Src Tyrosine Kinase Research Articles

Src-mediated phosphorylation of GAPDH regulates its nuclear localization and cellular response to DNA damage.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme involved in energy metabolism. Recently, GAPDH has been suggested to have extraglycolytic functions in DNA repair, but the underlying mechanism for the GAPDH response to DNA damage remains unclear. Here, we demonstrate that the tyrosine kinase Src is activated under DNA damage stress and phosphorylates GAPDH at Tyr41. This phosphorylation of GAPDH is essential for its nuclear translocation and DNA repair function. Blocking the nuclear import of GAPDH by suppressing Src signaling or through a GAPDH Tyr41 mutation impairs its response to DNA damage. Nuclear GAPDH is recruited to DNA lesions and associates with DNA polymerase β (Pol β) to function in DNA repair. Nuclear GAPDH promotes Pol β polymerase activity and increases base excision repair (BER) efficiency. Furthermore, GAPDH knockdown dramatically decreases BER efficiency and sensitizes cells to DNA damaging agents. Importantly, the knockdown of GAPDH in colon cancer SW480 cells and xenograft models effectively enhances their sensitivity to the chemotherapeutic drug 5-FU. In summary, our findings provide mechanistic insight into the new function of GAPDH in DNA repair and suggest a potential therapeutic target in chemotherapy.

Src Inhibitors Pyrazolo[3,4-d]pyrimidines, Si306 and Pro-Si306, Inhibit Focal Adhesion Kinase and Suppress Human Glioblastoma Invasion In Vitro and In Vivo.

Glioblastoma (GBM), as the most aggressive brain tumor, displays a high expression of Src tyrosine kinase, which is involved in the survival, migration, and invasiveness of tumor cells. Thus, Src emerged as a potential target for GBM therapy. The effects of Src inhibitors pyrazolo[3,4-d]pyrimidines, Si306 and its prodrug pro-Si306 were investigated in human GBM cell lines (U87 and U87-TxR) and three primary GBM cell cultures. Primary GBM cells were more resistant to Si306 and pro-Si306 according to the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. However, the ability of all GBM cells to degrade the extracellular matrix was considerably compromised after Si306 and pro-Si306 applications. Besides reducing the phosphorylation of Src and its downstream signaling pathway components, both compounds decreased the phosphorylated form of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) expression, showing the potential to suppress the aggressiveness of GBM. In vivo, Si306 and pro-Si306 displayed an anti-invasive effect against U87 xenografts in the zebrafish embryo model. Considering that Si306 and pro-Si306 are able to cross the blood–brain barrier and suppress the spread of GBM cells, we anticipate their clinical testing in the near future. Moreover, the prodrug showed similar efficacy to the drug, implying the rationality of its use in clinical settings.

SRC Kinase in Glioblastoma News from an Old Acquaintance.

Glioblastoma multiforme (GBM) is one of the most recalcitrant brain tumors characterized by a tumor microenvironment (TME) that strongly supports GBM growth, aggressiveness, invasiveness, and resistance to therapy. Importantly, a common feature of GBM is the aberrant activation of receptor tyrosine kinases (RTKs) and of their downstream signaling cascade, including the non-receptor tyrosine kinase SRC. SRC is a central downstream intermediate of many RTKs, which triggers the phosphorylation of many substrates, therefore, promoting the regulation of a wide range of different pathways involved in cell survival, adhesion, proliferation, motility, and angiogenesis. In addition to the aforementioned pathways, SRC constitutive activity promotes and sustains inflammation and metabolic reprogramming concurring with TME development, therefore, actively sustaining tumor growth. Here, we aim to provide an updated picture of the molecular pathways that link SRC to these events in GBM. In addition, SRC targeting strategies are discussed in order to highlight strengths and weaknesses of SRC inhibitors in GBM management, focusing our attention on their potentialities in combination with conventional therapeutic approaches (i.e., temozolomide) to ameliorate therapy effectiveness.

