created by feeding a nutritionally complete high fat diet for 14 weeks. Betaine also reduced liver injury as assessed by ALT and by liver histology. In vitro, betaine reversed insulin resistance by increasing insulin-stimulated tyrosine phosphorylation of IRS1 and activation of downstream proteins in the insulin signaling cascade in insulin resistant primary human hepatocytes.

BDNF/TrkB pathway activation in D1 receptor-expressing striatal projection neurons plays a protective role against L-DOPA-induced dyskinesia

L-DOPA-induced dyskinesia (LID) is a frequent adverse side effect of L-DOPA treatment in Parkinson's disease (PD). Understanding the mechanisms underlying the development of these motor disorders is needed to reduce or prevent them. We investigated the role of TrkB receptor in LID, in hemiparkinsonian mice treated by chronic L-DOPA administration. Repeated L-DOPA treatment for 10 days specifically increased full-length TrkB receptor mRNA and protein levels in the dopamine-depleted dorsal striatum (DS) compared to the contralateral non-lesioned DS or to the DS of sham-operated animals. Dopamine depletion alone or acute L-DOPA treatment did not significantly increase TrkB protein levels. In addition to increasing TrkB protein levels, chronic L-DOPA treatment activated the TrkB receptor as evidenced by its increased tyrosine phosphorylation. Using specific agonists for the D1 or D2 receptors, we found that TrkB increase is D1 receptor-dependent. To determine the consequences of these effects, the TrkB gene was selectively deleted in striatal neurons expressing the D1 receptor. Mice with TrkB floxed gene were injected with Cre-expressing adeno-associated viruses or crossed with Drd1-Cre transgenic mice. After unilateral lesion of dopamine neurons in these mice, we found an aggravation of axial LID compared to the control groups. In contrast, no change was found when TrkB deletion was induced in the indirect pathway D2 receptor-expressing neurons. Our study suggests that BDNF/TrkB signaling plays a protective role against the development of LID and that agonists specifically activating TrkB could reduce the severity of LID.

A novel interface between the N-terminal and coiled-coil domain of STAT1 functions in an auto-inhibitory manner

BackgroundSTAT1 is an intracellular signaling molecule that is crucially involved in the regulation of the innate immune system by activation of defense mechanisms against microbial pathogens. Phosphorylation-dependent activation of the STAT1 transcription factor is associated with a conversion from an antiparallel to parallel dimer configuration, which after nuclear import binds to DNA. However, not much is known about the specific intermolecular interactions that stabilize unphosphorylated, antiparallel STAT1 complexes prior to activation.ResultsIn this study, we identified a previously unknown interdimeric interaction site, which is involved in the termination of STAT1 signaling. Introduction of the glutamic acid-to-alanine point mutation E169A in the coiled-coil domain (CCD) by site-directed mutagenesis led to increased tyrosine phosphorylation as well as accelerated and prolonged nuclear accumulation in transiently transfected cells. In addition, DNA-binding affinity and transcriptional activity were strongly enhanced in the substitution mutant compared to the wild-type (WT) protein. Furthermore, we have demonstrated that the E169 residue in the CCD mediates the release of the dimer from the DNA in an auto-inhibitory manner.ConclusionBased on these findings, we propose a novel mechanism for the inactivation of the STAT1 signaling pathway, assigning the interface with the glutamic acid residue 169 in the CCD a crucial role in this process.9po3jP83sSjHVE3A4BbhA7Video

Corneal Edema in Inducible Slc4a11 Knockout Is Initiated by Mitochondrial Superoxide Induced Src Kinase Activation.

