The Experts below are selected from a list of 2310 Experts worldwide ranked by ideXlab platform

Akihiko Yoshimura - One of the best experts on this subject based on the ideXlab platform.

  • mek inhibition ameliorates social behavior phenotypes in a SPRED1 knockout mouse model for rasopathy disorders
    2021
    Co-Authors: Sarah C Borrie, Eric Legius, Akihiko Yoshimura, Ellen Plasschaert, Zsuzsanna Callaertsvegh, Rudi Dhooge, Ype Elgersma, Steven A Kushner, Hilde Brems
    Abstract:

    Background RASopathies are a group of disorders that result from mutations in genes coding for proteins involved in regulating the Ras-MAPK signaling pathway, and have an increased incidence of autism spectrum disorder (ASD). Legius syndrome is a rare RASopathy caused by loss-of-function mutations in the SPRED1 gene. The patient phenotype is similar to, but milder than, Neurofibromatosis type 1-another RASopathy caused by loss-of-function mutations in the NF1 gene. RASopathies exhibit increased activation of Ras-MAPK signaling and commonly manifest with cognitive impairments and ASD. Here, we investigated if a SPRED1-/- mouse model for Legius syndrome recapitulates ASD-like symptoms, and whether targeting the Ras-MAPK pathway has therapeutic potential in this RASopathy mouse model. Methods We investigated social and communicative behaviors in SPRED1-/- mice and probed therapeutic mechanisms underlying the observed behavioral phenotypes by pharmacological targeting of the Ras-MAPK pathway with the MEK inhibitor PD325901. Results SPRED1-/- mice have robust increases in social dominance in the automated tube test and reduced adult ultrasonic vocalizations during social communication. Neonatal ultrasonic vocalization was also altered, with significant differences in spectral properties. SPRED1-/- mice also exhibit impaired nesting behavior. Acute MEK inhibitor treatment in adulthood with PD325901 reversed the enhanced social dominance in SPRED1-/- mice to normal levels, and improved nesting behavior in adult SPRED1-/- mice. Limitations This study used an acute treatment protocol to administer the drug. It is not known what the effects of longer-term treatment would be on behavior. Further studies titrating the lowest dose of this drug that is required to alter SPRED1-/- social behavior are still required. Finally, our findings are in a homozygous mouse model, whereas patients carry heterozygous mutations. These factors should be considered before any translational conclusions are drawn. Conclusions These results demonstrate for the first time that social behavior phenotypes in a mouse model for RASopathies (SPRED1-/-) can be acutely reversed. This highlights a key role for Ras-MAPK dysregulation in mediating social behavior phenotypes in mouse models for ASD, suggesting that proper regulation of Ras-MAPK signaling is important for social behavior.

  • impaired instrumental learning in SPRED1 mice a model for a rare rasopathy
    2021
    Co-Authors: Sarah C Borrie, Eric Legius, Akihiko Yoshimura, Alexa E Horner, Maksym V Kopanitsa, Hilde Brems
    Abstract:

    RASopathies are neuro-cardio-facio-cutaneous disorders stemming from mutations in genes regulating the RAS-MAPK pathway. Legius syndrome is a rare RASopathy disorder caused by mutations in the SPRED1 gene. SPRED1 protein negatively regulates activation of Ras by inhibiting RAS/RAF and by its interaction with neurofibromin, a Ras GTPase-activating protein (RAS-GAP). Cognitive impairments have been reported in Legius syndrome as well as in other RASopathy disorders. Modelling these cognitive deficits in a SPRED1 mouse model for Legius syndrome has demonstrated spatial learning and memory deficits, but other cognitive domains remained unexplored. Here, we attempted to utilize a cognitive touchscreen battery to investigate if SPRED1-/- mice exhibit deficits in other cognitive domains. We show that SPRED1-/- mice had heterogeneous performance in instrumental operant learning, with a large subgroup (n = 9/20) failing to reach the standard criterion on touchscreen operant pretraining, precluding further cognitive testing. To examine whether targeting the RAS-MAPK signalling pathway could rescue these cognitive impairments, SPRED1-/- mice were acutely treated with the clinically relevant mitogen-activated protein kinase (MEK) inhibitor PD325901. However, MEK inhibition did not improve their instrumental learning. We conclude that SPRED1-/- mice can model severe cognitive impairments that cannot be reversed in adulthood.