ErbB1-dependent signalling and vesicular trafficking in primary afferent nociceptors associated with hypersensitivity in neuropathic pain

Effective analgesic treatment for neuropathic pain remains an unmet need, so previous evidence that epidermal growth factor receptor inhibitors (EGFRIs) provide unexpected rapid pain relief in a clinical setting points to a novel therapeutic opportunity. The present study utilises rodent models to address the cellular and molecular basis for the findings, focusing on primary sensory neurons because clinical pain relief is provided not only by small molecule EGFRIs, but also by the anti-EGFR antibodies cetuximab and panitumumab, which are unlikely to access the central nervous system in therapeutic concentrations. We report robust, rapid and dose-dependent analgesic effects of EGFRIs in two neuropathic pain models, matched by evidence with highly selective antibodies that expression of the EGFR (ErbB1 protein) is limited to small nociceptive afferent neurons. As other ErbB family members can heterodimerise with ErbB1, we investigated their distribution, showing consistent co-expression of ErbB2 but not ErbB3 or ErbB4, with ErbB1 in cell bodies of nociceptors, as well as providing evidence for direct molecular interaction of ErbB1 with ErbB2 in situ. Co-administration of selective ErbB1 and ErbB2 inhibitors produced clear evidence of greater-than-additive, synergistic analgesia; highlighting the prospect of a unique new combination therapy in which enhanced efficacy could be accompanied by minimisation of side-effects. Peripheral (intraplantar) administration of EGF elicited hypersensitivity only following nerve injury and this was reversed by local co-administration of selective inhibitors of either ErbB1 or ErbB2. Investigating how ErbB1 is activated in neuropathic pain, we found evidence for a role of Src tyrosine kinase, which can be activated by signals from inflammatory mediators, chemokines and cytokines during neuroinflammation. Considering downstream consequences of ErbB1 activation in neuropathic pain, we found direct recruitment to ErbB1 of an adapter for PI 3-kinase and Akt signalling together with clear Akt activation and robust analgesia from selective Akt inhibitors. The known Akt target and regulator of vesicular trafficking, AS160 was strongly phosphorylated at a perinuclear location during neuropathic pain in an ErbB1-, ErbB2- and Akt-dependent manner, corresponding to clustering and translocation of an AS160-partner, the vesicular chaperone, LRP1. Exploring whether neuronal ion channels that could contribute to hyperexcitability might be transported by this vesicular trafficking pathway we were able to identify Nav1.9, (Nav1.8) and Cav1.2 moving towards the plasma membrane or into proximal axonal locations – a process prevented by ErbB1 or Akt inhibitors. Overall these findings newly reveal both upstream and downstream signals to explain how ErbB1 can act as a signalling hub in neuropathic pain models and identify the trafficking of key ion channels to neuronal subcellular locations likely to contribute to hyperexcitability. The new concept of combined treatment with ErbB1 plus ErbB2 blockers is mechanistically validated as a promising strategy for the relief of neuropathic pain.

Temperature sensitivities of metazoan and pre-metazoan Src kinases

Homologous enzymes from different species display functional characteristics that correlate with the physiological and environmental temperatures encountered by the organisms. In this study, we have investigated the temperature sensitivity of the nonreceptor tyrosine kinase Src. We compared the temperature dependencies of c-Src and two Src kinases from single-celled eukaryotes, the choanoflagellate Monosiga brevicollis and the filasterean Capsaspora owczarzaki. Metazoan c-Src exhibits temperature sensitivity, with high activity at 30 °C and 37 °C. This sensitivity is driven by changes in substrate binding as well as maximal velocity, and it is dependent on the amino acid sequence surrounding tyrosine in the substrate. When tested with a peptide that displays temperature-dependent phosphorylation by c-Src, the enzymatic rates for the unicellular Src kinases show much less variation over the temperatures tested. The data demonstrate that unicellular Src kinases are temperature compensated relative to metazoan c-Src, consistent with an evolutionary adaptation to their environments.

