Activation Of Src Family Kinases Research Articles

Peroxiredoxin 6 triggers melanoma cell growth by increasing arachidonic acid-dependent lipid signalling.

Tumour cells are reported to display an imbalance in the levels of ROS (reactive oxygen species). Frequently, elevated ROS production goes along with compensatory up-regulation of antioxidant enzymes. Accordingly, we found in a previous study that protein levels of several peroxiredoxins, including PRDX6 (peroxiredoxin 6), are highly elevated in experimentally induced melanomas. In the present study, we investigated the functional role of PRDX6in human melanoma cells. PRDX6 is a bifunctional enzyme, which harbours iPLA2 (Ca(2+)-independent phospholipase A2) activity in addition to its peroxidase function. Our results show that PRDX6 is strongly expressed in most melanoma cells and its expression levels are maintained in a post-transcriptional manner, particularly by EGFR (epidermal growth factor receptor)-dependent signalling. PRDX6 enhances cell viability mainly by enhancing proliferation, which goes along with activation of Src family kinases. Interestingly, we were able to show that the phospholipase activity of the enzyme mediates the pro-proliferative effect of PRDX6. We identified AA (arachidonic acid) as a crucial effector of PRDX6-dependent proliferation and inducer of Src family kinase activation. These results support further the biological importance of the emerging field of lipid signalling in melanoma and highlight the particular functional relevance of PRDX6-dependent phospholipase activity.

Leptin activates chicken growth hormone promoter without chicken STAT3 in vitro.

Leptin is an adipocyte-derived hormone that not only regulates food intake and energy homeostasis but also induces growth hormone (GH) mRNA expression and release, thereby controlling growth and metabolism in mammals. The molecular mechanism of leptin-induced regulation of GH gene transcription is unclear. The current study investigated the effects of leptin on the chicken GH (cGH) promoter and the molecular mechanism underlying leptin-induced cGH gene expression in vitro. Leptin activated the cGH promoter in the presence of chPit-1α in CHO cells stably expressing the chicken leptin receptor. Promoter activation did not require STAT-binding elements in the cGH promoter or STAT3 activity. However, JAK2 activation was required for leptin-dependent activity. JAK2-dependent pathways include p42/44 MAPK and PI3K, and inhibition of these pathways partially blocked leptin-induced cGH gene transcription. Although CK2 directly activates JAK2, a CK2 inhibitor blocked leptin-dependent activation of the cGH gene without affecting JAK2 phosphorylation. The CK2 inhibitor suppressed Erk1/2 and Akt phosphorylation. Additional data implicate Src family kinases in leptin-dependent cGH gene activation. These results suggest that leptin activates the cGH gene in the presence of chPit-1α via several leptin-activated kinases. Although further study is required, we suggest that the leptin-induced JAK2/p42/44 MAPK and JAK2/PI3K cascades are activated by Src-meditated CK2, leading to CBP phosphorylation and interaction with chPit-1α, resulting in transactivation of the cGH promoter.

Prolactin and growth hormone affect metaphase-II chromosomes in aging oocytes via cumulus cells using similar signaling pathways.

General senescence of the adult organism is closely connected with reproductive one. Meanwhile, the age-related reduction in the female fertility is primarily associated with a decline in the gamete quality. Molecular and cellular changes in oocytes of old mammalian females are very similar to those occurring during aging of matured ova of their young counterparts, suggesting similarities in underlying mechanisms. The aim of the present work was to study actions of two related pituitary hormones, prolactin (PRL) and growth hormone (GH), on age-associated modifications of metaphase-II (M-II) chromosomes in bovine oocytes using a model of the prolonged culture. We analyzed: (1) effects of PRL and GH on abnormal changes in the chromosome morphology in aging matured oocytes and the role of cumulus cells in these effects and (2) signaling pathways involved in the hormone actions. During the prolonged culture of oocytes, a gradual rise in the frequency of destructive modifications of M-II chromosomes was revealed. In the case of cumulus-enclosed oocytes (CEOs), PRL and GH exerted dose-dependent biphasic effects on the frequency of these modifications. Both PRL (50 ng/ml) and GH (10 ng/ml) decelerated the abnormal chromosome changes in CEOs, but did not affect the chromosome configuration in denuded oocytes. Concurrently, the presence of PRL and GH receptors in cumulus cells surrounding matured oocytes was demonstrated. Attenuating effects of both hormones on the chromosome modifications in aging CEOs were abolished by PP2 (an inhibitor of Src-family tyrosine kinases), triciribine (an inhibitor of Akt kinase), and calphostin C (a protein kinase C inhibitor). Our findings indicate that PRL and GH can exert the similar decelerating action on age-associated alterations in the M-II chromosome morphology in bovine ova, which is mediated by cumulus cells and may be related to activation of Src-family tyrosine kinases as well as Akt- and protein kinase C-dependent signal pathways.

