Src Protein Tyrosine Kinase Research Articles

An effective cell-penetrating antibody delivery platform.

Despite their well-recognized success in the clinic, antibodies generally do not penetrate cellular membranes to target intracellular molecules, many of which underlie incurable diseases. Here we show that covalently conjugating phosphorothioated DNA oligonucleotides to antibodies enabled their efficient cellular internalization. Antibody cell penetration was partially mediated by membrane potential alteration. Moreover, without an antigen to bind, intracellular levels of the modified antibodies underwent cellular clearance, which involved efflux and lysosomal degradation, enabling detection of intended intracellular molecules as tested in fibroblasts, tumor cells, and T cells. This target-dependent cellular retention of modified antibodies extended to in vivo studies. Both local and systemic administrations of low doses of modified antibodies effectively inhibited intracellular targets, such as transcription factors Myc, interferon regulatory factor 4, and tyrosine-protein kinase SRC, and expression of their downstream genes in tumors, resulting in tumor cell apoptosis and tumor growth inhibition. This simple modification enables the use of antibodies to detect and modulate intracellular molecules in both cultured living cells and in whole animals, forming the foundation for a new paradigm for antibody-based research, diagnostics, and therapeutics.

Pharmacological preconditioning with phosphodiestrase inhibitor: an answer to stem cell survival against ischemic injury through JAK/STAT signaling.

Stem cell transplantation in regenerative medicine has been widely used in various disorders including cardiovascular diseases (CVD) and emerging next-generation therapy. However, transplanted stem cell encountered ischemia/reperfusion (IR) injury which is a major challenge for stem cell survival. During the acute phase after myocardial infarction (MI) cytokine-rich hostile microenvironment, extensive immune cell infiltration and lack of oxygen have been a bottleneck in cell-based therapy. During prolonged ischemia, intracellular pH and ATP level decrease results in anaerobic metabolism and lactate accumulation. Consequentially, ATPase-dependent ion transport becomes dysfunctional, contributing to calcium overload and cell death by apoptosis and necrosis. Although O2 level revitalizes upon reperfusion, a surge in the generation of reactive oxygen species (ROS) occurs with neutrophil infiltration in ischemic tissues further aggravating the injury. Ischemic preconditioning (IPC) of stem cells with a repeated short cycle of IR results in the release of chemical signals such as NO, ROS, and adenosine which triggers a cascade of signaling events that activates protein kinase C (PKC), Src protein tyrosine kinases, and nuclear factor κB (NF-κB) and subsequently increased synthesis of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), Heme oxygenase-1 [HO-1], aldose reductase, Mn superoxide dismutase, and anti-apoptotic genes (Mcl-1, BCl-xL, c-FLIPL, c-FLIPS). Pharmacological preconditioning uses a phosphodiestrase inhibitor, another mode of protecting stem cell or heart per se from impending ischemic injury in two phases. During the early phase of cardioprotection (2h), PC leads to increased expression of survival factors like BCl2/Bax ratio while late phase (24h) showed activation of the JAK/STAT survival pathway. Phosphorylation of STAT3 at two crucial residues, Tyr-705 and Ser-727, allows its entry inside the nucleus and upregulates the expression of protein kinase G-1 (PKG1) which evokes cardioprotective signaling. To confirm, heart-specific conditional STAT3 knockout mice undergone IR surgery, abolishing late-phase cardioprotective effects.

Small-Molecule Inhibition of the UNC-Src Interaction Impairs Dynamic Src Localization in Cells

Interference with the signaling activity of the N-myristoylated nonreceptor protein tyrosine kinase Src is considered a viable approach in anti-cancer drug discovery. However, ATP-competitive Src inhibitors have not reached the clinic yet and alternative approaches are in high demand. The UNC119A/B proteins bind the myristoylated N terminus of Src and thereby mediate energy-driven spatial cycles that maintain Src enrichment at the plasma membrane, which is critical for Src signaling activity. We describe the discovery of a potent and specific inhibitor of the UNC119-Src interaction with unprecedented chemotype. The inhibitor binds to UNC119 in cells, and induces redistribution of Src to endomembranes and reduction of activating Src autophosphorylation on Y419. UNC119 inhibition in Src-dependent colorectal cancer cells results in the specific reduction of cell growth and clonogenic potential. Our results demonstrate that small-molecule interference with the dynamics of the Src spatial cycle may provide an opportunityto impair oncogenic Src signaling.