Fundamental control of grade-specific colorectal cancer morphology by Src regulation of ezrin-centrosome engagement.

The phenotypic spectrum of colorectal cancer (CRC) is remarkably diverse, with seemingly endless variations in cell shape, mitotic figures and multicellular configurations. Despite this morphological complexity, histological grading of collective phenotype patterns provides robust prognostic stratification in CRC. Although mechanistic understanding is incomplete, previous studies have shown that the cortical protein ezrin controls diversification of cell shape, mitotic figure geometry and multicellular architecture, in 3D organotypic CRC cultures. Because ezrin is a substrate of Src tyrosine kinase that is frequently overexpressed in CRC, we investigated Src regulation of ezrin and morphogenic growth in 3D CRC cultures. Here we show that Src perturbations disrupt CRC epithelial spatial organisation. Aberrant Src activity suppresses formation of the cortical ezrin cap that anchors interphase centrosomes. In CRC cells with a normal centrosome number, these events lead to mitotic spindle misorientation, perturbation of cell cleavage, abnormal epithelial stratification, apical membrane misalignment, multilumen formation and evolution of cribriform multicellular morphology, a feature of low-grade cancer. In isogenic CRC cells with centrosome amplification, aberrant Src signalling promotes multipolar mitotic spindle formation, pleomorphism and morphological features of high-grade cancer. Translational studies in archival human CRC revealed associations between Src intensity, multipolar mitotic spindle frequency and high-grade cancer morphology. Collectively, our study reveals Src regulation of CRC morphogenic growth via ezrin-centrosome engagement and uncovers combined perturbations underlying transition to high-grade CRC morphology. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

SRC Tyrosine Kinase Inhibitor and X-rays Combined Effect on Glioblastoma Cell Lines.

Glioblastoma (GBM) is one of the most lethal types of tumor due to its high recurrence level in spite of aggressive treatment regimens involving surgery, radiotherapy and chemotherapy. Hypoxia is a feature of GBM, involved in radioresistance, and is known to be at the origin of treatment failure. The aim of this work was to assess the therapeutic potential of a new targeted c-SRC inhibitor molecule, named Si306, in combination with X-rays on the human glioblastoma cell lines, comparing normoxia and hypoxia conditions. For this purpose, the dose modifying factor and oxygen enhancement ratio were calculated to evaluate the Si306 radiosensitizing effect. DNA damage and the repair capability were also studied from the kinetic of γ-H2AX immunodetection. Furthermore, motility processes being supposed to be triggered by hypoxia and irradiation, the role of c-SRC inhibition was also analyzed to evaluate the migration blockage by wound healing assay. Our results showed that inhibition of the c-SRC protein enhances the radiotherapy efficacy both in normoxic and hypoxic conditions. These data open new opportunities for GBM treatment combining radiotherapy with molecularly targeted drugs to overcome radioresistance.

TASK channels: channelopathies, trafficking, and receptor-mediated inhibition

TWIK-related acid-sensitive K+ (TASK) channels contribute to the resting membrane potential in various kinds of cells, such as brain neurons, smooth muscle cells, and endocrine cells. Loss-of-function mutations at multiple sites in the KCNK3 gene encoding for TASK1 channels are one of the causes of pulmonary arterial hypertension in humans, whereas a mutation at only one site is reported for TASK3 channels, resulting in a syndrome of mental retardation, hypotonia, and facial dysmorphism. TASK channels are subject to regulation by G protein-coupled receptors (GPCRs). Two mechanisms have been proposed for the GPCR-mediated inhibition of TASK channels: a change in gating and channel endocytosis. The most feasible mechanism for altered gating is diacylglycerol binding to a site in the C-terminus, which is shared by TASK1 and TASK3. The inhibition of channel function by endocytosis requires the presence of a tyrosine residue subjected to phosphorylation by the non-receptor tyrosine kinase Src and a dileucine motif in the C-terminus of TASK1. Therefore, homomeric TASK1 and heteromeric TASK1-TASK3 channels, but not homomeric TASK3, are internalized by GPCR stimulation. Tyrosine phosphorylation by Src is expected to result in a conformational change in the C-terminus, allowing for AP-2, an adaptor protein for clathrin, to bind to the dileucine motif. It is likely that a raft membrane domain is a platform where TASK1 is located and the signaling molecules protein kinase C, Pyk2, and Src are recruited in sequence in response to GPCR stimulation.