Small molecule inhibition of Csk alters affinity recognition by T cells.

The C-terminal Src kinase (Csk), the primary negative regulator of Src-family kinases (SFK), plays a crucial role in controlling basal and inducible receptor signaling. To investigate how Csk activity regulates T cell antigen receptor (TCR) signaling, we utilized a mouse expressing mutated Csk (Csk(AS)) whose catalytic activity is specifically and rapidly inhibited by a small molecule. Inhibition of Csk(AS) during TCR stimulation led to stronger and more prolonged TCR signaling and to increased proliferation. Inhibition of Csk(AS) enhanced activation by weak but strictly cognate agonists. Titration of Csk inhibition revealed that a very small increase in SFK activity was sufficient to potentiate T cell responses to weak agonists. Csk plays an important role, not only in basal signaling, but also in setting the TCR signaling threshold and affinity recognition.

Inhibition of Src family kinases overcomes anoikis resistance induced by spheroid formation and facilitates cisplatin-induced apoptosis in human mesothelioma cells.

Malignant mesothelioma is an aggressive tumor arising from mesothelial cells of serous membranes, and forms spheroid-like cell aggregates in pleural and peritoneal effusions. We examined the levels of anoikis, apoptosis induced by the detachment of cells from the extracellur matrix, in suspension culture in the human mesothelioma cell line NCI-H2052. NCI-H2052 cells were adherent in conventional monolayer cultures, but were found to form spheroids in suspension cultures using dishes with ultra-low cell binding capacity. NCI-H2052 cells proliferated in both cultures, but the proliferation rate was markedly lower in suspension cultures than in monolayer cultures. In addition, NCI-H2052 cells in suspension cultures showed little apoptosis, suggesting that the suspension culture induces anoikis resistance. Western blot analysis revealed that suspension cultures induced activation of Src family kinases (SFK) after spheroid formation. Dasatinib, an inhibitor of multi-tyrosine kinases including SFK, abolished anoikis resistance in suspension cultures, indicating that SFK activated by spheroid formation are responsible for anoikis resistance. Cisplatin induced apoptosis in NCI-H2052 cells, but the apoptotic rate was significantly lower in suspension cultures than in monolayer cultures, suggesting that spheroid formation is involved in cisplatin resistance. Furthermore, a combination of dasatinib and cisplatin induced apoptosis more significantly than either alone in suspension cultures. These results suggest that spheroid formation induces resistance to anoikis and to cisplatin through SFK activation and that dasatinib facilitates cisplatin-induced apoptosis in human mesothelioma cells.

