WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways.

Wnt1-inducible signaling protein 1 (WISP1/CCN4) is a secreted matricellular protein that is implicated in lung and airway remodeling. The macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been associated with chronic lung diseases. In this study, we aimed to investigate the WISP1 signaling pathway and its ability to induce the expression of MIF in primary cultures of fibroblasts from normal human lungs (HLFs). Our results showed that WISP1 significantly stimulated the expression of MIF in a concentration- and time-dependent fashion. In WISP1-induced expression of MIF, αvß5-integrin and chondroitin sulfate proteoglycans as well as Src tyrosine kinases, MAP kinases, phosphatidylinositol 3-kinase/Akt, PKC, and NF-κB were involved. WISP1-induced expression of MIF was attenuated in the presence of the Src kinase inhibitor PP2 or the MIF tautomerase activity inhibitor ISO-1. Moreover, WISP1 significantly increased the phosphorylation and activation of EGF receptor (EGFR) through transactivation by Src kinases. WISP1 also induced the expression of MIF receptor CD74 and coreceptor CD44, through which MIF exerts its effects on HLFs. In addition, it was found that MIF induced its own expression, as well as its receptors CD74/CD44, acting in an autocrine manner. Finally, WISP1-induced MIF promoted the expression of cyclooxygenase 2, prostaglandin E2, IL-6, and matrix metalloproteinase-2 demonstrating the regulatory role of WISP1-MIF axis in lung inflammation and remodeling involving mainly integrin αvß5, Src kinases, PKC, NF-κB, and EGFR. The specific signaling pathways involved in WISP1-induced expression of MIF may prove to be excellent candidates for novel targets to control inflammation in chronic lung diseases.NEW & NOTEWORTHY The present study demonstrates for the first time that Wnt1-inducible signaling protein 1 (WISP1) regulates migration inhibitory factor (MIF) expression and activity and identifies the main signaling pathways involved. The newly discovered WISP1-MIF axis may drive lung inflammation and could result in the design of novel targeted therapies in inflammatory lung diseases.

Human dendritic cell maturation induced by amorphous silica nanoparticles is Syk-dependent and triggered by lipid raft aggregation.

BACKGROUND: Synthetic amorphous silica nanoparticles (SAS-NPs) are widely employed in pharmaceutics, cosmetics, food and concretes. Workers and the general population are exposed daily via diverse routes of exposure. SAS-NPs are generally recognized as safe (GRAS) by the Food and Drug Administration, but because of their nanoscale size and extensive uses, a better assessment of their immunotoxicity is required. In the presence of immune "danger signals", dendritic cells (DCs) undergo a maturation process resulting in their migration to regional lymph nodes where they activate naive T-cells. We have previously shown that fumed silica pyrogenic SAS-NPs promote the two first steps of the adaptative immune response by triggering DC maturation and T-lymphocyte response, suggesting that SAS-NPs could behave as immune "danger signals". The present work aims to identify the mechanism and the signalling pathways involved in DC phenotype modifications provoked by pyrogenic SAS-NPs. As a pivotal intracellular signalling molecule whose phosphorylation is associated with DC maturation, we hypothesized that Spleen tyrosine kinase (Syk) may play a central role in SAS-NPs-induced DC response. RESULTS: In human monocyte-derived dendritic cells (moDCs) exposed to SAS-NPs, Syk inhibition prevented the induction of CD83 and CD86 marker expression. A significant decrease in T-cell proliferation and IFN-γ, IL-17F and IL-9 production was found in an allogeneic moDC:T-cell co-culture model. These results suggested that the activation of Syk was necessary for optimal co-stimulation of T-cells. Moreover, Syk phosphorylation, observed 30 min after SAS-NP exposure, occurred upstream of the c-Jun N-terminal kinase (JNK) Mitogen-activated protein kinases (MAPK) and was elicited by the Src family of protein tyrosine kinases. Our results also showed for the first time that SAS-NPs provoked aggregation of lipid rafts in moDCs and that MßCD-mediated raft destabilisation altered Syk activation. CONCLUSIONS: We showed that SAS-NPs could act as an immune danger signal in DCs through a Syk-dependent pathway. Our findings revealed an original mechanism whereby the interaction of SAS-NPs with DC membranes promoted aggregation of lipid rafts, leading to a Src kinase-initiated activation loop triggering Syk activation and functional DC maturation.

Impact of lipid rafts on transient receptor potential channel activities.