Blocking immunosuppressive neutrophils deters pY696-EZH2-driven brain metastases.

The functions of immune cells in brain metastases are unclear because the brain has traditionally been considered "immune privileged." However, we found that a subgroup of immunosuppressive neutrophils is recruited into the brain, enabling brain metastasis development. In brain metastatic cells, enhancer of zeste homolog 2 (EZH2) is highly expressed and phosphorylated at tyrosine-696 (pY696)-EZH2 by nuclear-localized Src tyrosine kinase. Phosphorylation of EZH2 at Y696 changes its binding preference from histone H3 to RNA polymerase II, which consequently switches EZH2's function from a methyltransferase to a transcription factor that increases c-JUN expression. c-Jun up-regulates protumorigenic inflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF), which recruits Arg1+- and PD-L1+ immunosuppressive neutrophils into the brain to drive metastasis outgrowth. G-CSF-blocking antibodies or immune checkpoint blockade therapies combined with Src inhibitors impeded brain metastasis in multiple mouse models. These findings indicate that pY696-EZH2 can function as a methyltransferase-independent transcription factor to facilitate the brain infiltration of immunosuppressive neutrophils, which could be clinically targeted for brain metastasis treatment.

Focal adhesion kinase and Src mediate microvascular hyperpermeability caused by fibrinogen- γC- terminal fragments.

ObjectivesWe previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvβ3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation.Methods and resultsUsing intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition.ConclusionThe FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.

Oncogenic role of LYN in human gastric cancer via the Wnt/β-catenin and AKT/mTOR pathways.

LYN kinase (LYN) is a member of the Src tyrosine kinase family, which plays an important role in multiple tumor-related functions. The current study demonstrated that LYN functions as a pro-oncogene in AGS gastric cancer cells. It was found that LYN expression levels were significantly raised in gastric cancer tissue and were significantly associated with the pathological grades of patients with gastric cancer. This was accomplished by knocking down LYN in AGS cells using short hairpin RNA (shRNA) plasmid transfection, with reverse transcription-quantitative PCR detection verifying the effectiveness of RNA interference. It was found that the cell proliferation and colony formation abilities of AGS cells were significantly inhibited, using CCK-8 and clone formation assays, respectively. Furthermore, LYN knockdown was found to induce apoptosis and inhibit both migration and invasion in AGS cells, using flow cytometry and Transwell assays, respectively. A mechanical investigation further suggested that LYN knockdown resulted in the activation of the mitochondrial apoptotic pathway. Likewise, the Wnt/β-catenin pathway was inactivated by LYN knockdown, including decreased levels of Wnt3a, β-catenin, snail family transcriptional repressor (Snail)1 and Snail2. Epithelial-mesenchymal transition mesenchymal markers (including N-cadherin and vimentin) were also found to be downregulated, and E-cadherin was upregulated in LYN-silenced AGS cells. Finally, the AKT/mTOR pathway was found to be downregulated by LYN knockdown in AGS cells, including decreased levels of phosphorylated (p)-AKT (Ser473), p-mTOR (Ser2448), and the down-stream effector p70S6 kinase (p70S6K). Furthermore, the AKT pathway activator, insulin like growth factor-1 (IGF-1), was found to reverse the inhibitory effects of LYN knockdown on the proliferation, migration and invasion of AGS cells. In conclusion, the current study demonstrated that LYN plays an oncogenic role in the proliferation, survival and movement of human gastric cancer cells by activating the mitochondrial apoptotic pathway, and downregulating the Wnt/β-catenin and AKT/mTOR pathways. The current research provides a comprehensive insight into the regulation of LYN in gastric cancer and may help with the development of new tumor treatment strategies.