Inducible Slc4a11 KO leads to corneal edema by disruption of the pump and barrier functions of the corneal endothelium (CE). The loss of Slc4a11 NH3-activated mitochondrial uncoupling leads to mitochondrial membrane potential hyperpolarization-induced oxidative stress. The goal of this study was to investigate the link between oxidative stress and the failure of pump and barrier functions and to test different approaches to revert the process. Mice which were homozygous for Slc4a11 Flox and Estrogen receptor -Cre Recombinase fusion protein alleles at 8 weeks of age were fed Tamoxifen (Tm)-enriched chow (0.4 g/Kg) for 2 weeks, and controls were fed normal chow. During the initial 14 days, Slc4a11 expression, corneal thickness (CT), stromal [lactate], Na+-K+ ATPase activity, mitochondrial superoxide levels, expression of lactate transporters, and activity of key kinases were assessed. In addition, barrier function was assessed by fluorescein permeability, ZO-1 tight junction integrity, and cortical cytoskeleton F-actin morphology. Tm induced a rapid decay in Slc4a11 expression that was 84% complete at 7 days and 96% complete at 14 days of treatment. Superoxide levels increased significantly by day 7; CT and fluorescein permeability by day 14. Tight junction ZO-1 distribution and the cortical cytoskeleton were disrupted at day 14, concomitant with decreased expression of Cldn1, yet with increased tyrosine phosphorylation. Stromal lactate increased by 60%, Na+-K+ ATPase activity decreased by 40%, and expression of lactate transporters MCT2 and MCT4 significantly decreased, but MCT1 was unchanged at 14 days. Src kinase was activated, but not Rock, PKCα, JNK, or P38Mapk. Mitochondrial antioxidant Visomitin (SkQ1, mitochondrial targeted antioxidant) and Src kinase inhibitor eCF506 significantly slowed the increase in CT, with concomitant decreased stromal lactate retention, improved barrier function, reduced Src activation and Cldn1 phosphorylation, and rescued MCT2 and MCT4 expression. Slc4a11 KO-induced CE oxidative stress triggered increased Src kinase activity that resulted in perturbation of the pump components and barrier function of the CE.

Regulation of Cx43 Gap Junction Intercellular Communication by Bruton's Tyrosine Kinase and Interleukin-2-Inducible T-Cell Kinase.

T and B cell receptor signaling involves the activation of Akt, MAPKs, and PKC as well as an increase in intracellular Ca2+ and calmodulin activation. While these coordinate the rapid turnover of gap junctions, also implicated in this process is Src, which is not activated as part of T and B cell receptor signaling. An in vitro kinase screen identified that Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) phosphorylate Cx43. Mass spectroscopy revealed that BTK and ITK phosphorylate Cx43 residues Y247, Y265, and Y313, which are identical to the residues phosphorylated by Src. Overexpression of BTK or ITK in the HEK-293T cells led to increased Cx43 tyrosine phosphorylation as well as decreased gap junction intercellular communication (GJIC) and Cx43 membrane localization. In the lymphocytes, activation of the B cell receptor (Daudi cells) or T cell receptor (Jurkat cells) increased the BTK and ITK activity, respectively. While this led to increased tyrosine phosphorylation of Cx43 and decreased GJIC, the cellular localization of Cx43 changed little. We have previously identified that Pyk2 and Tyk2 also phosphorylate Cx43 at residues Y247, Y265, and Y313 with a similar cellular fate to that of Src. With phosphorylation critical to Cx43 assembly and turnover, and kinase expression varying between different cell types, there would be a need for different kinases to achieve the same regulation of Cx43. The work presented herein suggests that in the immune system, ITK and BTK have the capacity for the tyrosine phosphorylation of Cx43 to alter the gap junction function in a similar manner as Pyk2, Tyk2, and Src.

A strategy for uncovering germline variants altering anti-tumor CD8 T cell response

Among many factors known to alter the outcomes of T cell receptor (TCR)-induced proximal signaling, the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained challenging to address. Here, we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations (pTyr-SNVs) that directly impact TCR-induced proximal phosphotyrosine motif-based signaling pathways. We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin (OVA) antigens and retrovirally engineered syngeneic major histocompatibility complex (MHC) Class I restricted OVA TCR-transgenic CD8 T cells (OT-I). Using the synthetic version of pTyr-SNV rs1178800678-G/T-encoding integrin alpha 4 (ITGA4) p.S1027I variant as a prototype, we show that under identical TCR stimulation conditions, genetically determined membrane-proximal immunoreceptor tyrosin activation motif (ITAM) results in increased tyrosine phosphorylation of 70 kDa zeta-chain-associated protein (ZAP70) and the levels of cytotoxic effector molecule granzyme B (GZMB), which in turn result in enhanced cytotoxic activity against metastatic melanoma cell line. This strategy paves the way for rapid molecular and functional characterization of anti-tumor immune response-linked germline pTyr-SNVs so as to improve our understanding of the genetic basis of individual-to-individual differences in anti-tumor CD8 T cell response.