Dopaminergic and cholinergic regulation of Fyn tyrosine kinase phosphorylation in the rat striatum in vivo

Src and Fyn are two Src family kinase (SFK) members that are expressed in mammalian brains and play important roles in the regulation of a variety of neuronal and synaptic substrates. Here we investigated the responsiveness of these SFKs to changing dopamine receptor signals in dopamine responsive regions of adult rat brains in vivo. Pharmacological activation of dopamine D1 receptors (D1Rs) by a systemic injection of the selective agonist SKF81297 increased phosphorylation of SFKs at a conserved and activation-associated autophosphorylation site (Y416) in the striatum, indicating activation of SFKs following SKF81297 injection. The dopamine D2 receptor (D2R) agonist quinpirole had no effect. Blockade of D1Rs with an antagonist SCH23390 did not alter striatal Y416 phosphorylation, while the D2R antagonist eticlopride elevated it. Between Src and Fyn, SKF81297 seemed to preferentially facilitate Fyn phosphorylation. Activation of muscarinic acetylcholine M4 receptors (M4Rs) with a positive allosteric modulator VU0152100 suppressed SFK Y416 responses to SKF81297. Additionally, SKF81297 induced a correlated increase in phosphorylation of N-methyl-d-aspartate (NMDA) receptor GluN2B subunits at a Fyn site (Y1472), which was attenuated by VU0152100. SKF81297 also enhanced synaptic recruitments of active Fyn and GluN1/GluN2B-containing NMDA receptors. These data demonstrate that D1Rs regulate Fyn and downstream NMDA receptors in striatal neurons in vivo. Acetylcholine through activating M4Rs inhibits Fyn and NMDA receptors in their sensitivity to D1R signaling.

Ligands Binding to Cell Surface Ganglioside GD2 Cause Src-Dependent Activation of N-Methyl-D-Aspartate Receptor Signaling and Changes in Cellular Morphology.

Ganglioside GD2 is a plasma membrane glycosphinogolipid. In healthy adults it is expressed at low levels, but it is over-expressed in many cancers. For cancer therapy, GD2 is targeted with anti-GD2 monoclonal antibodies (mAbs), and one adverse side effect is severe visceral pain. Pain is not neuropathic, cannot be blocked with morphine, and stops on discontinuation of mAb therapy. Here, we provide evidence that ligand binding to cell surface GD2 induces rapid and transient activation of Src-family kinases, followed by Src-dependent phosphorylation of NMDA-receptor NR2B subunits selectively, activation of Ca++ fluxes, production of cAMP, and changes in cellular morphology. These GD2-ligand activated signals differ in kinetics and in pharmacology from activation of the same signals in the same cells by BDNF, the growth factor agonist of the TrkB receptor, suggesting biological specificity. Hence, cell surface GD2 regulates pathways that can be associated with neoplasia and with morphine-intractable pain; and this can explain why expression of GD2 correlates with these two pathologies.

Oxidative Stress Increases Surface Toll-Like Receptor 4 Expression in Murine Macrophages Via Ceramide Generation

Multiorgan failure is a major cause of late mortality following trauma. Oxidative stress generated during shock/resuscitation contributes to tissue injury by priming the immune system for an exaggerated response to subsequent inflammatory stimuli, such as lipopolysaccharide (LPS). We recently reported that oxidative stress causes rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts, thus increasing LPS responsiveness and cellular priming. We hypothesized that activation of Src family kinases by oxidants might contribute to these events. We utilized microscopy, flow cytometry, Western blotting, and thin-layer chromatography methods. Using hydrogen peroxide in vitro and hemorrhagic shock/resuscitation in vivo, oxidant-induced TLR4 translocation in macrophages occurred in an Src-dependent manner. Approaches supporting this conclusion included pharmacologic inhibition of the Src family kinases by PP2, Src inhibition by a molecular approach of cell transfection with Csk, and genetic inhibition of all Src kinases relevant to the monocyte/macrophage lineage in hckfgrlyn triple knockout mice. To evaluate the upstream molecules involved in Src activation, we evaluated the ability of oxidative stress to activate the bioactive lipid molecule ceramide. Oxidants induced ceramide generation in macrophages both in vitro and in vivo, an effect that appears to be due to activation of the acid sphingomyelinase. Using pharmacological approaches, ceramide was shown to be both necessary and sufficient to mediate TLR4 translocation to the plasma membrane in an Src-dependent manner. This study identifies a hierarchy of signaling molecules following oxidative stress that might represent novel targets for therapy in critical illness and organ injury.