Expression of a Constitutively Active Form of Hck in Chondrocytes Activates Wnt and Hedgehog Signaling Pathways, and Induces Chondrocyte Proliferation in Mice.

Runx2 is required for chondrocyte proliferation and maturation. In the search of Runx2 target genes in chondrocytes, we found that Runx2 up-regulated the expression of hematopoietic cell kinase (Hck), which is a member of the Src tyrosine kinase family, in chondrocytes, that Hck expression was high in cartilaginous limb skeletons of wild-type mice but low in those of Runx2–/– mice, and that Runx2 bound the promoter region of Hck. To investigate the functions of Hck in chondrocytes, transgenic mice expressing a constitutively active form of Hck (HckCA) were generated using the Col2a1 promoter/enhancer. The hind limb skeletons were fused, the tibia became a large, round mass, and the growth plate was markedly disorganized. Chondrocyte maturation was delayed until E16.5 but accelerated thereafter. BrdU-labeled, but not terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive, chondrocytes were increased. Furthermore, Hck knock-down reduced the proliferation of primary chondrocytes. In microarray and real-time RT-PCR analyses using hind limb RNA from HckCA transgenic mice, the expression of Wnt (Wnt10b, Tcf7, Lef1, Dkk1) and hedgehog (Ihh, Ptch1, and Gli1) signaling pathway genes was upregulated. These findings indicated that Hck, whose expression is regulated by Runx2, is highly expressed in chondrocytes, and that HckCA activates Wnt and hedgehog signaling pathways, and promotes chondrocyte proliferation without increasing apoptosis.

Investigation of type 1 angiotensin II receptor activation induced gene expression changes in rat vascular smooth muscle cells

Angiotensin II (AngII) mainly acts through type 1 angiotensin II receptor (AT1R) to promote a broad variety of biological effects. The vascular smooth muscle cells are one of the main target of AngII and its stimulation activates numerous signaling pathways which could result in gene expression changes in these cells.We carried out Affymetrix GeneChip experiments to analyze the effects of AngII stimulation on gene expression. More than 200 genes were upregulated in response to AngII stimulation of primary rat vascular smooth muscle cells in our experimental setup. We have selected several genes (DUSP5, DUSP6, DUSP10, Lmcd1, HbEGF, and endothelin) to further investigate the kinetics of the gene‐expression changes, and the possible signal transduction processes which lead to the altered expression changes after AngII stimulation. The results of the quantitative PCR measurements confirmed the AngII‐induced upregulation of the investigated genes. The maximal induction of the most genes appeared two hours after the AngII stimulation. Our data show that the upregulated genes can be stimulated through several, possibly parallel signal transduction pathways. We also examined the role of epidermal growth factor receptor (EGFR) transactivation in the upregulation of the investigated genes. According to our data the EGF receptor transactivation is not critical in response to AngII stimulation since silencing of EGF receptor did not significantly alter the AngII induced gene expression changes. Our pharmacological approach revealed that dasatinib, which is a potent inhibitor of ABL and Src family soluble tyrosine kinases, can significantly inhibit several gene expression changes. Interestingly, when we applied ABL kinase inhibitors, which have less potency toward Src tyrosine kinases such as imatinib or nilotinib did not evoke similar effects as dasatinib.Our data can help to understand the details of AngII‐induced long term effects and the pathophysiology of AT1R, moreover it can help to develop potential interventions in symptoms when this receptor over functions such as vascular remodeling or atherosclerosis.Support or Funding InformationThis work was supported by the National Research, Development and Innovation Fund (NKFI K116954 and NVKP_16‐1‐2016‐0039).