  • id 171 SPRED1 a suppressor of the ras erk pathway negatively regulates expansion and function of group 2 innate lymphoid cells
    2015
    Co-Authors: Mayu Suzuki, Akihiko Yoshimura
    Abstract:

    Cytokines from group 2 innate lymphoid cells (ILC2s) have been implicated in acute allergic responses, such as papain-induced lung inflammation. However, the means of homeostatic regulation of ILC2s have not been established. In this study, we demonstrated that SPRED1, a negative regulator of the Ras–ERK pathway, plays an important role in the proliferation and apoptosis of ILC2s and in cytokine secretion from ILC2s. Intranasal administration of papain stimulated IL-5 and IL-13 production in the lung, which was enhanced when SPRED1 was deleted. In vitro, SPRED1-deficient LC2s proliferated faster than wild type ILC2s did and produced higher levels of cytokines in response to IL-33. On the contrary, a MEK inhibitor suppressed ILC2 proliferation and cytokine production. SPRED1 deficiency resulted in stabilization of GATA3, which has been shown to play essential roles in the maintenance and cytokine production of ILC2. These data suggest that SPRED1 negatively regulates ILC2 development and functions through the suppression of the Ras–ERK pathway.

  • SPRED1 a suppressor of the ras erk pathway negatively regulates expansion and function of group 2 innate lymphoid cells
    2015
    Co-Authors: Mayu Suzuki, Yasuko Hirata, Rimpei Morita, Takashi Shichita, Akihiko Yoshimura
    Abstract:

    Cytokines from group 2 innate lymphoid cells (ILC2s) have been implicated in acute allergic responses, such as papain-induced lung inflammation. However, the means of homeostatic regulation of ILC2s have not been established. In this study, we demonstrated that SPRED1, a negative regulator of the Ras-ERK pathway, plays an important role in the proliferation and apoptosis of ILC2s and in cytokine secretion from ILC2s. Intranasal administration of papain stimulated IL-5 and IL-13 production in the lung, which was enhanced when SPRED1 was deleted. In vitro, SPRED1(-/-) ILC2s proliferated faster than wild type ILC2s did and produced higher levels of cytokines in response to IL-33. On the contrary, a MEK inhibitor suppressed ILC2 proliferation and cytokine production. SPRED1 deficiency resulted in stabilization of GATA3, which has been shown to play essential roles in the maintenance and cytokine production of ILC2. These data suggest that SPRED1 negatively regulates ILC2 development and functions through the suppression of the Ras-ERK pathway.

  • Spred-2 Deficiency Exacerbates Lipopolysaccharide-Induced Acute Lung Inflammation in Mice
    2014
    Co-Authors: Toshihiro Ito, Soichiro Fushimi, Sakuma Takahashi, Junya Itakura, Ryojiro Kimura, Miwa Sato, Megumi Mino, Akihiko Yoshimura, Akihiro Matsukawa
    Abstract:

    BackgroundAcute respiratory distress syndrome (ARDS) is a severe and life-threatening acute lung injury (ALI) that is caused by noxious stimuli and pathogens. ALI is characterized by marked acute inflammation with elevated alveolar cytokine levels. Mitogen-activated protein kinase (MAPK) pathways are involved in cytokine production, but the mechanisms that regulate these pathways remain poorly characterized. Here, we focused on the role of Sprouty-related EVH1-domain-containing protein (Spred)-2, a negative regulator of the Ras-Raf-extracellular signal-regulated kinase (ERK)-MAPK pathway, in lipopolysaccharide (LPS)-induced acute lung inflammation.MethodsWild-type (WT) mice and Spred-2−/− mice were exposed to intratracheal LPS (50 µg in 50 µL PBS) to induce pulmonary inflammation. After LPS-injection, the lungs were harvested to assess leukocyte infiltration, cytokine and chemokine production, ERK-MAPK activation and immunopathology. For exvivo experiments, alveolar macrophages were harvested from untreated WT and Spred-2−/− mice and stimulated with LPS. In invitro experiments, specific knock down of Spred-2 by siRNA or overexpression of Spred-2 by transfection with a plasmid encoding the Spred-2 sense sequence was introduced into murine RAW264.7 macrophage cells or MLE-12 lung epithelial cells.ResultsLPS-induced acute lung inflammation was significantly exacerbated in Spred-2−/− mice compared with WT mice, as indicated by the numbers of infiltrating leukocytes, levels of alveolar TNF-α, CXCL2 and CCL2 in a later phase, and lung pathology. U0126, a selective MEK/ERK inhibitor, reduced the augmented LPS-induced inflammation in Spred-2−/− mice. Specific knock down of Spred-2 augmented LPS-induced cytokine and chemokine responses in RAW264.7 cells and MLE-12 cells, whereas Spred-2 overexpression decreased this response in RAW264.7 cells.ConclusionsThe ERK-MAPK pathway is involved in LPS-induced acute lung inflammation. Spred-2 controls the development of LPS-induced lung inflammation by negatively regulating the ERK-MAPK pathway. Thus, Spred-2 may represent a therapeutic target for the treatment of ALI.

Akihiro Matsukawa - One of the best experts on this subject based on the ideXlab platform.

  • spred2 controls the severity of concanavalin a induced liver damage by limiting interferon gamma production by cd4 and cd8 t cells
    2021
    Co-Authors: Xu Yang, Masayoshi Fujisawa, Teizo Yoshimura, Toshiaki Ohara, Steven L Kunkel, Cuiming Sun, Qiuying Liu, Chen Cao, Akihiro Matsukawa
    Abstract:

    Abstract Introduction Mitogen-activated protein kinases (MAPKs) are involved in T cell-mediated liver damage. However, the inhibitory mechanism(s) that controls T cell-mediated liver damage remains unknown. Objectives We investigated whether Spred2 (Sprouty-related, EVH1 domain-containing protein 2) that negatively regulates ERK-MAPK pathway has a biological impact on T cell-mediated liver damage by using a murine model. Methods We induced hepatotoxicity in genetically engineered mice by intravenously injecting Concanavalin A (Con A) and analyzed the mechanisms using serum chemistry, histology, ELISA, qRT-PCR, Western blotting and flow cytometry. Results Spred2-deficient mice (Spred2-/-) developed more sever liver damage than wild-type (WT) mice with increased interferon-γ (IFNγ) production. Hepatic ERK phosphorylation was enhanced in Spred2-/- mice, and pretreatment of Spred2-/- mice with the MAPK/ERK inhibitor U0126 markedly inhibited the liver damage and reduced IFNγ production. Neutralization of IFNγ abolished the damage with decreased hepatic Stat1 activation in Spred2-/- mice . IFNγ was mainly produced from CD4+ and CD8+ T cells, and their depletion decreased liver damage and IFNγ production. Transplantation of CD4+ and/or CD8+ T cells from Spred2-/- mice into RAG1-/- mice deficient in both T and B cells caused more severe liver damage than those from WT mice. Hepatic expression of T cell attractants, CXCL9 and CXCL10, was augmented in Spred2-/- mice as compared to WT mice. Conversely, liver damage, IFNγ production and the recruitment of CD4+ and CD8+ T cells in livers after Con A challenge were lower in Spred2 transgenic mice, and Spred2-overexpressing CD4+ and CD8+ T cells produced lower levels of IFNγ than WT cells upon stimulation with Con A in vitro. Conclusion We demonstrated, for the first time, that Spred2 functions as an endogenous regulator of T cell IFNγ production and Spred2-mediated inhibition of ERK-MAPK pathway may be an effective remedy for T cell-dependent liver damage.