Members of the transient receptor potential (TRP) superfamily are cation channels that are expressed in nearly every mammalian cell type and respond as cellular sensors to various environmental stimuli. Light, pressure, osmolarity, temperature, and other stimuli can induce TRP calcium conductivity and correspondingly trigger many signaling processes in cells. Disruption of TRP channel activity, as a rule, harms cellular function. Despite numerous studies, the mechanisms of TRP channel regulation are not yet sufficiently clear, in part, because TRP channels are regulated by a broad set of ligands having diverse physical and chemical features. It is now known that some TRP members are located in membrane microdomains termed lipid rafts. Moreover, interaction between specific raft-associated lipids with channels may be a key regulation mechanism. This review examines recent findings related to the roles of lipid rafts in regulation of TRP channel activity. The mechanistic events of channel interactions with the main lipid raft constituent, cholesterol, are being clarified. Better understanding of mechanisms behind such interactions would help establish the key elements of TRP channel regulation and hence allow control of cellular responses to environmental stimuli.

Agitation-dependent biomechanical forces modulate GPVI receptor expression and platelet adhesion capacity during storage.

BACKGROUND: Continuous agitation during storage slows down the platelet storage lesions. However, in special circumstances, manual-mixing can be alternatively used to store products for short time periods without compromising platelet quality. Based on this finding, and given the role of shear stress in modulating receptor expression, we were interested in comparing the levels of platelet adhesion receptor, GPVI and platelet adhesion capacity under each storage condition. METHODS: Platelet concentrates (PCs) were divided into three groups: continuously-agitated PCs (CAG-PCs) with or without PP2 (Src kinase inhibitor) and manually-mixed PCs (MM-PCs). Platelet count/MPV, swirling, GPVI and P-selectin expression, GPVI shedding, platelet adhesion/spreading to collagen were examined during 5 days of storage. RESULTS: While MM- and CAG-PCs showed similar levels of P-selectin expression, GPVI expression was significantly elevated in MM-PCs with lower GPVI shedding/expression ratios, enhanced platelet adhesion/spreading and swirling in manually-mixed PCs. Of note, CAG-PCs treated with PP2 also demonstrated lower P-selectin expression and GPVI shedding, higher GPVI expression and attenuated swirling and spreading capability. CONCLUSION: Given the comparable platelet activation state in MM and CAG-PCs as indicated by P-selectin expression, enhanced platelet adhesion/spreading in MM-PCs, along with relatively higher GPVI expression here, supports previous studies demonstrating a role for biomechanical forces in modulating GPVI-dependent function. Thus, lower GPVI expression in CAG-PCs may be due to shear forces induced by agitation, which keeps this receptor down-regulated while also attenuating platelet adhesion/spreading capacities during storage. Low platelet function in PP2-CAG-PCs also highlights the importance of Src-kinases threshold activity in maintaining platelets quality.

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation.

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR-SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.

Targeting SRC Kinase Signaling in Pancreatic Cancer Stem Cells.

The proto-oncogene nonreceptor tyrosine-protein kinase SRC is a member of the SRC family of tyrosine kinases (SFKs), and its activation and overexpression have been shown to play a protumorigenic role in multiple solid cancers, including pancreatic ductal adenocarcinoma (PDAC). PDAC is currently the seventh-leading cause of cancer-related death worldwide, and, by 2030, it is predicted to become the second-leading cause of cancer-related death in the United States. PDAC is characterized by its high lethality (5-year survival of rate of <10%), invasiveness, and chemoresistance, all of which have been shown to be due to the presence of pancreatic cancer stem cells (PaCSCs) within the tumor. Due to the demonstrated overexpression of SRC in PDAC, we set out to determine if SRC kinases are important for PaCSC biology using pharmacological inhibitors of SRC kinases (dasatinib or PP2). Treatment of primary PDAC cultures established from patient-derived xenografts with dasatinib or PP2 reduced the clonogenic, self-renewal, and tumor-initiating capacity of PaCSCs, which we attribute to the downregulation of key signaling factors such as p-FAK, p-ERK1-2, and p-AKT. Therefore, this study not only validates that SRC kinases are relevant and biologically important for PaCSCs but also suggests that inhibitors of SRC kinases may represent a possible future treatment option for PDAC patients, although further studies are still needed.

On the activation and deactivation pathways of the Lck kinase domain: a computational study.