Inhibition of Src activation reverses pulmonary vascular remodeling in experimental pulmonary arterial hypertension via Akt/mTOR/HIF-1 signaling pathway

Pulmonary arterial hypertension (PAH) is a diffuse pulmonary microvascular remodeling disease accompanied by malignant proliferation of pulmonary artery smooth muscle cells (PASMCs), which causes persistent pulmonary artery pressure elevation, right ventricular hypertrophy (RVH) and death. However, current therapies targeting pulmonary vascular remodeling and RVH remain poorly effective in reversing PAH. Overactivation of the protein tyrosine kinase Src plays an important role in tumor cell growth, proliferation and invasion; we thus hypothesized that inhibitors targeting Src activation could reverse experimental PAH. We demonstrated that Src was markedly activated in hypoxia-stimulated PASMCs from donors and PASMCs isolated from PAH patients. We investigated the effects of the Src-selective inhibitor 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP1) and berberine (BBR) on PAH-PASMC proliferation and migration by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2′-deoxyuridine (EdU) and wound-healing assays. Our in vitro results showed that inhibition of Src (Tyr416) phosphorylation repressed PAH-PASMC proliferation and migration by inhibiting hypoxia-inducible factor-1α (HIF-1α) expression through Akt/mTOR signal pathway. In vivo, PP1 and BBR significantly alleviated distal pulmonary vascular remodeling and decreased right ventricular systolic pressure (RVSP) and RVH in Sugen (SU) 5416/hypoxia (SU-PAH) mice. These findings demonstrate that pharmacological (PP1 or BBR) inhibition of Src activation could be a novel means of treating severe pulmonary vascular remodeling and RVH in PAH patients.

Signaling Cross-Talk between MHC Class II Molecular Conformers in Resting Murine B Cells.

In addition to functioning as a ligand to engage TCRs and drive TCR signaling, MHC class II molecules are signaling molecules that generate a number of signals within APCs, such as B lymphocytes. Moreover, MHC class II signaling is critical for B cell activation and development of a robust humoral immune response. Murine class II molecules exist in two distinct conformational states, based primarily on the differential pairing of transmembrane domain GxxxG dimerization motifs (i.e., M1- and M2-paired class II). This laboratory has previously reported that the binding of a multimerized form of an anti-class II mAb that selectively recognizes M1-paired I-Ak class II drives intracellular calcium signaling in resting murine B cells and that this signaling is dependent on both src and Syk protein tyrosine kinase activity. In contrast, multimerized forms of two different anti-I-Ak mAbs that bind both M1- and M2-paired class II fail to elicit a response. In this report, a flow cytometry-based calcium flux assay is used to demonstrate that coligation of M1- and M2-paired I-Ak class II results in the active and selective inhibition of M1-paired I-Ak class II B cell calcium signaling by M2-paired class II molecules. Because M1- and M2-paired class II can be loaded with different sets of peptides derived from Ags acquired by distinct pathways of endocytosis, these findings suggest an MHC class II signaling-based mechanism by which CD4 T cells of differing specificities can either enhance or suppress B cell activation.

The Fyn kinase inhibitor, AZD0530, suppresses mouse alcohol self-administration and seeking.