  • Image_2_Spred2 Regulates High Fat Diet-Induced Adipose Tissue Inflammation, and Metabolic Abnormalities in Mice.JPEG
    2019
    Co-Authors: Takahiro Ohkura, Masayoshi Fujisawa, Teizo Yoshimura, Toshiaki Ohara, Rie Marutani, Kaya Usami, Akihiro Matsukawa
    Abstract:

    Chronic low-grade inflammation in visceral adipose tissues triggers the development of obesity-related insulin resistance, leading to the metabolic syndrome, a serious health condition with higher risk of cardiovascular disease, diabetes, and stroke. In the present study, we investigated whether Sprouty-related EVH1-domain-containing protein 2 (Spred2), a negative regulator of the Ras/Raf/ERK/MAPK pathway, plays a role in the development of high fat diet (HFD)-induced obesity, adipose tissue inflammation, metabolic abnormalities, and insulin resistance. Spred2 knockout (KO) mice, fed with HFD, exhibited an augmented body weight gain, which was associated with enhanced adipocyte hypertrophy in mesenteric white adipose tissue (mWAT) and deteriorated dyslipidemia, compared with wild-type (WT) controls. The number of infiltrating macrophages with a M1 phenotype, and the crown-like structures, composed of macrophages surrounding dead or dying adipocytes, were more abundant in Spred2 KO-mWAT compared to in WT-mWAT. Exacerbated adipose tissue inflammation in Spred2 KO mice led to aggravated insulin resistance and fatty liver disease. To analyze the mechanism(s) that caused adipose tissue inflammation, cytokine response in mWAT was investigated. Stromal vascular fraction that contained macrophages from Spred2 KO-mWAT showed elevated levels of tumor necrosis factor α (TNFα) and monocyte chemoattractant protein-1 (MCP-1/CCL2) compared with those from WT-mWAT. Upon stimulation with palmitate acid (PA), bone marrow-derived macrophages (BMDMs) derived from Spred2 KO mice secreted higher levels of TNFα and MCP-1 than those from WT mice with enhanced ERK activation. U0126, a MEK inhibitor, reduced the PA-induced cytokine response. Taken together, these results suggested that Spred2, in macrophages, negatively regulates high fat diet-induced obesity, adipose tissue inflammation, metabolic abnormalities, and insulin resistance by inhibiting the ERK/MAPK pathway. Thus, Spred2 represents a potential therapeutic tool for the prevention of insulin resistance and resultant metabolic syndrome.

  • spred2 deficiency exacerbates d galactosamine lipopolysaccharide induced acute liver injury in mice via increased production of tnfα
    2018
    Co-Authors: Xu Yang, Miwa Sato, Megumi Mino, Masayoshi Fujisawa, Teizo Yoshimura, Toshiaki Ohara, Thar Htet San, Tong Gao, Steven L Kunkel, Akihiro Matsukawa
    Abstract:

    Acute liver injury (ALI) is characterized by hepatocyte damage and inflammation. In the present study, we examined whether the absence of Sprouty-related EVH1-domain-containing protein 2 (Spred2), a negative regulator of the Ras/Raf/ERK/MAPK pathway, influences ALI induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). Compared to wild-type mice, Spred2−/− mice developed exacerbated liver injury represented by enhanced hepatocyte damage and inflammation. Enhanced ERK activation was observed in Spred2−/−-livers, and the MEK/ERK inhibitor U0126 ameliorated ALI. Hepatic tumour necrosis factor α (TNFα) and interleukin (IL)-1β levels were increased in Spred-2−/−-livers, and the neutralization of TNFα dramatically ameliorated ALI, which was associated with decreased levels of endogenous TNFα and IL-1β. When mice were challenged with D-GalN and TNFα, much severer ALI was observed in Spred2−/− mice with significant increases in endogenous TNFα and IL-1β in the livers. Immunohistochemically, Kupffer cells were found to produce TNFα, and isolated Kupffer cells from Spred2−/− mice produced significantly higher levels of TNFα than those from wild-type mice after LPS stimulation, which was significantly decreased by U0126. These results suggest that Spred2 negatively regulates D-GalN/LPS-induced ALI under the control of TNFα in Kupffer cells. Spred2 may present a therapeutic target for the treatment of ALI.