Phosphorylation and function of OATP1B1 with tyrosine kinase inhibitors.

IntroductionThe organic anion transporting polypeptide OATP1B1 regulates hepatic uptake of many therapeutics. As a result, drug‐drug interactions (DDI’s) are commonly mediated by OATP1B1 activity. Reduced drug elimination via this transporter can lead to increased rates of drug‐associated adverse reactions, such as statin‐induced rhabdomyolysis. Surprisingly, numerous tyrosine kinase inhibitors (TKIs) that are either weak or non‐substrates of OATP1B1 increase systemic concentrations of various known OATP1B1 substrates by an unknown mechanism. We hypothesize that OATP1B1 function is dependent on tyrosine phosphorylation, which can be reduced by specific TKIs.MethodsPrimary human hepatocytes and HEK293 cells transfected with OATP1B1, or tyrosine (Y) to phenylalanine (F) mutant variants of OATP1B1, were used to assess the uptake of 8‐[2‐[Fluoresceinyl]‐aminoethylthio)‐adenosine‐3′,5′‐cyclic‐monophosphate (5–40 μM) or estradiol‐b‐glucuronide (2 μM) in the presence or absence of FDA approved TKIs. A biotinylation labeling assay was utilized to measure surface expression of OATP1B1 treated with and without nilotinib (0–20 μM). Phosphorylation of OATP1B1 was assessed by Western blot and mass spectrometry‐based proteomic analysis. OATP1B1 protein was purified by immunoprecipitation from overexpressing cell lines in the presence or absence of nilotinib. siRNA was utilized to identify kinases that potentially mediate OATP1B1 function. Statistical analysis was performed with Graph‐pad Prism, and each experiment was repeated in triplicate.ResultsNine of 46 FDA approved TKIs inhibit OATP1B1 function over 80%, compared to untreated controls. Nilotinib, the most potent TKI among assessed, does not decrease OATP1B1 expression or membrane localization. Instead, it elicits inhibition of OATP1B1 through a non‐ or mixed‐competitive and reversible mechanism. Nilotinib also reduces OATP1B1‐mediated uptake in primary human hepatocytes (P < 0.001). Mass spectrometry analysis of OATP1B1 revealed a variety of tyrosine phosphorylation sites, among them, phosphorylation at position 625 is greatly reduced in the presence of nilotinib. Moreover, mutation of Y625F results in significantly reduced transport activity compared to the wild‐type OATP1B1 activity (P < 0.001). Finally, we confirmed that SiRNA knocking down of LYN kinase activity, a Src tyrosine kinase, significantly reduces OATP1B1 activity (P < 0.001).ConclusionWe found that OATP1B1 is tyrosine phosphorylated at numerous sites and that position 625 is highly sensitive to nilotinib exposure. The Y625F mutation of OATP1B1 results in decreased transport activity and similarly, loss of LYN expression also leads to lower OATP1B1 transport. These findings indicate that clinically relevant TKIs may promote drug‐drug interactions by interfering with a post‐translational event that is required for OATP1B1‐mediated transport.Support or Funding InformationUniversity at Buffalo School of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Seed Support Program

Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the channel

The voltage-gated potassium channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 channel. The low osmolarity-induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5 Our results further showed that the extracellular S1-S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1-S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1-S2 linker and made it no longer susceptible to proteinase K-mediated cleavage. In summary, the findings of our study indicate that the S1-S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this channel. By targeting the S1-S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic channel inhibition and results in an increase in IKv1.5.

