Promotes Tyrosine Phosphorylation Research Articles

AXL activates YAP through the EGFR-LATS1/2 axis and confers resistance to EGFR-targeted drugs in head and neck squamous cell carcinoma.

The Hippo signaling pathway and its downstream effector YAP play a central role in cell proliferation. Dysregulation of the Hippo pathway triggers YAP hyperactivation, thereby inducing head and neck squamous cell carcinoma (HNSCC). Recently, we reported that EGFR promotes tyrosine phosphorylation of MOB1 and subsequent LATS1/2 inactivation, which are core components of the Hippo pathway, resulting in YAP activation. However, EGFR-targeted monotherapy has shown a low response rate in HNSCC patients. Given that YAP is activated in patient samples refractory to EGFR-targeted therapy, EGFR inhibitors may temporarily inactivate YAP, but intrinsic hyperactivation or acquired reactivation of YAP may confer resistance to EGFR inhibitors in HNSCC cells. The mechanism by which YAP is activated in HNSCC resistant to EGFR inhibitors remains unclear. Comprehensive transcriptional analysis revealed that AXL activates YAP through a novel mechanism: AXL heterodimerizes with EGFR, thereby activating YAP via the EGFR-LATS1/2 axis. The combination of AXL and EGFR inhibitors synergistically inactivates YAP and suppresses the viability of HNSCC and lung adenocarcinoma cells. In turn, LATS1/2 knockout and YAP hyperactivation confer resistance to the synergistic effects of these inhibitors. Our findings suggest that co-targeting both AXL and EGFR represent a promising therapeutic approach in patients with EGFR-altered cancers.

Mass Spectrometry and Pharmacological Approaches to Measuring Cooption and Reciprocal Activation of Receptor Tyrosine Kinases.

Receptor tyrosine kinases (RTKs) can show extensive crosstalk, directly and indirectly. Elucidating RTK crosstalk remains an important goal in the clinical combination of anti-cancer therapies. Here, we present mass spectrometry and pharmacological approaches showing the hepatocyte growth factor receptor (MET)-promoting tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and other membrane receptors in MET-amplified H1993 NSCLC cells. Conversely, in H292 wt-EGFR NSCLC cells, EGFR promotes the tyrosine phosphorylation of MET. Reciprocal regulation of the EGFR and insulin receptor (IR) was observed in the GEO CRC cells, where inhibition of the EGFR drives tyrosine phosphorylation of the insulin receptor. Similarly, in platelet-derived growth factor receptor (PDGFR)-amplified H1703 NSCLC cells, inhibition of the EGFR promotes the tyrosine phosphorylation of the PDGFR. These RTK interactions are used to illustrate basic principles applicable to other RTK signaling networks. More specifically, we focus on two types of RTK interaction: (1) co-option of one RTK by another and (2) reciprocal activation of one receptor following the inhibition of a distinct receptor.

PRMT4-mediated arginine methylation promotes tyrosine phosphorylation of VEGFR-2 and regulates filopodia protrusions

SummaryThrough tightly controlled multilayer mechanisms, vascular endothelial growth factor receptor-2 (VEGFR-2) activation and its downstream signal transduction govern vasculogenesis and pathological angiogenesis, such as tumor angiogenesis. Therefore, it is critical to understand the molecular mechanisms governing VEGFR-2 signal transduction. We report that protein arginine methyltransferase 4 (PRMT4) via its highly conserved EVH1 and PH domain-like N-terminal domain binds to VEGFR-2 and mediates methylation of the juxtamembrane arginine 817 (R817) on VEGFR-2. Methylation of R817 selectively increases phosphorylation of tyrosine 820 (Y820). Phosphorylation of Y820 facilitates the c-Src binding with VEGFR-2 via Src homology domain 2 (SH2). Interfering with the methylation of R817 or phosphorylation of Y820 inhibits VEGFR-2-induced filopodia protrusions, a process that is critical for the core angiogenic responses of VEGFR-2. Methylation of R817 is an important previously unrecognized mechanism of the angiogenic signaling of VEGFR-2, with implications for the development of novel-targeted VEGFR-2 inhibitors.