  • Spred2-deficiecy Protects Mice from Polymicrobial Septic Peritonitis by Enhancing Inflammation and Bacterial Clearance
    2017
    Co-Authors: Junya Itakura, Toshihiro Ito, Soichiro Fushimi, Sakuma Takahashi, Miwa Sato, Megumi Mino, Teizo Yoshimura, Akihiro Matsukawa
    Abstract:

    Abstract Sepsis is an infection-induced systemic inflammatory syndrome and a major cause of death for critically ill patients. Here, we examined whether the absence of Sprouty-related EVH1-domain-containing protein 2 (Spred2), a negative regulator of the Ras/Raf/ERK/MAPK pathway, influences host defense against polymicrobial sepsis (PMS) induced by cecal ligation and puncture (CLP). Compared to wild-type mice, Spred2−/− mice exhibited higher survival rates with increased level of leukocyte infiltration and local chemokine production and reduced plasma and peritoneal bacterial loads after CLP. The MEK inhibitor U0126 significantly reduced LPS-induced chemokine production by Spred2−/− resident macrophages in vitro, and decreased CLP-induced leukocyte infiltration in vivo. Spred2−/− resident macrophages, but not neutrophils or elicited macrophages, exhibited increased phagocytic activity. Interestingly, surface expression of complement receptor 1/2 (CR1/2) was increased in Spred2−/− resident macrophages in response to lipopolysaccharide in a manner dependent on the ERK/MAPK pathway, and blocking CR1/2 in vivo resulted in reduced leukocyte infiltration and increased bacterial loads after CLP. Taken together, our results indicate that Spred2-deficiency protects mice from PMS via increased activation of the ERK/MAPK pathway and subsequent increase in innate immune responses. Thus, inhibiting Spred2 may present a novel means to prevent the development of PMS

  • Spred-2 Deficiency Exacerbates Lipopolysaccharide- Induced Acute Lung Inflammation in Mice
    2016
    Co-Authors: Toshihiro Ito, Soichiro Fushimi, Sakuma Takahashi, Junya Itakura, Ryojiro Kimura, Miwa Sato, Megumi Mino, Akihiro Matsukawa
    Abstract:

    Background: Acute respiratory distress syndrome (ARDS) is a severe and life-threatening acute lung injury (ALI) that is caused by noxious stimuli and pathogens. ALI is characterized by marked acute inflammation with elevated alveolar cytokine levels. Mitogen-activated protein kinase (MAPK) pathways are involved in cytokine production, but the mechanisms that regulate these pathways remain poorly characterized. Here, we focused on the role of Sprouty-related EVH1-domain-containing protein (Spred)-2, a negative regulator of the Ras-Raf-extracellular signal-regulated kinase (ERK)-MAPK pathway, in lipopolysaccharide (LPS)-induced acute lung inflammation. Methods: Wild-type (WT) mice and Spred-22/2 mice were exposed to intratracheal LPS (50 mg in 50 mL PBS) to induce pulmonary inflammation. After LPS-injection, the lungs were harvested to assess leukocyte infiltration, cytokine and chemokine production, ERK-MAPK activation and immunopathology. For ex vivo experiments, alveolar macrophages were harvested from untreated WT and Spred-22/2 mice and stimulated with LPS. In in vitro experiments, specific knock down o

Graeme R. Guy - One of the best experts on this subject based on the ideXlab platform.