Here we report the description of the conformational pathways connecting the Lck active and inactive states by means of all-atoms molecular dynamics simulations coupled to an enhancing sampling methodology. By such an approach, we describe the major structural determinants characterizing these large conformational transitions and compare such pathways to those obtained for a similar kinase, i.e. c-Src. Our results show that both the activation and deactivation processes could follow distinct pathways, differentiated by the order by which the A-loop and the C-helix regions rearrange.

Src-family tyrosine kinases and the Ca2+ signal.

In this review, we shall describe the rich crosstalk between non-receptor Src-family kinases (SFKs) and the Ca2+ transient generated in activated cells by a variety of extracellular and intracellular stimuli, resulting in diverse signaling events. The exchange of information between SFKs and Ca2+ is reciprocal, as it flows in both directions. These kinases are main actors in pathways leading to the generation of the Ca2+ signal, and reciprocally, the Ca2+ signal modulates SFKs activity and functions. We will cover how SFKs participate in the generation of the cytosolic Ca2+ rise upon activation of a series of receptors and the mechanism of clearance of this Ca2+ signal. The role of SFKs modulating Ca2+-translocating channels participating in these events will be amply discussed. Finally, the role of the Ca2+ sensor protein calmodulin on the activity of c-Src, and potentially on other SFKs, will be outlined as well. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts.

BACKGROUND: Pterygium is a condition characterized by epithelial overgrowth of the cornea, inflammatory cell infiltration and an abnormal extracellular matrix accumulation. Chronic UV exposure is considered as a pathogenic factor of this disease. Proteasome is an intracellular multi-subunit protease complex that degrades intracellular proteins. Among proteasome subunits the ß5 (PSMB5), bearing chymotrypsin-like activity. It is considered as the main proteasome subunit and its expression is mediated by Nrf2-ARE pathway in many cell types. This study investigates the expression of PSMB5 in pterygium and the effect of UVB irradiation on its expression and activity in pterygium fibroblasts. METHODS: Normal conjunctival and pterygium specimens were obtained from the bulbar conjunctiva of patients undergoing cataract surgery and from patients with pterygium undergoing surgical removal of primary tissue, respectively. Fibroblasts were isolated upon treatment of specimens with clostridium collagenase. The expression of PSMB5 and Nrf2 in tissues and cells was ascertained by RT-PCR analysis and western blotting. Cell survival was measured by the MTT method and the proteasome chymotrypsin-like activity was determined by fluorometry. RESULTS: RT-PCR analysis showed that the expression of PSMB5 was significantly lower in pterygium than in normal conjunctiva. The expression of PSMB5 was mediated by the Nrf2/ARE pathway as indicated by using the Nrf2 activator Oltipraz. The expression of PSMB5 and Nrf2 by pterygium fibroblasts was suppressed in a dose dependent manner following UVB radiation of 0-50 mJ/cm2 doses. The expression of PSMB5, but not of Nrf2, remained at almost the control levels, when UVB exposure was performed after pre-incubation of cells with the src kinases inhibitor PP2. UVB irradiation had very low deleterious effect on fibroblasts survival, while it did not affect the proteasome chymotrypsin-like activity. CONCLUSION: In pterygium fibroblasts, UVB exposure leads to down-regulation of Nrf2/ARE-mediated PSMB5 gene expression, in which src kinases may be implicated. This effect may be partially responsible for the lower expression of PSMB5 detected in pterygium as compared to normal conjunctiva.

Oxidation of KCNB1 Potassium Channels Causes Neurotoxicity and Cognitive Impairment in a Mouse Model of Traumatic Brain Injury.