Infection of humanized mice with a novel phlebovirus presented pathogenic features of severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne emerging phlebovirus with high mortality rates of 6.0 to 30%. SFTSV infection is characterized by high fever, thrombocytopenia, leukopenia, hemorrhage and multiple organ failures. Currently, specific therapies and vaccines remain elusive. Suitable small animal models are urgently needed to elucidate the pathogenesis and evaluate the potential drug and vaccine for SFTSV infection. Previous models presented only mild or no pathogenesis of SFTS, limiting their applications in SFTSV infection. Therefore, it is an urgent need to develop a small animal model for the investigation of SFTSV pathogenesis and evaluation of therapeutics. In the current report, we developed a SFTSV infection model based on the HuPBL-NCG mice that recapitulates many pathological characteristics of SFTSV infection in humans. Virus-induced histopathological changes were identified in spleen, lung, kidney, and liver. SFTSV was colocalized with macrophages in the spleen and liver, suggesting that the macrophages in the spleen and liver could be the principle target cells of SFTSV. In addition, histological analysis showed that the vascular endothelium integrity was severely disrupted upon viral infection along with depletion of platelets. In vitro cellular assays further revealed that SFTSV infection increased the vascular permeability of endothelial cells by promoting tyrosine phosphorylation and internalization of the adhesion molecule vascular endothelial (VE)-cadherin, a critical component of endothelial integrity. In addition, we found that both virus infection and pathogen-induced exuberant cytokine release dramatically contributed to the vascular endothelial injury. We elucidated the pathogenic mechanisms of hemorrhage syndrome and developed a humanized mouse model for SFTSV infection, which should be helpful for anti-SFTSV therapy and pathogenesis study.

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration.

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch‐TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch‐TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress‐induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp‐1, suggesting gain‐of‐function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity.

Miconazole inhibits signal transducer and activator of transcription 3 signaling by preventing its interaction with DNA damage-induced apoptosis suppressor.

DNA damage‐induced apoptosis suppressor (DDIAS) facilitates the survival of lung cancer by suppressing apoptosis. Moreover, DDIAS promotes tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) via their interaction. Here, we identified miconazole as an inhibitor of DDIAS/STAT3 interaction by screening a chemical library using a yeast two‐hybrid assay. Miconazole inhibited growth, migration and invasion of lung cancer cells. Furthermore, miconazole suppressed STAT3 tyrosine Y705 phosphorylation and the expression of its target genes, such as cyclin D1, survivin and snail but had no suppressive effect on the activation of ERK1/2 or AKT, which is involved in the survival of lung cancer. As expected, no interaction between DDIAS and STAT3 occurred in the presence of miconazole, as confirmed by immunoprecipitation assays. Mouse xenograft experiments showed that miconazole significantly suppressed both tumor size and weight in an NCI‐H1703 mouse model. Tyrosine phosphorylation of STAT3 at Y705 and expression of its targets, such as cyclin D1, survivin and snail, were decreased in miconazole‐treated tumor tissues, as compared with those in vehicle‐treated tumor tissues. These data suggest that miconazole exerts an anti–cancer effect by suppressing STAT3 activation through inhibiting DDIAS/STAT3 binding.

Correcting abnormalities in miR-124/PTPN1 signaling rescues tau pathology in Alzheimer's disease.