Abstract 1854: Evidence that RARs interact with Src family kinases and that inhibition of Src family kinase activity can affect the growth response of SKOV3 ovarian cancer cell to atRA

Abstract Chemotherapy resistance is a major cause of ovarian cancer treatment failure. All trans-retinoic acid (atRA) is a promising therapeutic agent due to its growth suppressing activity however, not all ovarian carcinomas are growth inhibited by atRA. Understanding the underlying mechanisms of atRA action in ovarian cancer is crucial to overcome chemotherapy resistance. Recent studies have reported novel nongenomic actions of atRA and retinoic acid receptors (RARs). The main goal of this work was to study nongenomic actions of RARs. A SH3 domain phage display was performed to identify proteins that interact with the proline rich region of RARγ. Our results indicated that RARγ proline rich region binds to the SH3 domain of Src family kinase proteins (SFKs) Lyn, Src and Yes. Immunofluorescence assays showed that RARγ colocalizes in the cell cytoplasm with Lyn, Src and Yes. Proximity ligation assays were performed to determine whether RARs and Lyn, Src and Yes interact in vivo. Our results showed that RARα and RARγ but not RARβ form a complex with both Lyn and Src. No interaction was found between any of the RARs and Yes. Interaction between RARs and SFKs was found to be directly correlated with phosphorylation of Y419 in Src and Y397 in Lyn. We have previously shown that the ovarian cancer cell line CAOV3 is sensitive to growth inhibition by atRA while the ovarian cancer cell line SKOV3 is resistance to atRA growth repression. Since SFKs such as Src and Lyn are known to regulate cancer cell growth, we next investigated the effect of inhibiting SFKs activity on the atRA regulated growth response of these cell lines. CAOV3 and SKOV3 cells were treated with atRA (1μM) alone or in combination with varying doses of SFKs inhibitors PP1 and PP2 and analyzed for cell growth inhibition. CAOV3 but not SKOV3 cells were growth arrested by atRA. Both PP1 and PP2 caused growth inhibition in both cell lines. Interestingly, when atRA was combined with either of the SFKs inhibitors, SKOV3 but not CAOV3 cells exhibited growth inhibition greater than that observed with PP1 or PP2 alone. These data demonstrate that inhibition of SFKs activity converts the growth response of SKOV3 cells from atRA resistant to atRA sensitive. Analysis of cell cycle regulators in atRA and PP1 treated CAOV3 cells showed that both p27 and p130 are up-regulated while CDK6 is down-regulated. On the other hand, in SKOV3 cells treated with atRA and PP1, p130 is unaffected while p27, cyclin A, CDK1, CDK2 and CDK6 are down-regulated. In conclusion, we have confirmed the interaction between RARγ and Src and describe for the first time the interaction between RARα and both Lyn and Src, and RARγ and Lyn. We also provide evidence that reduction of SFKs activity and atRA treatment but not atRA treatment alone can inhibit the growth of SKOV3 cells. This suggests that SFKs activity must be reduced in order for atRA to inhibit cell growth in at least some ovarian carcinomas. Citation Format: Thais Acquafreda Lakind, Kenneth Soprano, Dianne Soprano. Evidence that RARs interact with Src family kinases and that inhibition of Src family kinase activity can affect the growth response of SKOV3 ovarian cancer cell to atRA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1854. doi:10.1158/1538-7445.AM2015-1854

EGFR signaling upregulates surface expression of the GluN2B-containing NMDA receptor and contributes to long-term potentiation in the hippocampus.