Fyn is a member of the Src family of protein tyrosine kinases (PTKs) that plays an important role not only in normal synaptic functions but also in brain pathologies including alcohol use disorder. We previously reported that repeated cycles of binge drinking and withdrawal activate Fyn in the dorsomedial striatum (DMS) of rodents, and that Fyn signaling in the DMS contributes to rat alcohol intake and relapse. Here, we used AZD0530, a CNS penetrable inhibitor of Src PTKs developed for the treatment of Alzheimer disease and cancer and tested its efficacy to suppress alcohol-dependent molecular and behavioral effects. We show that systemic administration of AZD0530 prevents alcohol-induced Fyn activation and GluN2B phosphorylation in the DMS of mice. We further report that a single dose of AZD0530 reduces alcohol operant self-administration and promotes extinction of alcohol self-administration without altering basal and dopamine D1 receptor-dependent locomotion. Together, our findings suggest that AZD0530, through its inhibitory actions on Fyn kinase, dampens alcohol seeking and drinking.

Oncolytic adenovirus Delta-24-RGD induces a widespread glioma proteotype remodeling during autophagy

Adenovirus Delta-24-RGD has shown a remarkable efficacy in a phase I clinical trial for glioblastoma. Delta-24-RGD induces autophagy in glioma cells, however, the molecular derangements associated with Delta-24-RGD infection remains poorly understood. Here, proteomics was applied to characterize the glioma metabolic disturbances soon after Delta-24-RGD internalization and late in infection. Minutes post-infection, a rapid survival reprogramming of glioma cells was evidenced by an early c-Jun activation and a time-dependent dephosphorylation of multiple survival kinases. At 48 h post-infection (hpi), a severe intracellular proteostasis impairment was characterized, detecting differentially expressed proteins related to mRNA splicing, cytoskeletal organization, oxidative response, and inflammation. Specific kinase-regulated protein interactomes for Delta-24-RGD-modulated proteome revealed interferences with the activation dynamics of protein kinases C and A (PKC, PKA), tyrosine-protein kinase Src (c-Src), glycogen synthase kinase-3 (GSK-3) as well as serine/threonine-protein phosphatases 1 and 2A (PP1, PP2A) at 48hpi, in parallel with adenoviral protein overproduction. Moreover, the late activation of the nuclear factor kappa B (NF-κB) correlates with the extracellular increment of specific cytokines involved in migration, and activation of different inflammatory cells. Taken together, our integrative analysis provides further insights into the effects triggered by Delta-24-RGD in the modulation of tumor suppression and immune response against glioma. SignificanceThe current study provides new insights regarding the molecular mechanisms governing the glioma metabolism during Delta-24-RGD oncolytic adenoviral therapy. The compilation and analysis of intracellular and extracellular proteomics have led us to characterize: i) the cell survival reprogramming during Delta-24-RGD internalization, ii) the proteostatic disarrangement induced by Delta-24-RGD during the autophagic stage, iii) the protein interactomes for Delta-24-RGD-modulated proteome, iv) the regulatory effects on kinase dynamics induced by Delta-24-RGD late in infection, and v) the overproduction of multitasking cytokines upon Delta-24-RGD treatment. We consider that the quantitative molecular maps generated in this study may establish the foundations for the development of complementary adenoviral based-vectors to increase the potency against glioma.

Proteomic Array Analysis of an Epithelial Cell Model Infected with Cryptosporidium

Cryptosporidiosis is a human diarrheal disease caused by the etiological agent Cryptosporidium. This parasite abounds in the environment in resistant oocyst form and can cause life-threatening consequences in immunosuppressed individuals. By evaluating the fundamental mechanisms of pathogenesis in the host, it is helpful to deduce proteins involved in virulence. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured human ileocecal carcinoma epithelial (HCT-8) cells inoculated with C. parvum sporozoites. Although many efforts have been made to characterize gene expression changes, these changes may not necessarily reflect changes in protein expression within an infected cell. New applications in proteomic arrays have the added benefit of studying changes in protein expression and functionality. In this study, we use a protein array platform to observe changes in protein expression and function when host cells are infected with C. parvum sporozoites. Previous studies have shown that the invasion of epithelial cells involves host-signaling whereby host cell actin reorganization allows for the enveloping of the parasite. A major signaling pathway implicated in actin polymerization involves the activation and deactivation of proteins containing SH2 domains, such as CRKL, PIK3, ABL, and the Src protein tyrosine kinase (PTK) family. Our studies show that a phospho-proteomic array of HCT-8 cells inoculated with the pathogen have a 0.87-fold increase in the phosphorylated form of c-Abl. Moreover, an SH2 proteomic array overlayed with protein lysates from uninfected and infected cells revealed that infected protein lysates had decreased binding (66%) to the SH2 domain of CRKL and increased binding (45%) to the SH2 domain of PIK3. These findings demonstrate a prominent role for proteins that may play a role in formation and stabilization of microvillar protrusions which envelope the pathogen.