MicroRNAs have been implicated in diverse physiological and pathological processes. We previously reported that aberrant microRNA-124 (miR-124)/non-receptor-type protein phosphatase 1 (PTPN1) signaling plays an important role in the synaptic disorders associated with Alzheimer's disease (AD). In this study, we further investigated the potential role of miR-124/PTPN1 in the tau pathology of AD. We first treated the mice with intra-hippocampal stereotactic injections. Then, we used quantitative real-time reverse transcription PCR (qRT-PCR) to detect the expression of microRNAs. Western blotting was used to measure the level of PTPN1, the level of tau protein, the phosphorylation of tau at AD-related sites, and alterations in the activity of glycogen synthase kinase 3β (GSK-3β) and protein phosphatase 2 (PP2A). Immunohistochemistry was also used to detect changes in tau phosphorylation levels at AD-related sites and somadendritic aggregation. Soluble and insoluble tau protein was separated by 70% formic acid (FA) extraction to examine tau solubility. Finally, behavioral experiments (including the Morris water maze, fear conditioning, and elevated plus maze) were performed to examine learning and memory ability and emotion-related behavior. We found that artificially replicating the abnormalities in miR-124/PTPN1 signaling induced AD-like tau pathology in the hippocampus of wild-type mice, including hyperphosphorylation at multiple sites, insolubility and somadendritic aggregation, as well as learning/memory deficits. We also found that disruption of miR-124/PTPN1 signaling was caused by the loss of RE1-silencing transcription factor protein, which can be initiated by Aβ insults or oxidative stress, as observed in the brains of P301S mice. Correcting the deregulation of miR-124/PTPN1 signaling rescued the tau pathology and learning/memory impairments in the P301S mice. We also found that miR-124/PTPN1 abnormalities induced activation of glycogen synthase kinase 3 (GSK-3) and inactivation of protein phosphatase 2A (PP2A) by promoting tyrosine phosphorylation, implicating an imbalance in tau kinase/phosphatase. Thus, targeting the miR-124/PTPN1 signaling pathway is a promising therapeutic strategy for AD.

Tranexamic Acid Inhibits Angiogenesis and Melanogenesis in Vitro by Targeting VEGF Receptors.

Melasma is a common but complex skin condition concerning cosmetic problems. Tranexamic acid (TA) has been proved to be effective in treatment of melasma with still unclear mechanisms. Here, we show that VEGF165 enhanced the expression of VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-2 and NRP-1) in human umbilical vein endothelial cells (HUVECs), which was attenuated by TA. VEGF165 also promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in HUVECs, which was again abolished by TA. TA further showed similar effects to neutralization of VEGFR-1 and VEGFR-2 in inhibiting cell proliferation, migration, invasion and tube formation of HUVECs induced by VEGF165, suggesting that TA could inhibit angiogenesis by targeting VEGFRs in HUVECs. In addition, VEGF165 enhanced the expression of VEGFRs and promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in normal human melanocytes, which were also attenuated by TA. Furthermore, TA showed similar effects to neutralization of VEGFR-1 and VEGFR-2 in inhibiting tyrosinase activity, melanin production and even melanogenic proteins induced by VEGF165, suggesting that TA could reduce melanogenesis via inhibiting activation of VEGFRs and subsequent expression of melanogenic proteins in melanocytes. Taken together, we demonstrate that TA can inhibit angiogenesis and melanogenesis in vitro at least in part by targeting VEGFRs, which may offer a new understanding of the pathogenesis of melasma as well as the molecular mechanism for TA in treatment of the disease.

DDIAS promotes STAT3 activation by preventing STAT3 recruitment to PTPRM in lung cancer cells

DNA damage-induced apoptosis suppressor (DDIAS) regulates cancer cell survival. Here we investigated the involvement of DDIAS in IL-6–mediated signaling to understand the mechanism underlying the role of DDIAS in lung cancer malignancy. We showed that DDIAS promotes tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is constitutively activated in malignant cancers. Interestingly, siRNA protein tyrosine phosphatase (PTP) library screening revealed protein tyrosine phosphatase receptor mu (PTPRM) as a novel STAT3 PTP. PTPRM knockdown rescued the DDIAS-knockdown-mediated decrease in STAT3 Y705 phosphorylation in the presence of IL-6. However, PTPRM overexpression decreased STAT3 Y705 phosphorylation. Moreover, endogenous PTPRM interacted with endogenous STAT3 for dephosphorylation at Y705 following IL-6 treatment. As expected, PTPRM bound to wild-type STAT3 but not the STAT3 Y705F mutant. PTPRM dephosphorylated STAT3 in the absence of DDIAS, suggesting that DDIAS hampers PTPRM/STAT3 interaction. In fact, DDIAS bound to the STAT3 transactivation domain (TAD), which competes with PTPRM to recruit STAT3 for dephosphorylation. Thus we show that DDIAS prevents PTPRM/STAT3 binding and blocks STAT3 Y705 dephosphorylation, thereby sustaining STAT3 activation in lung cancer. DDIAS expression strongly correlates with STAT3 phosphorylation in human lung cancer cell lines and tissues. Thus DDIAS may be considered as a potential biomarker and therapeutic target in malignant lung cancer cells with aberrant STAT3 activation.