N-methyl-d-aspartate receptors (NMDARs) have been known to be regulated by various receptor tyrosine kinases. Activation of epidermal growth factor receptor (EGFR) specifically increases NMDAR-mediated currents and enhances long-term potentiation (LTP) in the hippocampus. However, the mechanism through which EGFR regulates NMDARs remains to be elucidated. In this study we found that EGFR was highly expressed in the hippocampus and mainly localized in the non-synaptic region including the soma and neurites of cultured hippocampal neurons. EGFR activation led to an increase in ifenprodil-sensitive NMDAR currents. Consistent with this, we also observed that surface expression of GluN2B-containing NMDAR was upregulated. Our biochemical data from hippocampal slices and hippocampal cultured neurons demonstrated that EGF treatment in vitro significantly increased phosphorylation of the GluN2B subunit at Y1472 with a coincidental activation of Src family kinases (SFKs). EGFR blockade with a specific antagonist BIBX-1382 attenuated an increase of GluN2B in the postsynaptic density during high-frequency stimulation (HFS)-induced LTP. Moreover, BIBX blockade significantly impaired HFS-induced LTP. In conclusion, our findings suggest that EGFR signaling upregulates NMDARs through modification of the GluN2B subunit, and is required for HFS-induced LTP in the hippocampus.

The focal adhesion kinase Pyk2 links Ca2+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin-stimulated platelets.

In blood platelets, stimulation of G protein-coupled receptors (GPCRs) by thrombin triggers the activation of Src family kinases (SFKs), resulting in the tyrosine-phosphorylation of multiple substrates, but the mechanism underlying this process is still poorly understood. In the present study, we show that the time-dependent protein-tyrosine phosphorylation triggered by thrombin in human or murine platelets was totally suppressed only upon concomitant chelation of intracellular Ca(2+) and inhibition of SFKs. Thrombin-induced activation of SFKs was regulated by intracellular Ca(2+) and accordingly the Ca(2+) ionophore A23187 was sufficient to stimulate SFKs. A23187 also triggered the phosphorylation and activation of the Ca(2+)-dependent focal adhesion kinase Pyk2 and Pyk2 activation by thrombin was Ca(2+)-dependent. Stimulation of SFKs by thrombin or A23187 was strongly reduced in platelets from Pyk2 knockout (KO) mice, as was the overall pattern of protein-tyrosine phosphorylation. By immunoprecipitation experiments, we demonstrate that Lyn and Fyn, but not Src, were activated by Pyk2. Inhibition of SFKs by PP2 also reduced the phosphorylation of Pyk2in thrombin or A23187-stimulated platelets. Analysis of KO mice demonstrated that Fyn, but not Lyn, was required for complete Pyk2 phosphorylation by thrombin. Finally, PP2 reduced aggregation of murine platelets to a level comparable to that of Pyk2-deficient platelets, but did not have further effects in the absence of Pyk2. These results indicate that in thrombin-stimulated platelets, stimulation of Pyk2 by intracellular Ca(2+) initiates SFK activation, establishing a positive loop that reinforces the Pyk2/SFK axis and allows the subsequent massive tyrosine phosphorylation of multiple substrates required for platelet aggregation.

CagA of Helicobacter pylori interacts with and inhibits the serine-threonine kinase PRK2.

CagA is a multifunctional toxin of Helicobacter pylori that is secreted into host epithelial cells by a type IV secretion system. Following host cell translocation, CagA interferes with various host-cell signalling pathways. Most notably this toxin is involved in the disruption of apical-basolateral cell polarity and cell adhesion, as well as in the induction of cell proliferation, migration and cell morphological changes. These are processes that also play an important role in epithelial-to-mesenchymal transition and cancer cell invasion. In fact, CagA is considered as the only known bacterial oncoprotein. The cellular effects are triggered by a variety of CagA activities including the inhibition of serine-threonine kinase Par1b/MARK2 and the activation of tyrosine phosphatase SHP-2. Additionally, CagA was described to affect the activity of Src family kinases and C-terminal Src kinase (Csk) suggesting that interference with multiple cellular kinase- and phosphatase-associated signalling pathways is a major function of CagA. Here, we describe the effect of CagA on protein kinase C-related kinase 2 (PRK2), which acts downstream of Rho GTPases and is known to affect cytoskeletal rearrangements and cell polarity. CagA interacts with PRK2 and inhibits its kinase activity. Because PRK2 has been linked to cytoskeletal rearrangements and establishment of cell polarity, we suggest that CagA may hijack PRK2 to further manipulate cancer-related signalling pathways.