The delayed rectifier potassium (K+) channel KCNB1 (Kv2.1), which conducts a major somatodendritic current in cortex and hippocampus, is known to undergo oxidation in the brain, but whether this can cause neurodegeneration and cognitive impairment is not known. Here, we used transgenic mice harboring human KCNB1 wild-type (Tg-WT) or a nonoxidable C73A mutant (Tg-C73A) in cortex and hippocampus to determine whether oxidized KCNB1 channels affect brain function. Animals were subjected to moderate traumatic brain injury (TBI), a condition characterized by extensive oxidative stress. Dasatinib, a Food and Drug Administration-approved inhibitor of Src tyrosine kinases, was used to impinge on the proapoptotic signaling pathway activated by oxidized KCNB1 channels. Thus, typical lesions of brain injury, namely, inflammation (astrocytosis), neurodegeneration, and cell death, were markedly reduced in Tg-C73A and dasatinib-treated non-Tg animals. Accordingly, Tg-C73A mice and non-Tg mice treated with dasatinib exhibited improved behavioral outcomes in motor (rotarod) and cognitive (Morris water maze) assays compared to controls. Moreover, the activity of Src kinases, along with oxidative stress, were significantly diminished in Tg-C73A brains. Together, these data demonstrate that oxidation of KCNB1 channels is a contributing mechanism to cellular and behavioral deficits in vertebrates and suggest a new therapeutic approach to TBI. SIGNIFICANCE STATEMENT: This study provides the first experimental evidence that oxidation of a K+ channel constitutes a mechanism of neuronal and cognitive impairment in vertebrates. Specifically, the interaction of KCNB1 channels with reactive oxygen species plays a major role in the etiology of mouse model of traumatic brain injury (TBI), a condition associated with extensive oxidative stress. In addition, a Food and Drug Administration-approved drug ameliorates the outcome of TBI in mouse, by directly impinging on the toxic pathway activated in response to oxidation of the KCNB1 channel. These findings elucidate a basic mechanism of neurotoxicity in vertebrates and might lead to a new therapeutic approach to TBI in humans, which, despite significant efforts, is a condition that remains without effective pharmacological treatments.

Structural studies and SH3 domain binding properties of a human antiviral salivary proline-rich peptide.

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.

Btk-dependent epithelial cell rearrangements contribute to the invagination of nearby tubular structures in the posterior spiracles of Drosophila.

The Drosophila respiratory system consists of two connected organs, the tracheae and the spiracles. Together they ensure the efficient delivery of air-borne oxygen to all tissues. The posterior spiracles consist internally of the spiracular chamber, an invaginated tube with filtering properties that connects the main tracheal branch to the environment, and externally of the stigmatophore, an extensible epidermal structure that covers the spiracular chamber. The primordia of both components are first specified in the plane of the epidermis and subsequently the spiracular chamber is internalized through the process of invagination accompanied by apical cell constriction. It has become clear that invagination processes do not always or only rely on apical constriction. We show here that in mutants for the src-like kinase Btk29A spiracle cells constrict apically but do not complete invagination, giving rise to shorter spiracular chambers. This defect can be rescued by using different GAL4 drivers to express Btk29A throughout the ectoderm, in cells of posterior segments only, or in the stigmatophore pointing to a non cell-autonomous role for Btk29A. Our analysis suggests that complete invagination of the spiracular chamber requires Btk29A-dependent planar cell rearrangements of adjacent non-invaginating cells of the stigmatophore. These results highlight the complex physical interactions that take place among organ components during morphogenesis, which contribute to their final form and function.

Adenosine A2A receptors permit mGluR5-evoked tyrosine phosphorylation of NR2B (Tyr1472) in rat hippocampus: a possible key mechanism in NMDA receptor modulation.

A great body of evidence points toward a functional interaction between metabotropic glutamate 5 receptors (mGluR5) and NMDA receptors (NMDAR) that enhances synaptic plasticity and cognition. However, the molecular mechanism underlying this interaction remains unclear. Here, we show that co-activation of mGluR5 and NMDAR in hippocampal slices synergistically leads to a robust phosphorylation of NR2B (Tyr1472), which is Src kinase dependent and is enabled by endogenous adenosine acting on A2A receptors. As it is well known, NR2B (Tyr1472) phosphorylation anchors NR2B-containing NMDARs to the surface of post-synaptic membranes, preventing their internalization. This is supported by our electrophysiological experiments showing that co-activation of mGluR5 and NMDARs robustly enhances NMDAR-dependent neuronal excitability recorded in CA1 hippocampal region, which temporally coincides with the robust increase in NR2B (Tyr1472) phosphorylation, depends on Src kinases and is also permitted by A2A receptors. Thus, we strongly suggest that NR2B (Tyr1472) phosphorylation constitutes, at least to some extent, the molecular mechanism underlying the mGluR5-mediated enhancement of NMDAR-dependent responses, which is modulated by A2A receptors. A better understanding of the molecular basis of mGluR5/NMDAR interaction would elucidate their role in synaptic plasticity processes as well as in pathological conditions. We propose the following molecular mechanism by which metabotropic Glutamate Receptor 5 (mGluR5) potentiate ionotropic Glutamate N-Methyl-D-Aspartate Receptor (NMDAR) responses in rat hippocampus. Co-activation of mGLUR5/NMDAR activates Src kinases, leading to NR2B(Tyr1472) phosphorylation, which anchors NR2B-containing NMDAR to the plasma membrane, thus inducing a robust increase in the NMDA-dependent excitability. Interestingly, adenosine A2A receptors license the mGluR5-induced NR2B(Tyr1472) phosphorylation.