Activation of VEGF receptors in response to UVB promotes cell proliferation and melanogenesis of normal human melanocytes

VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and signaling via VEGFRs extends beyond the classical roles in blood vessel formation. We previously showed VEGFRs were also expressed in epidermal keratinocytes and activation of VEGFR-2 by ultraviolet B (UVB) was involved in the pro-survival mechanism. Here, we show that both VEGF165 and UVB enhanced the expression of VEGFRs (including VEGFR-1, VEGFR-2 and NRP-1) in normal human melanocytes, and increased expression of VEGFRs by UVB was mediated through hypoxia and oxidative stress. Also, VEGF165 and UVB promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2, and UVB-induced phosphorylation of VEGFR-1 and VEGFR-2 required PKA but not P38 MAPK. In addition, UVB and VEGF165 contributed to the over-expression of melanogenic proteins in melanocytes, which could be reduced by neutralization of VEGFR-1 and/or VEGFR-2. UVB, but not VEGF165 promoted cell proliferation, while neutralization of VEGFR-1 and/or VEGFR-2 abolished this effect. UVB showed stronger than VEGF165 in promoting tyrosinase activity and melanin production, while neutralization of VEGFR-2 was stronger in reducing these effects than that of VEGFR-1. Furthermore, tranexamic acid (TA) decreased tyrosinase activity and melanin production via inhibiting activation of VEGFRs and subsequent expression of melanogenic proteins in melanocytes. Taken together, we demonstrate that VEGFRs are functionally involved in UVB-induced melanogenesis, and TA can inhibit melanogenesis at least in part by targeting VEGFRs in melanocytes.

CD25 and Protein Phosphatase 2A Cooperate to Enhance IL-2R Signaling in Human Regulatory T Cells.

Low-dose IL-2 therapy is a direct approach to boost regulatory T cells (Tregs) and promote immune tolerance in autoimmune patients. However, the mechanisms responsible for selective response of Tregs to low-dose IL-2 is not fully understood. In this study we directly assessed the contribution of CD25 and protein phosphatase 2A (PP2A) in promoting IL-2R signaling in Tregs. IL-2-induced tyrosine phosphorylation of STAT5 (pSTAT5) was proportional to CD25 levels on human CD4+ T cells and YT human NK cell line, directly demonstrating that CD25 promotes IL-2R signaling. Overexpression of the PP2A catalytic subunit (PP2Ac) by lentiviral transduction in human Tregs increased the level of IL-2R subunits and promoted tyrosine phosphorylation of Jak3 and STAT5. Interestingly, increased expression of CD25 only partially accounted for this enhanced activation of pSTAT5, indicating that PP2A promotes IL-2R signaling through multiple mechanisms. Consistent with these findings, knockdown of PP2Ac in human Tregs and impaired PP2Ac activity in mouse Tregs significantly reduced IL-2-dependent STAT5 activation. In contrast, overexpression or knockdown of PP2Ac in human T effector cells did not affect IL-2-dependent pSTAT5 activation. Overexpression of PP2Ac in human Tregs also increased the expressions of proteins related to survival, activation, and immunosuppressive function, and upregulated several IL-2-regulated genes. Collectively, these findings suggest that CD25 and PP2A cooperatively enhance the responsiveness of Tregs to IL-2, which provide potential therapeutic targets for low-dose IL-2 therapy.

Regulation of Insulin Receptor Pathway and Glucose Metabolism by CD36 Signaling.

During reduced energy intake, skeletal muscle maintains homeostasis by rapidly suppressing insulin-stimulated glucose utilization. Loss of this adaptation is observed with deficiency of the fatty acid transporter CD36. A similar loss is also characteristic of the insulin-resistant state where CD36 is dysfunctional. To elucidate what links CD36 to muscle glucose utilization, we examined whether CD36 signaling might influence insulin action. First, we show that CD36 deletion specific to skeletal muscle reduces expression of insulin signaling and glucose metabolism genes. It decreases muscle ceramides but impairs glucose disposal during a meal. Second, depletion of CD36 suppresses insulin signaling in primary-derived human myotubes, and the mechanism is shown to involve functional CD36 interaction with the insulin receptor (IR). CD36 promotes tyrosine phosphorylation of IR by the Fyn kinase and enhances IR recruitment of P85 and downstream signaling. Third, pretreatment for 15 min with saturated fatty acids suppresses CD36-Fyn enhancement of IR phosphorylation, whereas unsaturated fatty acids are neutral or stimulatory. These findings define mechanisms important for muscle glucose metabolism and optimal insulin responsiveness. Potential human relevance is suggested by genome-wide analysis and RNA sequencing data that associate genetically determined low muscle CD36 expression to incidence of type 2 diabetes.