Role of Src homology/collagen adaptor protein p66Shc in Pyk2 translocation into mitochondria under Gq protein–coupled receptor stimulation

IntroductionMitochondrial Ca2+ (mtCa2+) uptake via a main mtCa2+ channel, mtCa2+ uniporter (MCU), mediates cell survival and death via its contribution of mitochondrial ATP synthesis and apoptotic signaling activation, respectively. Our group previously showed that Gq protein–coupled receptor (GqPCR) stimulation promotes activation of a tyrosine kinase named proline‐rich tyrosine kinase 2 (Pyk2), and its translocation from cytosol to the mitochondrial matrix, which leads to the tyrosine phosphorylation of MCU, and mtCa2+ overload, and activation of apoptotic signaling in the cardiomyocytes. However, it is still unclear how Pyk2 access from cytosol to mitochondrial matrix under GqPCR stimulation. The Src homology/collagen (Shc) adaptor protein, p66Shc, is also known to be capable to translocate to the mitochondria where it promotes increased ROS production and apoptosis. Importantly, p66Shc can bind to another tyrosine kinase Src, but its association to Pyk2 has not been well investigated.Hypothesisp66Shc serves as an important chaperon for Pyk2 translocation into the mitochondrial matrix.Aimto test whether p66Shc can associate with Pyk2.MethodsMammalian expression plasmids containing p66Shc or Pyk2 were transformed in to bacteria and purified. Overexpression of p66Shc and/or Pyk2 in HEK293T cells were performed by transient plasmid transfection. Whole cell lysates were collected and used for biochemical assay including Western blotting and co‐immunoprecipitation assay.ResultsWe confirmed that transient transfections of the plasmids containing His‐tagged p66Shc and GFP‐tagged Pyk2 were successfully yield overexpression of these proteins in HEK293Tcells. Co‐immunoprecipitation assay using GFP antibody showed that p66Shc and Pyk2 forms complex in the whole cell lysates from cells co‐transfected with two plasmids.Conclusionp66Shc can bind Pyk2 and may contribute to the mechanism for Pyk2 translocation to mitochondria. Pur next plan is to test whether the removal (overexpression) of p66Shc blocks (Pyk2 transportation and prevent excessive calcium uptake by the MCU under GqPCR stimulation both in heterologous expression system and primary cardiomyocytes.Support or Funding InformationApart of this research was supported by the Lillehei Heart Institute Summer Research Fellowship (to G.D.), American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.

Src mediates β-adrenergic receptor induced YAP tyrosine phosphorylation.

The Hippo pathway is a newly identified pathway and evolutionarily conserved from flies to humans mainly regulating cell proliferation. Transcriptional co-activator Yes-associated protein (YAP) functions as a major downstream effector and key node of the Hippo pathway. Phosphorylation of YAP is critical to regulate YAP activity and its corresponding functions. β-adrenergic receptor (β-AR), a typical G protein coupled receptor (GPCR), mediates proliferation in various cell types and regulates multiple physical and pathological processes. However, the role of β-AR in regulating YAP remains elusive. Here, we report that β-AR can obviously stimulate YAP tyrosine phosphorylation. The mechanism is that β-AR stimulation results in tyrosine kinase Src activation and Src phosphorylates YAP tyrosine at Y357. Further studies demonstrate that inhibition of Src kinase activity can obviously alleviate β-AR induced YAP tyrosine phosphorylation and cell proliferation. We conclude that β-AR can induce YAP tyrosine phosphorylation and also establish the Src/YAP pathway as a critical signaling branch downstream of GPCR.

Gene expression profiling of epithelium-associated FcRL4+ B cells in primary Sjögren's syndrome reveals a pathogenic signature