Src family kinases involved in CXCL12-induced loss of acute morphine analgesia.

Functional interactions between the chemokine receptor CXCR4 and opioid receptors have been reported in the brain, leading to a decreased morphine analgesic activity. However the cellular mechanisms responsible for this loss of opioid analgesia are largely unknown. Here we examined whether Src family-kinases (SFK)-linked mechanisms induced by CXCR4 contributed to the loss of acute morphine analgesia and could represent a new physiological anti-opioid signaling pathway. In this way, we showed by immunohistochemistry and western blot that CXCL12 rapidly activated SFK phosphorylation in vitro in primary cultured lumbar rat dorsal root ganglia (DRG) but also in vivo in the DRG and the spinal cord. We showed that SFK activation occurred in a sub population of sensory neurons, in spinal microglia but also in spinal nerve terminals expressing mu-(MOR) and delta-opioid (DOR) receptor. In addition we described that CXCR4 is detected in MOR- and DOR-immunoreactive neurons in the DRG and spinal cord. In vivo, we demonstrated that an intrathecal administration of CXCL12 (1µg) significantly attenuated the subcutaneous morphine (4mg/kg) analgesia. Conversely, pretreatment with a potent CXCR4 antagonist (5µg) significantly enhanced morphine analgesia. Similar effects were obtained after an intrathecal injection of a specific SFK inhibitor, PP2 (10µg). Furthermore, PP2 abrogated CXCL12-induced decrease in morphine analgesia by suppressing SFK activation in the spinal cord. In conclusion, our data highlight that CXCL12-induced loss of acute morphine analgesia is linked to Src family kinases activation.

Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2, and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand.

BACKGROUND: Clustering of the receptors glycoprotein receptor VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), low-affinity immunoglobulin γ Fc region receptor II-a (FcγRIIA), and platelet endothelial aggregation receptor 1 (PEAR1) leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signaling cascades. GPVI, CLEC-2, and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase. Current ligands for these receptors have an undefined valency and show significant batch variation and, for some, uncertain specificity. OBJECTIVES: We have raised nanobodies against each of these receptors and multimerized them to identify the minimum number of epitopes to achieve robust activation of human platelets. METHODS: Divalent and trivalent nanobodies were generated using a flexible glycine-serine linker. Tetravalent nanobodies utilize a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerize the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, adenosine triphosphate secretion, and protein phosphorylation were analyzed using standardized methods. RESULTS: Multimerization of the nanobodies led to a stepwise increase in affinity with divalent and higher-order nanobody oligomers having sub-nanomolar affinity. The trivalent nanobodies to GPVI, CLEC-2, and PEAR1 stimulated powerful and robust platelet aggregation, secretion, and protein phosphorylation at low nanomolar concentrations. A tetravalent nanobody was required to activate FcγRIIA with the concentration-response relationship showing a greater variability and reduced sensitivity compared with the other nanobody-based ligands, despite a sub-nanomolar binding affinity. CONCLUSION: The multivalent nanobodies represent a series of standardized, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays.

Bosutinib Stimulates Macrophage Survival, Phagocytosis, and Intracellular Killing of Bacteria.

Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.

Lck Function and Modulation: Immune Cytotoxic Response and Tumor Treatment More Than a Simple Event.

Lck, a member of the Src kinase family, is a non-receptor tyrosine kinase involved in immune cell activation, antigen recognition, tumor growth, and cytotoxic response. The enzyme has usually been linked to T lymphocyte activation upon antigen recognition. Lck activation is central to CD4, CD8, and NK activation. However, recently, it has become clearer that activating the enzyme in CD8 cells can be independent of antigen presentation and enhance the cytotoxic response. The role of Lck in NK cytotoxic function has been controversial in a similar fashion as the role of the enzyme in CAR T cells. Inhibiting tyrosine kinases has been a highly successful approach to treating hematologic malignancies. The inhibitors may be useful in treating other tumor types, and they may be useful to prevent cell exhaustion. New, more selective inhibitors have been documented, and they have shown interesting activities not only in tumor growth but in the treatment of autoimmune diseases, asthma, and graft vs. host disease. Drug repurposing and bioinformatics can aid in solving several unsolved issues about the role of Lck in cancer. In summary, the role of Lck in immune response and tumor growth is not a simple event and requires more research.

Phosphorylation of mammalian mitochondrial EF-Tu by Fyn and c-Src kinases.