Pseudothrombocytopenia induced by incubation at 37 ℃ in ethylenediaminetetraacetic acid tubes

Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant currently used for cell blood counts. Several studies showed that exposure of platelets (PLTs) to EDTA for a short period of time promote tyrosine phosphorylation of certain proteins, events that are related to activation. Spuriously low PLT counts were observed in a patient with cold agglutinins after the blood being warmed at 37 ℃ in EDTA tubes. Some cloudy polymers were found on peripheral blood smear after incubation at 37 ℃ for 15 minutes. These data suggest that PLT were activated after being warmed to 37 ℃ with EDTA. This process initiated coagulation and generated fibrin and PLT polymers from PLT agglutination and release. This case report hence describes a very rare phenomenon, which produces a spuriously low PLT counts caused by warming of EDTA tubes at 37 ℃.

Papaverine-sensitive phosphodiesterase activity is measured in bovine spermatozoa.

Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well-known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP-PDE activity in bovine spermatozoa. Total cAMP-PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2±49.5 and 1252.6±86.5 fmoles/min/106 spermatozoa, respectively. Using different family-specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP-PDE activity was papaverine-sensitive (44.5% and 57.5%, respectively, at 400nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post-acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.

Voltage-Dependent Accessibility of Phosphorylation Sites at the Carboxy-Terminal Domain of Kv1.3 Channels Determines Kv1.3-Induced Cell Proliferation

The plasticity of vascular smooth muscle cells that can switch from a contractile to a proliferative phenotype associates with several cardiovascular diseases. We have previously demonstrated that changes in the expression of voltage-dependent potassium (Kv) channels associated with this proliferative phenotype. These results were validated in transfected HEK293 cells: Kv1.5 decreased proliferation and Kv1.3 increased cell proliferation independently of ion flux (Cidad et al. ATVB 2012, 32:1299-307). Our data also suggested that the movement of the voltage-sensor of Kv1.3 channels could be coupled to signaling pathways leading to proliferation.Here we explore Kv1.3 regions involved in the signaling mechanism(s) associated with proliferation, by constructing chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels without consensus phosphorylation sites. These constructs were expressed as GFP- or cherry-fusion proteins, and we studied their functional expression, their effects on proliferation and their phosphorylation state in HEK cells combining immunocytochemical, electrophysiological and EdU incorporation studies.The molecular determinants of Kv1.3-induced proliferation are located at the carboxy-terminal domain. We identified two close residues (Y447 and S459) whose mutation abolished proliferation. Moreover, proliferation was increased with chimeric Kv1.5 mutants containing this segment (YS segment) but only when located at specific place within the channel molecule. Besides the selective MEK/ERK blocker PD98059 was able to inhibit Kv1.3-induced proliferation and tyrosine phosphorylation of the channel, having no effect on Y447A mutant channel. Finally, voltage-dependent transitions of Kv1.3 from close to open conformation increased pTyr labelling.We conclude that the signaling pathway linking Kv1.3 channels with proliferation involves voltage-induced conformational changes promoting tyrosine phosphorylation. Both phosphorylation and proliferation increase by facilitating the closed to open transition, being diminished in mutant channels that reside in the inactivated state. Supported by ISCIII-FEDER RD12/0042/0006, and BFU2013-45867-R grants.

Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation

The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT.