Tat-Src reduced NR2B tyrosine phosphorylation and its interaction with NR2B in levodopa-induced dyskinetic rats model

Augmented function of N-methyl-d-aspartate receptor subunit 2B (NR2B) and Src protein tyrosine kinase have been demonstrated to get involved in the pathological mechanisms of dyskinesia. In view of functional interactions between NR2B and Src, we investigated the effects of uncoupling NR2B and Src interactions on dyskinesia by using the Src-derived interfering peptide (Tat-Src). In the present study, valid 6-hydroxydopamine-lesioned parkinsonian rats were treated with levodopa intraperitoneally for 22 days to induce dyskinetic rats model. On day 23, dyskinetic rats received either Tat-Src or Tat-sSrc or vehicle with each levodopa dose. The data showed that in dyskinetic rats model intraperitoneal microinjection of Tat-Src improved dyskinetic behaviors and decreased NR2B tyrosine phosphorylation and the interactions of Src with NR2B induced by chronic levodopa treatment. Besides, Tat-Src also attenuated S-nitrosylation (SNO-Src) and the autophosphorylation (p-Src) of Src, which catalyzed NR2B phosphorylation. These findings suggest that targeting NR2B/Src complexes can be one potential treatment for dyskinesia in Parkinson’s disease.

Abstract 1830: A clinical paired resistant model elucidated novel dual salvage signaling that confers alectinib resistance in ALK-rearranged lung cancer

Abstract Conventional drug-resistant cancer cell line models have contributed to the elucidation of drug-resistant mechanisms. However, whether or not these models reflect patients in clinical settings is often controversial. We herein report a patient-derived drug resistant model system named the “clinical paired resistant model”. This model consists of 2 cell lines derived from a treatment-naïve patient (drug sensitive cell line model) and again after disease progression (drug resistant cell line model). The clinical paired resistant model of alectinib, the second generation ALK inhibitor, revealed the properties that cancer cells acquired during treatment using phosphoproteome and immunoblotting analyses; the proto-oncogene protein tyrosine kinase Src and hepatocyte growth factor receptor MET were activated after the development of alectinib resistance. No secondary mutations were detected in the coding region of ALK tyrosine kinase in the alectinib resistant model. In this alectinib-resistant model, the inhibition of Src and MET using saracatinib and PHA-665752 significantly restored alectinib sensitivity in vitro (17.2-fold change in IC50). Downstream signaling molecules for proliferation and survival, phosphorylation of Akt and ERK1/2, were inhibited and caspase 3/7 activity was significantly increased when the cells were treated with all three inhibitors (saracatinib, PHA-665752, and alectinib). Combined knockdown of SRC and MET restored alectinib sensitivity and inhibited downstream signaling in combination with ALK inhibition using alectinib, suggesting that the dual salvage signaling of MET and Src conferred alectinib resistance. A xenograft generated from our paired resistant model (N=5-6, in each group) indicated that combination therapy with a saracatinib and crizotinib, the first generation ALK inhibitor which also inhibits MET, significantly decreased tumor size in vivo as compared with saracatinib or crizotinib monotherapy. We also established a conventional alectinib resistant cell line model in vitro by exposing NCI-H2228 cells (EML4-ALK variant 3a/b) to 300 nM of alectinib for 3 months and found MET and Src were also activated in the model. Our clinical paired resistant model permits the detection of drug-resistant mechanisms without exploring the common characteristics of numerous drug-resistant patients. Our results demonstrate that MET and Src are potential therapeutic targets in patients with alectinib resistance and that the clinical paired resistant model may be a new strategy to elucidate drug-resistant mechanisms in relatively rare cancers. Citation Format: Takahiro Tsuji, Hiroaki Ozasa, Takashi Nomizo, Tomoko Funazo, Yuto Yasuda, Yuichi Sakamori, Hironori Yoshida, Kiyoshi Uemasu, Hitomi Ajimizu, Ryoko Okutani, Shunsuke Aburaya, Wataru Aoki, Mitsuyoshi Ueda, Koh Furugaki, Yasushi Yoshimura, Toyohiro Hirai, Young Hak Kim. A clinical paired resistant model elucidated novel dual salvage signaling that confers alectinib resistance in ALK-rearranged lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1830.