Effects of activation of Src-family tyrosine kinases on human LECs apoptosis and EMT under the high glucose environment

Background Diabetic cataract is one of the major ocular complications in diabetes mellitus, including cortical cataract, nuclear cataract, subcapsular cataract and mixed cataract, and different cataractogenesis may be associated with lens epithelial cells (LECs). Subcapsular cataract is one of diabetic cataracts.Studying the biological behavior of LECs in subcapsular cataract is crucial for prevention and treatment. Objective This study was to investigate the effects of Src-family tyrosine kinases (SFKs) on apoptosis and epithelial-mesenchymal transition (EMT) of human LECs cultured by high glucose. Methods Human LECs (HLE-B3) were cultured for 24 hours with DMEM containing 5.5 mmol/L glucose (normal control group), DMEM containing 35.5 mmol/L glucose (high glucose group) and DMEM containing 35.5 mmol/L glucose + 10 μmol/L PP1, a specific inhibitor of SFKs (PP1 group). In 3, 6, 12 and 24 hours after culture, the apoptosis of human LECs was detected by flow cytometry assay; morphological change of human LECs was observed under the inverted microscope, and the expressions of the markers of EMT, E-cadherin and α-smooth muscle actin (α-SMA), in the cells were detected by immnofluorescence staining.In addition, the alternations of p-Src418 (active c-Src), bcl-xl, survivin, caspase-3, E-cadherin and α-SMA proteins were assayed by Western blot analysis. Results An elevated expression level of p-Src418 was found in LECs in the high glucose group and peaked 6 hours after cultured.The expressions of p-Src418 (grey levels) were 0.125±0.036 in the high glucose group, and which was significantly higher than 0.042±0.011 in the normal control group and 0.035±0.018 in the PP1 group, respectively (both at P 0.05). The apoptotic rates of human LECs were(6.433±2.084)%, (10.333±2.610)% and (8.033±2.967)% in the PP1 group, which were higher than (3.233±1.320)%, (3.533±1.159)%, (5.733±0.230)% in the high glucose group and (3.133±1.170)%, (2.833±0.751)%, (3.333±1.201)% in the normal control group (all at P 0.05). In 6 hours and 12 hours after cell culture, the expression levels of bcl-xl and survivin (grey values) in human LECs were significantly declined, but the expression of caspase-3 was increased in the PP1 group compared with the the high glucose group and the normal control group (all at P<0.05). The LECs showed slender in shape 24 hours after culture in the high glucose group, but the cell shape was close to the normal in the PP1 group.Western blot and immunofluorescence assay revealed that the expression of E-cadherin in human LECs was significantly reduced and that of α-SMA was significantly increased 6 hours after culture in the high glucose group compared with the PP1 group and the normal control group (all at P<0.05). Conclusions High glucose activates c-Src kinase of LECs in high glucose environment and therefore induces EMT and inhibits apoptosis.However, PP1 impedes the biological process of EMT and apoptosis of LECs to maintain the epithelial characteristics even under the stress of high glucose. Key words: Glucose; Human; Epithelial cell, Lens; Apoptosis; Src-family tyrosine kinases; Epithelial-mesenchymal transition; Signaling pathway

GRID and docking analyses reveal a molecular basis for flavonoid inhibition of Src family kinase activity