The noncatalytic regions of the tyrosine kinase Tnk1 are important for activity and substrate specificity

Human Tnk1 (thirty-eight negative kinase 1) is a member of the Ack family of nonreceptor tyrosine kinases. Tnk1 contains a sterile alpha motif, a tyrosine kinase catalytic domain, an SH3 (Src homology 3) domain, and a large C-terminal region that contains a ubiquitin association domain. However, specific physiological roles for Tnk1 have not been characterized in depth. Here, we expressed and purified Tnk1 from Sf9 insect cells and established an invitro assay system using a peptide substrate derived from the Wiskott-Aldrich Syndrome Protein (WASP). By Tnk1 expression in mammalian cells, we found that the N-terminal SAM domain is important for self-association and kinase activity. We also studied a fusion protein, originally discovered in a Hodgkin's Lymphoma cell line, that contains an unrelated sequence from the C17ORF61 gene fused to the C-terminus of Tnk1. Cells expressing the fusion protein showed increased tyrosine phosphorylation of cellular substrates relative to cells expressing WT Tnk1. A truncated Tnk1 construct (residues 1-465) also showed enhanced phosphorylation, indicating that the C17ORF61 sequence was dispensable for the effect. Additionally, invitro kinase assays with the WASP peptide substrate showed no increase in intrinsic Tnk1 activity in C-terminally truncated constructs, suggesting that the truncations did not simply remove an autoinhibitory element. Fluorescence microscopy experiments demonstrated that the C-terminus of Tnk1 plays an important role in the subcellular localization of the kinase. Taken together, our data suggest that the noncatalytic regions of Tnk1 play important roles in governing activity and substrate phosphorylation.

Can we Make SpermFAST? Improving Sperm Function to Increase Fertilisation Rates after in vitro Fertilisation

Background: Intracytoplasmic sperm injection (ICSI) usage ([Formula: see text]64% of autologous cycles) has increased in Australia as an attempt to avoid low fertilisation and total fertilisation failure after standard in vitro fertilization (IVF). ICSI and to a lesser extent IVF, bypass key sperm maturation (hyperactivation, capacitation/acrosome reaction) events that naturally occur in the female tract and are vital for successful fertilisation. Current commercial sperm preparation media are not fully designed to induce these changes in sperm prior to insemination. Aim: To improve fertilisation rates following IVF by increasing sperm capacitation and hyperactivation between sperm preparation and insemination ultilising a new sperm medium (SpermFAST). Method: Sperm from 12 consenting normospermic men were incubated in either G-IVF+ (Vitrolife) or SpermFAST (UoA/Monash IVF) following a direct swim-up. Measures of capacitation, hyperactivation, sperm binding, acrosome reaction and oxidative DNA damage were assessed. Further, sperm from male CBAF1 mice (N=8) were incubated in either G-IVF+ or SpermFAST prior to IVF insemination. Fertilisation rates, embryo development, blastocyst cell numbers and DNA damage were assessed. Results: Incubation of human sperm in SpermFAST increased tyrosine phosphorylation (15.8% vs 9.5%, P[Formula: see text]0.05), hyperactive motility (38.3% vs 14.9%, P[Formula: see text]0.01), sperm binding (73.1% vs 47.7%, P[Formula: see text]0.01), while having no impact on sperm acrosomal status or oxidative DNA damage levels. Following IVF in the mouse, sperm incubated in SpermFAST increased fertilisation rates (94% vs 88%, P[Formula: see text]0.05), blastocyst total cell (92.2% vs 77.4%, P[Formula: see text]0.05), inner cell mass (14.9% vs 18.9%, P[Formula: see text]0.01) and epiblast cell numbers (3.7% vs 1.6%, P[Formula: see text]0.01), while the proportion of DNA damaged cells decreased in blastocysts (2.3% vs 4.8%, P[Formula: see text]0.001). Conclusion: Sperm function and fertilisation rates are improved when the sperm medium better mimics the environment of the female reproductive tract during natural conception. Improving IVF culture media to better meet the physiological needs of sperm could potentially improve outcomes following IVF.