Redox tuning of Ca2+ signaling in microglia drives glutamate release during hypoxia.

Hypoxia causes oxidative stress and excitotoxicity, culminating in neuronal damage during brain ischemia. Hypoxia also activates microglia, the myeloid resident cells of the brain parenchyma. Upon activation, microglia release high amounts of the neurotransmitter glutamate, contributing for neuronal excitotoxicity during brain insults. Here, we reveal a signaling pathway controlling glutamate release from human microglia during hypoxia. We show that hypoxia-mediated redox imbalance promotes the activation of endoplasmic reticulum inositol 1,4,5-trisphosphate (InsP3) receptors leading to Ca2+ mobilization into the cytosol. Increasing cytosolic Ca2+ signaling in microglia activates the non-receptor protein tyrosine kinase Src at the plasma membrane. Src activation enhances the permeability of microglial gap junctions promoting the release of glutamate during hypoxia. Preventing the hypoxia-triggered redox imbalance, using the dietary antioxidants neochlorogenic acid or vitamin C, inhibits InsP3-dependent Ca2+ signaling and abrogates the release of glutamate. Overall, modulating microglial Ca2+ signaling in response to changes in the redox microenvironment might be critical for controlling glutamate excitotoxicity during hypoxia.

Prefrontal AMPA receptors are involved in the effect of methylphenidate on response inhibition in rats.

Response inhibition is a critical executive control function in many species. Deficits in response inhibition have been observed in many disorders, eg, attention deficit/hyperactivity disorder (ADHD). The stop-signal task (SST) is a unique behavior task for evaluating response inhibition via measuring the covert latency of a stop process, and it is widely used in studies of humans, nonhuman primates and rodents. Methylphenidate (MPH; Ritalin®) is a psychostimulant that is widely used for the treatment of ADHD and that effectively improves response inhibition in individuals with ADHD and normal subjects. However, its mechanism of improving response inhibition remains unknown. In this study we adopted a rodent nose-poking version of the SST to examine response inhibition by estimating the stop signal reaction time (SSRT) in rats. Administration of MPH (1 mg/kg, sc) 25 min before the SST test exerted a baseline-dependent effect of MPH on response inhibition, ie, it shortened the SSRTs only in the rats with larger baseline SSRTs, thereby improving response inhibition in these rats. The effect of MPH on response inhibition remained 3 h after MPH administration. Co-administration of PP2 (1 mg/kg, sc), a Src-protein tyrosine kinase (Src-PTKs) inhibitor that inhibited the upregulation of glutamate receptor expression on the plasma membrane of the prefrontal cortex (PFC), abolished the MPH-caused improvement in response inhibition. Furthermore, intra-PFC infusion of a selective AMPAR antagonist.NASPM (0.3 mmol/L, per side) via stainless guide cannulas implanted earlier abolished the effect of MPH on SSRT. These results suggest that AMPA receptors in the PFC are involved in the effect of MPH on response inhibition in rats.

CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell‐Specific Adaptor Protein

The T cell-specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline-rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte-specific protein tyrosine kinase and interleukin 2-inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull-down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr)629 and linker of activated T cells (LAT) pTyr171 , pTyr191 and pTyr226 .