Alternate RASSF1 Transcripts Control SRC Activity, E-Cadherin Contacts, and YAP-Mediated Invasion

SummaryTumor progression to invasive carcinoma is associated with activation of SRC family kinase (SRC, YES, FYN) activity and loss of cellular cohesion. The hippo pathway-regulated cofactor YAP1 supports the tumorigenicity of RAS mutations but requires both inactivation of hippo signaling and YES-mediated phosphorylation of YAP1 for oncogenic activity. Exactly how SRC kinases are activated and hippo signaling is lost in sporadic human malignancies remains unknown. Here, we provide evidence that hippo-mediated inhibition of YAP1 is lost upon promoter methylation of the RAS effector and hippo kinase scaffold RASSF1A. We find that RASSF1A promoter methylation reduces YAP phospho-S127, which derepresses YAP1, and actively supports YAP1 activation by switching RASSF1 transcription to the independently transcribed RASSF1C isoform that promotes Tyr kinase activity. Using affinity proteomics, proximity ligation, and real-time molecular visualization, we find that RASSF1C targets SRC/YES to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, β-catenin, and YAP1. RASSF1A restricts SRC activity, preventing motility, invasion, and tumorigenesis in vitro and in vivo, with epigenetic inactivation correlating with increased inhibitory pY527-SRC in breast tumors. These data imply that distinct RASSF1 isoforms have opposing functions, which provide a biomarker for YAP1 activation and explain correlations of RASSF1 methylation with advanced invasive disease in humans. The ablation of epithelial integrity together with subsequent YAP1 nuclear localization allows transcriptional activation of β-catenin/TBX-YAP/TEAD target genes, including Myc, and an invasive phenotype. These findings define gene transcript switching as a tumor suppressor mechanism under epigenetic control.

Parafibromin Is a Component of IFN-γ-Triggered Signaling Pathways That Facilitates JAK1/2-Mediated Tyrosine Phosphorylation of STAT1.

IFN-γ (also known as type II IFN) is a cytokine that is critically involved in antiviral and immunomodulatory effects. IFN-γ activates JAK1 and JAK2, which lead to the phosphorylation and activation of the transcription factor STAT1. Whether and how additional molecules are involved in the process are not fully clear. In this study, we identified parafibromin as an important component of the IFN-γ-triggered signaling pathways. Overexpression of parafibromin promoted IFN-γ-triggered phosphorylation of STAT1 at Tyr(701), subsequent expression of downstream genes, and cellular antiviral response, whereas knockdown of parafibromin had opposite effects. Parafibromin interacted with JAK1/2, promoted the interactions of JAK1-JAK2 and JAK1/2-STAT1, and promoted tyrosine phosphorylation of STAT1 by JAKs after IFN-γ stimulation. Our results reveal a previously uncharacterized role of parafibromin in mediating IFN-γ-triggered signaling and cellular effects.

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser51 phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells.

Histidine-domain-containing protein tyrosine phosphatase regulates platelet-derived growth factor receptor intracellular sorting and degradation

Histidine domain-containing protein tyrosine phosphatase (HD-PTP) is a putative phosphatase that has been shown to affect the signaling and downregulation of certain receptor tyrosine kinases. To investigate if HD-PTP affects platelet-derived growth factor receptor β (PDGFRβ) signaling, we employed the overexpression of HA-tagged HD-PTP, as well as siRNA-mediated and lentivirus shRNA-mediated silencing of HD-PTP in NIH3T3 cells. We found that HD-PTP was recruited to the PDGFRβ in a ligand-dependent manner. Depletion of HD-PTP resulted in an inability of PDGF-BB to promote tyrosine phosphorylation of the ubiquitin ligases c-Cbl and Cbl-b, with a concomitant missorting and reduction of the degradation of activated PDGFRβ. In contrast, ligand-induced internalization of PDGFRβ was unaffected by HD-PTP silencing. Furthermore, the levels of STAM and Hrs of the ESCRT0 machinery were decreased, and immunofluorescence staining showed that in HD-PTP-depleted cells, PDGFRβ accumulated in large aberrant intracellular structures. After the reduction of HD-PTP expression, an NIH3T3-derived cell line that has autocrine PDGF-BB signaling (sis-3T3) showed increased ability of anchorage-independent growth. However, exogenously added PDGF-BB promoted efficient additional colony formation in control cells, but was not able to do so in HD-PTP-depleted cells. Furthermore, cells depleted of HD-PTP migrated faster than control cells. In summary, HD-PTP affects the intracellular sorting of activated PDGFRβ and the migration, proliferation and tumorigenicity of cells stimulated by PDGF.