  • sprouty related ena vasodilator stimulated phosphoprotein homology 1 domain containing protein SPRED1 a tyrosine protein phosphatase non receptor type 11 shp2 substrate in the ras extracellular signal regulated kinase erk pathway
    2011
    Co-Authors: Martina Quintanaraudelo, Permeen Yusoff, Saravanan Sinniah, Sumana Chandramouli, Graeme R. Guy
    Abstract:

    SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway.

  • direct association of sprouty related protein with an evh1 domain spred 1 or spred2 with dyrk1a modifies substrate kinase interactions
    2010
    Co-Authors: Rebecca A. Jackson, Permeen Yusoff, Graeme R. Guy
    Abstract:

    The mammalian SPRED (Sprouty-related protein with an EVH1 domain) proteins include a family of three members, SPRED1–3. Currently, little is known about their biochemistry. The best described, SPRED1, has been shown to inhibit the Ras/ERK pathway downstream of Ras. All three SPREDs have a cysteine-rich domain (CRD) that has high homology to the CRD of the Sprouty family of proteins, several of which are also Ras/ERK inhibitors. In the belief that binding partners would clarify SPRED function, we assayed for their associated proteins. Here, we describe the direct and endogenous interaction of SPRED1 and SPRED2 with the novel kinase, DYRK1A. DYRK1A has become the subject of recent research focus as it plays a central role in Caenorhabditis elegans oocyte maturation and egg activation, and there is strong evidence that it could be involved in Down syndrome in humans. Both SPRED1 and SPRED2 inhibit the ability of DYRK1A to phosphorylate its substrates, Tau and STAT3. This inhibition occurs via an interaction of the CRD of the SPREDs with the kinase domain of DYRK1A. DYRK1A substrates must bind to the kinase to enable phosphorylation, and SPRED proteins compete for the same binding site to modify this process. Our accumulated evidence indicates that the SPRED proteins are likely physiological modifiers of DYRK1A.

Frank Mccormick - One of the best experts on this subject based on the ideXlab platform.

  • Abstract 3765: c-Kit mediated phosphorylation of SPRED1 regulates Neurofibromin-SPRED1 interaction
    2020
    Co-Authors: Claire Lorenzo, Anatoly Urisman, Lucy C. Young, Alexandra Tannka, Frank Mccormick
    Abstract:

    SPRED1 negatively regulates Ras/MAPK signaling following growth factor stimulation. SPRED1 inhibits Ras by binding and localizing Neurofibromin, a RasGAP, to the plasma membrane to accelerate Ras GTPase activity. c-Kit, a receptor tyrosine kinase (RTK), is known to interact with SPRED1 but the consequence of this interaction is unknown. Here we demonstrate that c-Kit signaling regulates Neurofibromin-SPRED1 interaction. Stimulation with c-Kit ligand, SCF, results in a transient disruption in Neurofibromin-SPRED1 binding which corresponds to increased Ras signaling, followed by restoration of Neurofibromin-Spred binding which corresponds to nearly basal levels of Ras signaling. Mass spectrometry analysis identified potential phosphorylation sites on SPRED1 that correspond to the initial disruption and later restoration of Neurofibromin-SPRED1 binding. Phosphomimetic and phosphodeficient mutants affect the interaction. Our findings provide a potential mechanism by which RTK signaling regulates negative feedback to allow transient activation and subsequent termination of Ras signaling. Citation Format: Claire Lorenzo, Lucy C. Young, Alexandra Tannka, Anatoly Urisman, Frank McCormick. c-Kit mediated phosphorylation of SPRED1 regulates Neurofibromin-SPRED1 interaction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3765.

  • Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR.
    2020
    Co-Authors: Wupeng Yan, Frank Mccormick, Klaus Scheffzek, Matthew Drew, Evan Markegard, Srisathiyanarayanan Dharmaiah, Anatoly Urisman, Dominic Esposito, Dwight V. Nissley, Dhirendra K. Simanshu
    Abstract:

    Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.

  • abstract 5520 oncogenic rtk signaling inhibits SPRED1 nf1 to sustain constitutive ras mapk signaling
    2018
    Co-Authors: Evan Markegard, Anatoly Urisman, Ellen L. Mercado, Jacqueline Galeas, Pau Castel, Jillian M Silva, Frank Mccormick
    Abstract:

    Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that oncogenic RTK signaling leads to phosphorylation of SPRED1(S105) in the EVH domain, which disrupts SPRED1-NF1 binding and function. Phosphomimetic SPRED1 is unable to suppress Ras-GTP following growth factor stimulation and cancer cell proliferation. The SPRED1(S105) kinase is likely to be a CDK based on in vitro kinase and in vivo cell line assays. Our findings provide one potential mechanism by which oncogenic RTK signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Pau Castel, Jillian Silva, Jacqueline Galeas, Kathy Li, Anatoly Urisman, Frank McCormick. Oncogenic RTK signaling inhibits SPRED1/NF1 to sustain constitutive Ras/MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5520.

  • abstract 1370 egfr mediated SPRED1 phosphorylation inhibits nf1 to sustain constitutive ras mapk signaling
    2017
    Co-Authors: Evan Markegard, Anatoly Urisman, Ellen L. Mercado, Jacqueline Galeas, Marena I Trinidad, Jillian M Silva, Frank Mccormick
    Abstract:

    Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that EGFR signaling disrupts SPRED1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on SPRED1 and NF1 that could disrupt SPRED1-NF1 binding by steric hindrance. A serine phosphorylation site on SPRED1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased SPRED1-NF1 binding, respectively. Phosphomimetic SPRED1 is unable to suppress Ras-GTP following EGF stimulation. Therefore, phosphorylation of SPRED1 at this site by a serine kinase downstream of EGFR may disrupt SPRED1-NF1 binding. To identify the SPRED1 kinase we are performing an in vitro kinase assay and an unbiased CRISPRa screen. Our findings provide one potential mechanism by which EGFR signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Jillian M. Silva, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. EGFR-mediated SPRED1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1370. doi:10.1158/1538-7445.AM2017-1370

  • abstract 1874 oncogenic egfr signaling inhibits the SPRED1 nf1 interaction to sustain constitutive ras signaling
    2016
    Co-Authors: Evan Markegard, Anatoly Urisman, Ellen L. Mercado, Jacqueline Galeas, Marena I Trinidad, Frank Mccormick
    Abstract:

    Spred proteins negatively regulate Ras/MAPK signaling following mitogen stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Loss-of-function SPRED1 and NF1 mutations occur across multiple cancer types including melanoma, non-small cell lung carcinoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that oncogenic EGFR signaling disrupts SPRED1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on SPRED1 and NF1 that could disrupt SPRED1-NF1 binding by steric hindrance. A serine phosphorylation site on SPRED1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased SPRED1-NF1 binding, respectively. Therefore, phosphorylation of SPRED1 at this site by a serine kinase downstream of oncogenic EGFR may disrupt SPRED1-NF1 binding. Our findings provide one potential mechanism by which oncogenic EGFR signaling disrupts negative feedback to allow for constitutive Ras signaling. Furthermore, this work may elucidate a novel kinase therapeutic target for restoring NF1 mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. Oncogenic EGFR signaling inhibits the SPRED1-NF1 interaction to sustain constitutive Ras signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1874.

Elizabeth Conibear - One of the best experts on this subject based on the ideXlab platform.