Dopamine promotes NMDA receptor hypofunction in the retina through D1 receptor-mediated Csk activation, Src inhibition and decrease of GluN2B phosphorylation

Dopamine and glutamate are critical neurotransmitters involved in light-induced synaptic activity in the retina. In brain neurons, dopamine D1 receptors (D1Rs) and the cytosolic protein tyrosine kinase Src can, independently, modulate the behavior of NMDA-type glutamate receptors (NMDARs). Here we studied the interplay between D1Rs, Src and NMDARs in retinal neurons. We reveal that dopamine-mediated D1R stimulation provoked NMDAR hypofunction in retinal neurons by attenuating NMDA-gated currents, by preventing NMDA-elicited calcium mobilization and by decreasing the phosphorylation of NMDAR subunit GluN2B. This dopamine effect was dependent on upregulation of the canonical D1R/adenylyl cyclase/cAMP/PKA pathway, of PKA-induced activation of C-terminal Src kinase (Csk) and of Src inhibition. Accordingly, knocking down Csk or overexpressing a Csk phosphoresistant Src mutant abrogated the dopamine-induced NMDAR hypofunction. Overall, the interplay between dopamine and NMDAR hypofunction, through the D1R/Csk/Src/GluN2B pathway, might impact on light-regulated synaptic activity in retinal neurons.

Inhibition of Src and forkhead box O1 signaling by induced pluripotent stem-cell therapy attenuates hyperoxia-augmented ventilator-induced diaphragm dysfunction

Mechanical ventilation (MV) with hyperoxia is required for providing life support to patients with acute lung injury (ALI). However, MV may cause diaphragm weakness through muscle injury and atrophy, an effect termed ventilator-induced diaphragm dysfunction (VIDD). Src protein tyrosine kinase and class O of forkhead box 1 (FoxO1) mediate acute inflammatory responses and muscle protein degradation induced by oxidative stress. Induced pluripotent stem cells (iPSCs) have been reported to improve hyperoxia-augmented ALI; however, the mechanisms regulating the interactions among VIDD, hyperoxia, and iPSCs are unclear. In this study, we hypothesized that iPSC therapy can ameliorate hyperoxia-augmented VIDD by suppressing the Src-FoxO1 pathway. Male C57BL/6 mice, either wild-type or Src-deficient, aged between 6 and 8weeks were exposed to MV (6 or 10mL/kg) with or without hyperoxia for 2-8h after the administration of 5 × 10(7) cells/kg Oct4/Sox2/Parp1 mouse iPSCs or iPSC-derived conditioned medium (iPSC-CM). Nonventilated mice were used as controls. MV during hyperoxia aggravated VIDD, as demonstrated by the increases in Src activation, FoxO1 dephosphorylation, malondialdehyde, caspase-3, atrogin-1 and muscle ring finger-1 production, microtubule-associated protein light chain 3-II, disorganized myofibrils, disrupted mitochondria, autophagy, and myonuclear apoptosis; however, MV with hyperoxia reduced mitochondrial cytochrome C, diaphragm muscle fiber size, and contractility (P<0.05). Hyperoxia-exacerbated VIDD was attenuated in Src-deficient mice and by iPSCs and iPSC-CM (P<0.05). Our data indicate that iPSC therapy attenuates MV-induced diaphragmatic injury that occurs during hyperoxia-augmented VIDD by inhibiting the Src-FoxO1 signaling pathway.

Activity-dependent dephosphorylation of paxillin contributed to nociceptive plasticity in spinal cord dorsal horn.