Impact of 5'-AMP-activated protein kinase (AMPK) on Epithelial Sodium Channels (ENaCs) in human sperm

Hyperpolarization is associated with decreased intracellular Na+ concentration through the closure of the epithelial Na+ channels (ENaCs) during capacitation. 5'-AMP-activated protein kinase (AMPK) is involved in the regulation of Na+ transport by reducing ENaC-β abundance in the plasma membrane in somatic cells. However, it is not known whether AMPK acts on ENaCs in sperm. The aim of the present study was to analyze the role of AMPK activation in the regulation of ENaC and to examine its relationship with capacitation-associated hyperpolarization of human sperm. Human sperm were treated with AICAR (AMPK activator) in non-capacitating and capacitating conditions. AMPK activity and ENaC-β concentration were evaluated by ELISA. Flow cytometry was used to measure tyrosine phosphorylation, hyperpolarization, intracellular Na+ concentration and acrosome reaction. Immunofluorescence staining was carried out to analyze the distribution of ENaC-β and CD46 in sperm. We found that induction of capacitation triggered AMPK phosphorylation. AMPK activation by AICAR increased tyrosine phosphorylation. AICAR decreased ENaC-β levels, mainly localized at the principal-piece of the flagellum, resulting in lower intracellular Na+ concentration and increased hyperpolarization of the plasma membrane. Altogether, these data provide evidence that AMPK activation is involved in capacitation-associated hyperpolarization by reducing ENaC abundance in human sperm.

Adding of neurotensin to non-small cell lung cancer cells increases tyrosine phosphorylation of HER3

Neurotensin (NTS) receptor 1 regulates the growth non-small cell lung cancer (NSCLC) cells. NTS binds with high affinity to NTSR1, leading to increased tyrosine phosphorylation of the EGFR and HER2. Using Calu3, NCI-H358, or NCI-H441 cells, the effects of NTS on HER3 transactivation were investigated. HER3 tyrosine phosphorylation was increased by NTS or neuregulin (NRG1) addition to NSCLC cells. NCI-H358, NCI-H441, and Calu-3 cells have HER3, NTSR1 and neuregulin (NRG)1 protein. NTSR1 regulation of HER3 transactivation was impaired by SR48692 (NTSR1 antagonist) or monoclonal antibody (mAb)3481 (HER3 blocker). Immunoprecipitation experiments indicated that NTS addition to NCI-H441cells resulted in the formation of EGFR/HER3 and HER2/HER3 heterodimers. The ability of NTS to increase HER3 tyrosine phosphorylation was impaired by GM6001 (MMP inhibitor), PP2 (Src inhibitor), Tiron (superoxide scavenger), or N-acetylcysteine (antioxidant). Adding NTS to NSCLC cells increased phosphorylation of ERK, HER3, and AKT. NTS or NRG1 increased colony formation of NSCLC cells which was strongly inhibited by SR48692 and mAb3481. The results indicate that NTSR1 regulates HER3 transactivation in NSCLC cells leading to increased proliferation.

Abstract 2688: Bombesin receptors regulate transactivation of HER4

Abstract Bombesin (BB) is an autocrine growth factor for non-small cell lung cancer (NSCLC) cells. BB stimulates whereas the BB2 receptor (R) antagonist PD176252 inhibits NSCLC growth (Moody et al., Eur. J. Pharmacol; 2003; 474:21). The BB2R regulates the transactivation of the EGFR, HER2 and HER3 (Lee et al., BBAMCR 2020; 1867: 118625). HER4 increases the proliferation of NSCLC cells (Mota et al., Oncotarget 2017; 8: 89284). It is unknown if BB2R stimulation increases tyrosine phosphorylation of HER4 and this was investigated using NSCLC cells. BB or neuregulin (NRG1) addition to NSCLC cells increased the phosphorylation of Tyr1284-HER4. By RT-PCR and western blot, mRNA and protein for BB2R, HER4 and NRG1 was present in NCI-H522 and NCI-H661 NSCLC cells. The addition of BB to NCI-H522 or NCI-H661 cells increased phosphorylation of HER4, ERK and AKT which was blocked by PD176252. By immunoprecipitation, HER4 heterodimerized with HER2 and the EGFR after addition of BB to NSCLC cells. PP2 and N-acetylcysteine impaired the ability of BB to increase P-Tyr1284-HER4 in NSCLC cells, which indicates that SRC and reactive oxygen species are essential. Ibrutinib, a tyrosine kinase inhibitor (Rauf et al., Oncogene 2018; 37: 2237), impaired the ability of BB2Rs to regulate HER4 transactivation. Ibrutinib or PD176252 inhibited the growth of NSCLC cells whereas BB or NRG1 increased NSCLC colony formation. The results indicate that peptide receptors for BB regulate HER4 transactivation and the proliferation of NSCLC cells. Citation Format: Terry W. Moody, Irene Ramos-Alvarez, Samuel A. Mantey, Robert T. Jensen. Bombesin receptors regulate transactivation of HER4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2688.