In primary Sjögren's syndrome (pSS), FcRL4+ B cells are present in inflamed salivary gland tissue, within or in close proximity to ductal epithelium. FcRL4 is also expressed by nearly all pSS-related mucosa-associated lymphoid tissue (MALT) B cell lymphomas, linking FcRL4 expression to lymphomagenesis. Whether glandular FcRL4+ B cells are pathogenic, how these cells originate, and how they functionally differ from FcRL4- B cells in pSS is unclear. This study aimed to investigate the phenotype and function of FcRL4+ B cells in the periphery and parotid gland tissue of patients with pSS. First, circulating FcRL4+ B cells from 44 pSS and 54 non–SS–sicca patients were analyzed by flow cytometry. Additionally, RNA sequencing of FcRL4+ B cells sorted from parotid gland cell suspensions of 6 pSS patients was performed. B cells were sorted from cell suspensions as mini bulk (5 cells/well) based on the following definitions: CD19+CD27−FcRL4- (‘naive’), CD19+CD27+FcRL4- (‘memory’), and CD19+FcRL4+ B cells. We found that, although FcRL4+ B cells were not enriched in blood in pSS compared with non-SS sicca patients, these cells generally exhibited a pro-inflammatory phenotype. Genes coding for CD11c (ITGAX), T-bet (TBX21), TACI (TNFRSF13B), Src tyrosine kinases and NF-κB pathway-related genes were, among others, significantly upregulated in glandular FcRL4+ B cells versus FcRL4- B cells. Pathway analysis showed upregulation of B cell activation, cell cycle and metabolic pathways. Thus, FcRL4+ B cells in pSS exhibit many characteristics of chronically activated, pro-inflammatory B cells and their gene expression profile suggests increased risk of lymphomagenesis. We postulate that these cells contribute significantly to the epithelial damage seen in the glandular tissue and that FcRL4+ B cells are an important treatment target in pSS.

Ubiquitination of Src promotes its secretion via small extracellular vesicles

Upregulation of the Src tyrosine kinase is implicated in the progression of cancer. The oncogenic potential of Src is suppressed via several negative regulation systems including degradation via the ubiquitin–proteasome pathway. Here, we show that ubiquitination of Src promotes its secretion via small extracellular vesicles (sEVs) to suppress its oncogenic potential. In MDCK cells expressing a modified Src that can be activated by hydroxytamoxifen, activated Src was transported to late endosomes/lysosomes and secreted via sEVs. The secretion of Src was suppressed by ablation of Cbl E3-ligase, suggesting the contribution of ubiquitination to this process. Activated Src was ubiquitinated at multiple sites, and Lys429 was identified as a critical site for sEV-mediated secretion. Mutation of Src at Lys429 (R429) caused resistance to ubiquitination and decreased its secretion via sEVs. The activated R429 mutant was also transported to late endosomes/lysosomes, whereas its incorporation into intraluminal vesicles was reduced. Activation of the R429 mutant induced a greater FAK activation than that of wild-type Src, thereby potentiating Src-induced invasive phenotypes, such as invadopodia formation and invasive activity. These findings demonstrate that ubiquitination of activated Src at Lys429 promotes its secretion via sEVs, suggesting a potential strategy to suppress the oncogenic function of upregulated Src.

Src Inhibition Attenuates Neuroinflammation and Protects Dopaminergic Neurons in Parkinson's Disease Models.

Chronic neuroinflammation is of great importance in the pathogenesis of Parkinson’s disease (PD). During the process of neuroinflammation, overactivated microglia release many proinflammatory factors, which eventually induce neurodegeneration. Inhibition of excessive microglial activation is regarded as a promising strategy for PD treatment. Src is a non-receptor tyrosine kinase that is closely related to tumors. Recently, some reports indicated that Src is a central mediator in multiple signaling pathways including neuroinflammation. The aim of our study was to demonstrate the role of Src in microglial regulation and neuroinflammation. The lipopolysaccharide (LPS)-stimulated BV2 microglia model and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model were applied in this study. The results showed that inhibition of Src could significantly relieve microgliosis and decrease levels of inflammatory factors. Besides, inhibition of Src function reduced the loss of dopaminergic neurons and improved the motor behavior of the MPTP-treated mice. Thus, this study not only verified the critical role of Src tyrosine kinase in neuroinflammation but also further proved that interfering neuroinflammation is beneficial for PD treatment. More importantly, this study shed a light on the hypothesis that Src tyrosine kinase might be a potential therapeutic target for PD and other neuroinflammation-related diseases.