The enzymatic activity of protein tyrosine kinase Src is subjected to the regulation by C-terminal Src kinase (CSK) and protein tyrosine phosphatases (PTPs). Aberrant Src activation in the spinal cord dorsal horn is pivotal for the induction and development of nociceptive behavioral sensitization. In this study, we found that paxillin, one of the well-characterized cell adhesion components involved in cell migration and survival, integrated CSK and PTPs' signaling to regulate Src-dependent nociceptive plasticity. Paxillin localized at excitatory glutamatergic synapses in the spinal dorsal horn of mice, and the phosphorylation of Tyr118 on paxillin was necessary to associate with and target CSK at synapses. After peripheral tissue injury, the enhanced neuronal activity stimulated N-methyl-D-aspartate (NMDA) subtype glutamate receptors, which initiated PTPs' signaling to catalyze Tyr118 dephosphorylation. The reduced Tyr118 phosphorylation disrupted paxillin interaction with CSK, leading to the dispersal of CSK out of synapses. With the loss of CSK-mediated inhibition, Src activity was persistently increased. The active Src potentiated the synaptic transmission specifically mediated by GluN2B subunit-containing NMDA receptors. The active Src also facilitated the induction of long-term potentiation of C fiber-evoked field potentials and exaggerated painful responses. In complete Freund's adjuvant-injected mice, viral expression of phosphomimicking paxillin mutant to resume CSK synaptic localization repressed Src hyperactivity. Meanwhile, this phosphomimicking paxillin mutant blunted NMDA receptor-mediated synaptic transmission and alleviated chronic inflammatory pain. These data showed that PTPs-mediated dephosphorylation of paxillin at Tyr118 was involved in the modification of nociceptive plasticity through CSK-Src signaling.

Measurement of Mechanical Tension at Cell-cell Junctions Using Two-photon Laser Ablation.

The cortical actomyosin cytoskeleton is found in all non-muscle cells where a key function is to control mechanical force (Salbreux et al., 2012). When coupled to E-cadherin cell-cell adhesion, cortical actomyosin generates junctional tension that influences many aspects of tissue function, organization and morphogenesis (Lecuit and Yap, 2015). Uncovering the molecular mechanisms underlying the generation of junctional tension requires tools for measuring it in live cells with a high spatio-temporal resolution. For this, we have set up a technique of laser ablation, in which we use the high power output of a two-photon laser to physically cut the actin cortex at the sites of cell-cell adhesion labeled with E-cadherin-GFP. Tension, thus is visualized as the outwards recoil of the vertices that define a junction after this was ablated/cut. Analysis of recoil versus time allows extracting parameters related to the amount of contractile force that is applied to the junction before ablation (initial recoil) and the ratio between elasticity of the junction and viscosity of the media (cytoplasm) in which the junctional cortex is immersed. Using this approach we have discovered how Src protein-tyrosine kinase (Gomez et al., 2015); actin-binding proteins such as tropomyosins (Caldwell et al., 2014) and N-WASP (Wu et al., 2014); Myosin II (Priya et al., 2015) and coronin-1B (Michael et al., 2016) contribute to the molecular apparatus responsible for generating tension at the cell-cell junctions. This protocol describes the experimental procedure for setting up laser ablation experiments and how to optimize ablation and acquisition conditions for optimal measurements of junctional tension. It also provides a full description, step by step, of the post-acquisition analysis required to evaluate changes in contractile force as well as cell elasticity and/or cytoplasm viscosity.

Epibrassinolide alters PI3K/MAPK signaling axis via activating Foxo3a-induced mitochondria-mediated apoptosis in colon cancer cells

Epibrassinolide (EBR), a steroid-derived plant growth regulator, has been recently suggested as an apoptotic inducer in different cancer cells. In this experimental study, we investigated the potential apoptotic effect of EBR on stress-related and survival signaling molecules in colon carcinoma cells. EBR decreased cell viability and colony formation in HCT 116 and HT-29 colon carcinoma cells. The inactivation of PI3K/AKT by EBR treatment led to upregulation of Foxo3a, which in turn induced apoptosis in HCT 116 and HT-29 cells. In addition, the upstream non-receptor protein tyrosine kinase Src was found elevated allowing to the upregulation of p38, stress-activated protein kinase/Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 and their target genes c-jun, c-fos and c-myc in a time-dependent manner in HCT 116 cells within 48h. The alterations in PA metabolism caused intracellular PA pool decrease. The upregulation of pro-apoptotic Bak, Bax, Puma and Bim were accompanied with the decrease in Mcl-1 in HCT 116 and Bcl-xL expression profiles in HT-29 following 48h EBR treatment. We suggest that the upregulation of Bim expression levels might be related with one of the PI3K/AKT target transcription factor Foxo3a, which was dephosphorylated by EBR treatment in HCT 116 and HT-29 cells.