Flavonoids reduce cardiovascular disease risk through anti-inflammatory, anti-coagulant and anti-platelet actions. One key flavonoid inhibitory mechanism is blocking kinase activity that drives these processes. Flavonoids attenuate activities of kinases including phosphoinositide-3-kinase, Fyn, Lyn, Src, Syk, PKC, PIM1/2, ERK, JNK and PKA. X-ray crystallographic analyses of kinase–flavonoid complexes show that flavonoid ring systems and their hydroxyl substitutions are important structural features for their binding to kinases. A clearer understanding of structural interactions of flavonoids with kinases is necessary to allow construction of more potent and selective counterparts.We examined flavonoid (quercetin, apigenin and catechin) interactions with Src family kinases (Lyn, Fyn and Hck) applying the Sybyl docking algorithm and GRID. A homology model (Lyn) was used in our analyses to demonstrate that high-quality predicted kinase structures are suitable for flavonoid computational studies. Our docking results revealed potential hydrogen bond contacts between flavonoid hydroxyls and kinase catalytic site residues. Identification of plausible contacts indicated that quercetin formed the most energetically stable interactions, apigenin lacked hydroxyl groups necessary for important contacts and the non-planar structure of catechin could not support predicted hydrogen bonding patterns. GRID analysis using a hydroxyl functional group supported docking results. Based on these findings, we predicted that quercetin would inhibit activities of Src family kinases with greater potency than apigenin and catechin. We validated this prediction using in vitro kinase assays.We conclude that our study can be used as a basis to construct virtual flavonoid interaction libraries to guide drug discovery using these compounds as molecular templates.

Src Family Kinases (SFK) Mediate Angiotensin II-Induced Myosin Light Chain Phosphorylation and Hypertension.

Angiotensin (Ang) II is the major bioactive peptide of the renin–angiotensin system (RAS); it contributes to the pathogenesis of hypertension by inducing vascular contraction and adverse remodeling, thus elevated peripheral resistance. Ang II also activates Src family kinases (SFK) in the vascular system, which has been implicated in cell proliferation and migration. However, the role of SFK in Ang II-induced hypertension is largely unknown. In this study, we found that administration of a SFK inhibitor SU6656 markedly lowered the level of systemic BP in Ang II-treated mice, which was associated with an attenuated phosphorylation of the smooth-muscle myosin-light-chain (MLC) in the mesenteric resistant arteries. In the cultured human coronary artery smooth muscle cells (SMCs), pretreatment with SU6656 blocked Ang II-induced MLC phosphorylation and contraction. These results for the first time demonstrate that SFK directly regulate vascular contractile machinery to influence BP. Thus our study provides an additional mechanistic link between Ang II and vasoconstriction via SFK-enhanced MLC phosphorylation in SMCs, and suggests that targeted inhibition of Src may provide a new therapeutic opportunity in the treatment of hypertension.

Regulation of RPTPα-c-Src signalling pathway by miR-218.

Receptor protein tyrosine phosphatase alpha (RPTPα), an activator of Src family kinases, is found significantly overexpressed in human cancer tissues. However, little is known about the regulation of RPTPα expression. miRNAs target multiple genes and play important roles in many cancer processes. Here, we identified a miRNA, miR-218 that binds directly to the 3'-UTR of RPTPα. Ectopic overexpression of miR-218 decreased RPTPα protein leading to decreased dephosphorylation of c-Src and decreased tumour growth invitro and invivo. A feedback loop between c-Src and miR-218 was revealed where c-Src inhibits transcription of SLIT2, which intronically hosts miR-218. These results show a novel regulatory pathway for RPTPα-c-Src signalling.

The anti-inflammatory effect of 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol by targeting Lyn kinase in human neutrophils

The undesirable respiratory burst in neutrophils can lead to inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol (honokiol), a lignan extracted from the stem bark of Magnolia officinalis Rehd. et Wils (Magnoliaceae), on N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)-induced respiratory burst in human neutrophils. Signaling pathways regulated by honokiol which modulate fMLP-induced respiratory burst and cathepsin G release were evaluated by phosphorylation of Src family kinase induced by fMLP, Src family kinases activities and by immunoblotting analysis of the downstream targets of Src kinase. Briefly, honokiol inhibited fMLP-induced superoxide anion production (IC50=9.80±0.21μM, n=4), cathepsin G release (IC50=14.23±1.43μM, n=4) and migration (IC50=5.69±1.51μM, n=4) in a concentration dependent manner. Further, honokiol specifically suppresses fMLP-induced Lyn (a member of the Src kinase family) phosphorylation, by inhibiting Lyn kinase activity. Consequently, honokiol attenuated the downstream targets of Lyn kinase, such as Tec translocation from the cytosol to the inner leaflet of the plasma membrane, phosphorylation of AKT, P38, PLCγ2, protein kinase C and membrane localization of p47phox. On the other hand, fMLP-induced phosphorylation of Hck, Fgr kinase activity (other members of Src kinase), downstream phosphorylation of Vav1 and extracellular signal-regulated kinase remained unaffected. In addition, honokiol neither inhibited NADPH oxidase activity nor increased cyclic AMP levels. Honokiol is not a competitive or allosteric antagonist of fMLP. In conclusion, honokiol specifically modulates fMLP-mediated neutrophil activation by inhibiting Lyn activation which subsequently interferes with the activation of PLCγ2, AKT, p38, protein kinase C, and p47phox.