Src Family Kinases (SFKs) are tyrosine kinases known to regulate glucose and fatty acid metabolism as well as oxidative phosphorylation (OXPHOS) in mammalian mitochondria. We and others discovered the association of the SFK kinases Fyn and c-Src with mitochondrial translation components. This translational system is responsible for the synthesis of 13 mitochondrial (mt)-encoded subunits of the OXPHOS complexes and is, thus, essential for energy generation. Mitochondrial ribosomal proteins and various translation elongation factors including Tu (EF-Tumt) have been identified as possible Fyn and c-Src kinase targets. However, the phosphorylation of specific residues in EF-Tumt by these kinases and their roles in the regulation of protein synthesis are yet to be explored. In this study, we report the association of EF-Tumt with cSrc kinase and mapping of phosphorylated Tyr (pTyr) residues by these kinases. We determined that a specific Tyr residue in EF-Tumt at position 266 (EF-Tumt-Y266), located in a highly conserved c-Src consensus motif is one of the major phosphorylation sites. The potential role of EF-Tumt-Y266 phosphorylation in regulation of mitochondrial translation investigated by site-directed mutagenesis. Its phosphomimetic to Glu residue (EF-Tumt-E266) inhibited ternary complex (EF-Tumt•GTP•aatRNA) formation and translation in vitro. Our findings along with data mining analysis of the c-Src knock out (KO) mice proteome suggest that the SFKs have possible roles for regulation of mitochondrial protein synthesis and oxidative energy metabolism in animals.

Apoptosis regulation by the tyrosine-protein kinase CSK.

C-terminal Src kinase (CSK) is a cytosolic tyrosine-protein kinase with an important role in regulating critical cellular decisions, such as cellular apoptosis, survival, proliferation, cytoskeletal organization and many others. Current knowledge on the CSK mechanisms of action, regulation and functions is still at an early stage, most of CSK's known actions and functions being mediated by the negative regulation of the SRC family of tyrosine kinases (SFKs) through phosphorylation. As SFKs play a vital role in apoptosis, cell proliferation and survival regulation, SFK inhibition by CSK has a pro-apoptotic effect, which is mediated by the inhibition of cellular signaling cascades controlled by SFKs, such as the MAPK/ERK, STAT3 and PI3K/AKT signaling pathways. Abnormal functioning of CSK and SFK activation can lead to diseases such as cancer, cardiovascular and neurological manifestations. This review describes apoptosis regulation by CSK, CSK inhibition of the SFKs and further explores the clinical relevance of CSK in important pathologies, such as cancer, autoimmune, autoinflammatory, neurologic diseases, hypertension and HIV/AIDS.

Maternal Ethanol Exposure Acutely Elevates Src Family Kinase Activity in the Fetal Cortex.

Fetal alcohol syndrome (FAS) is characterized by disrupted fetal brain development and postnatal cognitive impairment. The targets of alcohol are diverse, and it is not clear whether there are common underlying molecular mechanisms producing these disruptions. Prior work established that acute ethanol exposure causes a transient increase in tyrosine phosphorylation of multiple proteins in cultured embryonic cortical cells. In this study, we show that a similar tyrosine phosphorylation transient occurs in the fetal brain after maternal dosing with ethanol. Using phospho-specific antibodies and immunohistochemistry, we mapped regions of highest tyrosine phosphorylation in the fetal cerebral cortex and found that areas of dendritic and axonal growth showed elevated tyrosine phosphorylation 10 min after maternal ethanol exposure. These were also areas of Src expression and Src family kinase (SFK) activation loop phosphorylation (pY416) expression. Importantly, maternal pretreatment with the SFK inhibitor dasatinib completely prevents both the pY416 increase and the tyrosine phosphorylation response. The phosphorylation response was observed in the perisomatic region and neurites of immature migrating and differentiating primary neurons. Importantly, the initial phosphotyrosine transient (~ 30 min) targets both Src and Dab1, two critical elements in Reelin signaling, a pathway required for normal cortical development. This initial phosphorylation response is followed by sustained reduction in Ser3 phosphorylation of n-cofilin, a critical actin severing protein and an identified downstream effector of Reelin signaling. This biochemical disruption is associated with sustained reduction of F-actin content and disrupted Golgi apparatus morphology in developing cortical neurons. The finding outlines a model in which the initial activation of SFKs by ethanol has the potential to disrupt multiple developmentally important signaling systems for several hours after maternal exposure.