The clinically approved drugs dasatinib and bosutinib induce anti-inflammatory macrophages by inhibiting the salt-inducible kinases.

Macrophages switch to an anti-inflammatory, ‘regulatory’-like phenotype characterized by the production of high levels of interleukin (IL)-10 and low levels of pro-inflammatory cytokines to promote the resolution of inflammation. A potential therapeutic strategy for the treatment of chronic inflammatory diseases would be to administer drugs that could induce the formation of ‘regulatory’-like macrophages at sites of inflammation. In the present study, we demonstrate that the clinically approved cancer drugs bosutinib and dasatinib induce several hallmark features of ‘regulatory’-like macrophages. Treatment of macrophages with bosutinib or dasatinib elevates the production of IL-10 while suppressing the production of IL-6, IL-12p40 and tumour necrosis factor α (TNFα) in response to Toll-like receptor (TLR) stimulation. Moreover, macrophages treated with bosutinib or dasatinib express higher levels of markers of ‘regulatory’-like macrophages including LIGHT, SPHK1 and arginase 1. Bosutinib and dasatinib were originally developed as inhibitors of the protein tyrosine kinases Bcr-Abl and Src but we show that, surprisingly, the effects of bosutinib and dasatinib on macrophage polarization are the result of the inhibition of the salt-inducible kinases. Consistent with the present finding, bosutinib and dasatinib induce the dephosphorylation of CREB-regulated transcription co-activator 3 (CRTC3) and its nuclear translocation where it induces a cAMP-response-element-binding protein (CREB)-dependent gene transcription programme including that of IL-10. Importantly, these effects of bosutinib and dasatinib on IL-10 gene expression are lost in macrophages expressing a drug-resistant mutant of salt-inducible kinase 2 (SIK2). In conclusion, our study identifies the salt-inducible kinases as major targets of bosutinib and dasatinib that mediate the effects of these drugs on the innate immune system and provides novel mechanistic insights into the anti-inflammatory properties of these drugs.

Missing-in-Metastasis regulates cell motility and invasion via PTPδ-mediated changes in SRC activity.

MIM (Missing-in-Metastasis), also known as MTSS1 (metastasis suppressor 1), is a scaffold protein that is down-regulated in multiple metastatic cancer cell lines compared with non-metastatic counterparts. MIM regulates cytoskeletal dynamics and actin polymerization, and has been implicated in the control of cell motility and invasion. MIM has also been shown to bind to a receptor PTP (protein tyrosine phosphatase), PTPδ, an interaction that may provide a link between tyrosine-phosphorylation-dependent signalling and metastasis. We used shRNA-mediated gene silencing to investigate the consequences of loss of MIM on the migration and invasion of the MCF10A mammary epithelial cell model of breast cancer. We observed that suppression of MIM by RNAi enhanced migration and invasion of MCF10A cells, effects that were associated with increased levels of PTPδ. Furthermore, analysis of human clinical data indicated that PTPδ was elevated in breast cancer samples when compared with normal tissue. We demonstrated that the SRC protein tyrosine kinase is a direct substrate of PTPδ and, upon suppression of MIM, we observed changes in the phosphorylation status of SRC; in particular, the inhibitory site (Tyr527) was hypophosphorylated, whereas the activating autophosphorylation site (Tyr416) was hyperphosphorylated. Thus the absence of MIM led to PTPδ-mediated activation of SRC. Finally, the SRC inhibitor SU6656 counteracted the effects of MIM suppression on cell motility and invasion. The present study illustrates that both SRC and PTPδ have the potential to be therapeutic targets for metastatic tumours associated with loss of MIM.