EGFR-activated Src family kinases maintain GAB1-SHP2 complexes distal from EGFR.

Complexes of signaling proteins that are nucleated upon activation of receptor tyrosine kinases are dynamic macromolecular assemblies held together by interactions, such as the recognition of phosphotyrosines by Src homology 2 (SH2) domains. We predicted that reversible binding and phosphatase activity enable dynamic regulation of these protein complexes, which could affect signal transduction. We explored how dynamics in the interactions among the epidermal growth factor (EGF) receptor (EGFR), GRB2-associated binder protein 1 (GAB1), and SH2 domain-containing phosphatase 2 (SHP2) affected EGFR signaling output, specifically SHP2 binding to tyrosine-phosphorylated GAB1, which relieves the autoinhibition of SHP2. Among the effects of activated SHP2 is increased extracellular signal-regulated kinase (ERK) activity. We found that in H1666 lung adenocarcinoma cells, EGFR-activated Src family kinases (SFKs) counteracted repeated GAB1 dephosphorylation events and maintained the association of SHP2 with phosphorylated GAB1 at a cytosolic site distal from EGFR. A computational model predicted that an experimentally verified delay in SFK inactivation after EGFR inactivation, combined with an amplification of GAB1 phosphorylation in cells with proteins in a specific range of concentrations, enabled GAB1 phosphorylation and GAB1-SHP2 complexes to persist longer than EGFR phosphorylation persisted in response to EGF. This SFK-dependent mechanism was specific to EGFR and did not occur in response to activation of the receptor tyrosine kinase c-MET. Thus, our results quantitatively describe a regulatory mechanism used by some receptor tyrosine kinases to remotely control the duration of a signal by regulating the persistence of a signaling protein complex.

Systematic investigation of CMTM family genes suggests relevance to glioblastoma pathogenesis and CMTM1 and CMTM3 as priority targets.

The novel CKLF-like Marvel Transmembrane Domain-containing gene family (CMTM) consists of 8 members (CMTM1-8). As little is known about the oncogenic impact of these genes, we aimed to systematically investigate the relevance of CMTMs to glioblastoma pathogenesis. We performed mRNA expression analyses and survival correlations in glioblastoma patients. Moreover, we analyzed the impact of RNAi-based silencing and overexpression of CMTM family genes on tumor cell proliferation and invasion in vitro. CMTMs appeared to be widely regulated in the group of glioblastomas relative to non-neoplastic brain (NB) tissue (significant upregulation for CMTM2, 3, and 6 and significant downregulation for CMTM 4 and 8). For CMTM1, 5 and 7, we found aberrant expression levels in individual tumors. Functionally, CMTM1, 3, and 7 promoted tumor cell invasion, while CMTM1 additionally enhanced cell proliferation. In a large clinically annotated dataset, higher CMTM1 and 3 expression was significantly correlated with shorter overall survival. Our data thus suggest CMTM1 and 3 as priority targets in glioblastomas. Using a human phosphokinase protein expression profiling assay, we can provide first insights into signalling of these two genes that might be conveyed by growth factor receptor, Src family kinase and WNT activation.

Neuroblastoma tyrosine kinase signaling networks involve FYN and LYN in endosomes and lipid rafts.

Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.