Peptides Regulating Proliferative Activity and Inflammatory Pathways in the Monocyte/Macrophage THP-1 Cell Line.

This study evaluates the effects of five different peptides, the Epitalon® tetrapeptide, the Vilon® dipeptide, the Thymogen® dipeptide, the Thymalin® peptide complex, and the Chonluten® tripeptide, as regulators of inflammatory and proliferative processes in the human monocytic THP-1, which is a human leukemia monocytic cell line capable of differentiating into macrophages by PMA in vitro. These peptides (Khavinson Peptides®), characterized by Prof. Khavinson from 1973 onwards, were initially isolated from animal tissues and found to be organ specific. We tested the capacity of the five peptides to influence cell cultures in vitro by incubating THP-1 cells with peptides at certain concentrations known for being effective on recipient cells in culture. We found that all five peptides can modulate key proliferative patterns, increasing tyrosine phosphorylation of mitogen-activated cytoplasmic kinases. In addition, the Chonluten tripeptide, derived from bronchial epithelial cells, inhibited in vitro tumor necrosis factor (TNF) production of monocytes exposed to pro-inflammatory bacterial lipopolysaccharide (LPS). The low TNF release by monocytes is linked to a documented mechanism of TNF tolerance, promoting attenuation of inflammatory action. Therefore, all peptides inhibited the expression of TNF and pro-inflammatory IL-6 cytokine stimulated by LPS on terminally differentiated THP-1 cells. Lastly, by incubating the THP1 cells, treated with the peptides, on a layer of activated endothelial cells (HUVECs activated by LPS), we observed a reduction in cell adhesion, a typical pro-inflammatory mechanism. Overall, the results suggest that the Khavinson Peptides® cooperate as natural inducers of TNF tolerance in monocyte, and act on macrophages as anti-inflammatory molecules during inflammatory and microbial-mediated activity.

Direct Intracellular Delivery of Benzene Diazonium Ions As Observed by Increased Tyrosine Phosphorylation.

A challenge within the field of bioconjugation is developing probes to uncover novel information on proteins and other biomolecules. Intracellular delivery of these probes offers the promise of giving relevant context to this information, and these probes can serve as hypothesis-generating tools within complex systems. Leveraging the utility of triazabutadiene chemistry, herein, we discuss the development of a probe that undergoes reduction-mediated deprotection to rapidly deliver a benzene diazonium ion (BDI) into cells. The intracellular BDI resulted in an increase in global tyrosine phosphorylation levels. Seeing phosphatase dysregulation as a potential source of this increase, a tyrosine phosphatase (PTP1B) was tested and shown to be both inhibited and covalently modified by the BDI. In addition to the expected azobenzene formation at tyrosine side chains, key reactive histidine residues were also modified.

The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor

Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRβ) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRβ′s tyrosine kinase activity and directly promotes IRβ interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.

Abstract 809: Neurotensin increases tyrosine phosphorylation of HER3

Abstract Neurotensin (NTS) is an autocrine growth factor for non-small cell lung cancer cells. NTS stimulates and the NTSR1 antagonist SR48692 inhibits NSCLC growth (Moody et al., Life Sci 2014; 100: 25). NSCLC patients, whose tumors have high NTSR1, have significantly lower relapse-free survival than those with low NTSR1 (Younes et al., Oncotarget 2014; 5: 8252). NTS addition to NSCLC cells increases the tyrosine phosphorylation of the EGFR and HER2 (Moody et al., Eur J Pharmacol 2019; 865:172735). Activation of HER3 in NSCLC mediates aggressive growth and drug resistance. Therefore the ability of NTSR1 to regulate transactivation of HER3 was investigated. By RT-PCR, HER3 and NTSR1 but not NTSR2 mRNA was present in NSCLC cells. NTS addition to NCI-H358 or H441 cells increased HER3 and ERK tyrosine phosphorylation by 210% and 240%, respectively and the increase caused by NTS was antagonized by SR48692 or mAb3481 (HER3 mAb). The increase is phosphorylation of Tyr1289-HER3 caused by NTS or neuregulin-1 addition to NSCLC cells was due to the formation of HER2-HER3 and EGFR-HER3 heterodimers. NTSR1 regulation of HER3 or Akt phosphorylation was impaired by PP2 (Src inhibitor) or GM6001 (MMP inhibitor). Because N-acetylcysteine (antioxidant) or Tiron (superoxide scavenger) inhibited the ability of NTS to increase HER3 phosphorylation, reactive oxygen species (ROS) are essential. Using a clonogenic assay, NTS increased NSCLC colony number whereas SR48692 or mAb3481 reduced colony number. The results indicate that NTS increased HER3 tyrosine phosphorylation in a ROS-/Src-/MMP-dependent manner. In summary, NTSR1 transactivates HER3 resulting in ERK and Akt phosphorylation increasing the proliferation and survival of NSCLC cells. Citation Format: Terry W. Moody, Irene Ramosalvarez, Robert T. Jensen. Neurotensin increases tyrosine phosphorylation of HER3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 809.