  • the palmitoyl acyltransferase hip14 shares a high proportion of interactors with huntingtin implications for a role in the pathogenesis of huntington s disease
    2014
    Co-Authors: Stefanie L. Butland, Mandi E. Schmidt, Seanpatrick Riechers, Dale D O Martin, Erich E. Wanker, Roshni R. Singaraja, Kuljeet Vaid, Shaun S. Sanders, Rona K. Graham, Elizabeth Conibear
    Abstract:

    HIP14 is the most highly conserved of 23 human palmitoyl acyltransferases (PATs) that catalyze the post-translational addition of palmitate to proteins, including huntingtin (HTT). HIP14 is dysfunctional in the presence of mutant HTT (mHTT), the causative gene for Huntington disease (HD), and we hypothesize that reduced palmitoylation of HTT and other HIP14 substrates contributes to the pathogenesis of the disease. Here we describe the yeast two-hybrid (Y2H) interactors of HIP14 in the first comprehensive study of interactors of a mammalian PAT. Unexpectedly, we discovered a highly significant overlap between HIP14 interactors and 370 published interactors of HTT, 4-fold greater than for control proteins (P = 8 × 10−5). Nearly half of the 36 shared interactors are already implicated in HD, supporting a direct link between HIP14 and the disease. The HIP14 Y2H interaction set is significantly enriched for palmitoylated proteins that are candidate substrates. We confirmed that three of them, GPM6A, and the Sprouty domain-containing proteins SPRED1 and SPRED3, are indeed palmitoylated by HIP14; the first enzyme known to palmitoylate these proteins. These novel substrates functions might be affected by reduced palmitoylation in HD. We also show that the vesicular cargo adapter optineurin, an established HTT-binding protein, co-immunoprecipitates with HIP14 but is not palmitoylated. mHTT leads to mislocalization of optineurin and aberrant cargo trafficking. Therefore, it is possible that optineurin regulates trafficking of HIP14 to its substrates. Taken together, our data raise the possibility that defective palmitoylation by HIP14 might be an important mechanism that contributes to the pathogenesis of HD.

  • the palmitoyl acyltransferase hip14 shares a high proportion of interactors with huntingtin implications for a role in the pathogenesis of huntington s disease
    2014
    Co-Authors: Stefanie L. Butland, Mandi E. Schmidt, Seanpatrick Riechers, Dale D O Martin, Erich E. Wanker, Roshni R. Singaraja, Kuljeet Vaid, Shaun S. Sanders, Rona K. Graham, Elizabeth Conibear
    Abstract:

    HIP14 is the most highly conserved of 23 human palmitoyl acyltransferases (PATs) that catalyze the post-translational addition of palmitate to proteins, including huntingtin (HTT). HIP14 is dysfunctional in the presence of mutant HTT (mHTT), the causative gene for Huntington disease (HD), and we hypothesize that reduced palmitoylation of HTT and other HIP14 substrates contributes to the pathogenesis of the disease. Here we describe the yeast two-hybrid (Y2H) interactors of HIP14 in the first comprehensive study of interactors of a mammalian PAT. Unexpectedly, we discovered a highly significant overlap between HIP14 interactors and 370 published interactors of HTT, 4-fold greater than for control proteins (P = 8 × 10−5). Nearly half of the 36 shared interactors are already implicated in HD, supporting a direct link between HIP14 and the disease. The HIP14 Y2H interaction set is significantly enriched for palmitoylated proteins that are candidate substrates. We confirmed that three of them, GPM6A, and the Sprouty domain-containing proteins SPRED1 and SPRED3, are indeed palmitoylated by HIP14; the first enzyme known to palmitoylate these proteins. These novel substrates functions might be affected by reduced palmitoylation in HD. We also show that the vesicular cargo adapter optineurin, an established HTT-binding protein, co-immunoprecipitates with HIP14 but is not palmitoylated. mHTT leads to mislocalization of optineurin and aberrant cargo trafficking. Therefore, it is possible that optineurin regulates trafficking of HIP14 to its substrates. Taken together, our data raise the possibility that defective palmitoylation by HIP14 might be an important mechanism that contributes to the pathogenesis of HD.