Integrin alpha6-Deletion Induces Apoptosis and Src Signaling Changes in Acute Lymphoblastic Leukemia Cells

After induction chemotherapy, the detection of minimal residual disease (MRD) in the bone marrow of patients with acute lymphoblastic leukemia (ALL) is a negative prognostic factor. Coustan-Smith et al recently identified integrin alpha6 (Itga6) as a novel MRD marker which was included in a panel of 22 flow cytometry markers for MRD ALL cells. Our data supports a role of Itga6 as a critical molecule in drug resistance of ALL. In a xenograft model of primary pre-B-ALL, a novel non-humanized antibody targeting Itga6, P5G10, significantly prolonged survival of leukemic mice after combination with chemotherapy (Vincristine, Dexamethasone, L-Asparaginase) (n=6) compared to chemotherapy only treated mice (n=6) (MST= 185 days vs. MST=71 days; p=0.0012). In vitro treatment of primary pre-B-ALL cells adhering to laminin-1 with the anti-human Itga6 P5G10 antibody caused loss of viability, suggesting that Itga6 signaling has a pro-survival function for these cells. To investigate this further, we established murine ERT2-Cre x Itga6fl/fl BCR-ABL1+ pre-B ALL cells in which Itga6 function can be ablated by tamoxifen (TAM) treatment in vitro and in vivo after injection in NSG mice. In vivo ablation of Itga6 prolonged survival of recipient leukemic mice compared to Itga6 competent leukemia recipient mice. Combination of tyrosine kinase inhibition with Itga6 ablation in vivo prolonged survival significantly compared to tyrosine kinase only treated mice (MST=127 days (n=6) vs. MST of 39.5 days (n=5); p=0.0018). When ERT2-Cre x Itga6fl/fl B-ALL cells were treated with TAM in vitro, Itga6 expression was lost over at least 5 days. Interestingly, this corresponded with a progressive decrease in cell viability. As Src-family kinases and other tyrosine kinases play important roles in signaling of integrins, we investigated specific changes in total tyrosine phosphorylation in Itga6 deleting cells to understand the underlying mechanism for Itga6 deletion associated apoptosis. Compared to empty vector x Itga6fl/fl pre-B ALL cells, cells losing Itga6 had increased signals on pTyr-containing proteins within 24 hours. To identify the affected proteins, we performed proteomic pTyr analysis and identified increased tyrosine phosphorylation on the docking protein CASL, which assembles signal transduction complexes including integrins, and decreases in phosphorylation of specific Y residues for Src family kinase substrates. Interestingly, increases in pY508 and pY531 in the non-receptor hematopoietic tyrosine kinases Lyn and Fyn compared to non-Itga6-deleted ALL cells were also noted. Lyn and Fyn are tyrosine residues and their phosphorylation inhibits the activity of these tyrosine kinases. Thus, kinases with increased phosphorylation on these residues have reduced tyrosine kinase activity. Treatment of Itga6fl/fl B-ALL cells with nilotinib, to inhibit the tyrosine kinase activity of BCR/ABL1 expressed in these cells, also caused a marked increase in pY531 Fyn levels. To further address the significance of Fyn and Lyn, we treated WT and Itga6-ablated B-ALL cells with siRNA against Lyn and Fyn. Knockdown of Lyn significantly decreased viability of Itga6 WT B-ALL cells, and combined loss of Itga6 and Lyn further lowered viability. Taken together, these results implicate Lyn and Fyn with decreased viability that accompanies Itga6 deletion. Identification of Lyn and Fyn critical for transducing Itga6 signals in B-ALL will not only yield new information on the specific function Itga6, which may be useful to others who study malignancies that involve Itga6, but will also provide a rationale on which to base possible combination treatments with Itga6 antibodies. DisclosuresNo relevant conflicts of interest to declare.