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Heba Alshaker - One of the best experts on this subject based on the ideXlab platform.
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leptin induces upregulation of sphingosine Kinase 1 in oestrogen receptor negative breast cancer via src family Kinase mediated janus Kinase 2 independent pathway
Breast Cancer Research, 2014Co-Authors: Heba Alshaker, Jonathan Krell, Adam E Frampton, Jonathan Waxman, Oleg Blyuss, Alexey Zaikin, Mathias Winkler, Justin Stebbing, Ernesto YagueAbstract:Introduction: Obesity is a known risk factor for breast cancer. Sphingosine Kinase 1 (SK1) is an oncogenic lipid Kinase that is overexpressed in breast tumours and linked with poor prognosis, however, its role in obesity-driven breast cancer was never elucidated. Methods: Human primary and secondary breast cancer tissues were analysed for SK1 and leptin receptor expression using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Leptin-induced signalling was analysed in human oestrogen receptor (ER)-positive and negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Results: Our findings show for the first time that human primary breast tumours and associated lymph node metastases exhibit a strong correlation between SK1 and leptin receptor expression (Pearson R= 0.78 and R = 0.77, respectively, P <0.001). Both these genes are elevated in metastases of ER-negative patients and show a significant increase in patients with higher body mass index (BMI). Leptin induces SK1 expression and activation in ER-negative breast cancer cell lines MDAMB-231 and BT-549, but not in ER-positive cell lines. Pharmacological inhibition and gene knockdown showed that leptin-induced SK1 activity and expression are mediated by activation of extracellular signal-regulated Kinases 1/2 (ERK1/2) and Src family Kinase (SFK) pathways, but not by the major pathways downstream of leptin receptor (LEPR) - janus Kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Src-homology 2 domain-containing phosphatase 2 (SHP2) appeared to be key to SK1 activation, and may function as an adaptor protein between SFKs and LEPR. Importantly, leptin-induced breast cancer cell proliferation was abrogated by SK1-specific small interfering RNA (siRNA). Conclusions: Overall, our findings demonstrate a novel SFK/ERK1/2-mediated pathway that links leptin signalling and expression of oncogenic enzyme SK1 in breast tumours and suggest the potential significance of this pathway in ER-negative breast cancer.
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Leptin induces upregulation of sphingosine Kinase 1 in oestrogen receptor-negative breast cancer via Src family Kinase-mediated, janus Kinase 2-independent pathway
Breast Cancer Research, 2014Co-Authors: Heba Alshaker, Jonathan Krell, Adam E Frampton, Jonathan Waxman, Oleg Blyuss, Alexey Zaikin, Mathias Winkler, Justin Stebbing, Ernesto Yague, Dmitri PchejetskiAbstract:Introduction Obesity is a known risk factor for breast cancer. Sphingosine Kinase 1 (SK1) is an oncogenic lipid Kinase that is overexpressed in breast tumours and linked with poor prognosis, however, its role in obesity-driven breast cancer was never elucidated. Methods Human primary and secondary breast cancer tissues were analysed for SK1 and leptin receptor expression using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Leptin-induced signalling was analysed in human oestrogen receptor (ER)-positive and negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Results Our findings show for the first time that human primary breast tumours and associated lymph node metastases exhibit a strong correlation between SK1 and leptin receptor expression (Pearson R = 0.78 and R = 0.77, respectively, P
Ernesto Yague - One of the best experts on this subject based on the ideXlab platform.
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leptin induces upregulation of sphingosine Kinase 1 in oestrogen receptor negative breast cancer via src family Kinase mediated janus Kinase 2 independent pathway
Breast Cancer Research, 2014Co-Authors: Heba Alshaker, Jonathan Krell, Adam E Frampton, Jonathan Waxman, Oleg Blyuss, Alexey Zaikin, Mathias Winkler, Justin Stebbing, Ernesto YagueAbstract:Introduction: Obesity is a known risk factor for breast cancer. Sphingosine Kinase 1 (SK1) is an oncogenic lipid Kinase that is overexpressed in breast tumours and linked with poor prognosis, however, its role in obesity-driven breast cancer was never elucidated. Methods: Human primary and secondary breast cancer tissues were analysed for SK1 and leptin receptor expression using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Leptin-induced signalling was analysed in human oestrogen receptor (ER)-positive and negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Results: Our findings show for the first time that human primary breast tumours and associated lymph node metastases exhibit a strong correlation between SK1 and leptin receptor expression (Pearson R= 0.78 and R = 0.77, respectively, P <0.001). Both these genes are elevated in metastases of ER-negative patients and show a significant increase in patients with higher body mass index (BMI). Leptin induces SK1 expression and activation in ER-negative breast cancer cell lines MDAMB-231 and BT-549, but not in ER-positive cell lines. Pharmacological inhibition and gene knockdown showed that leptin-induced SK1 activity and expression are mediated by activation of extracellular signal-regulated Kinases 1/2 (ERK1/2) and Src family Kinase (SFK) pathways, but not by the major pathways downstream of leptin receptor (LEPR) - janus Kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Src-homology 2 domain-containing phosphatase 2 (SHP2) appeared to be key to SK1 activation, and may function as an adaptor protein between SFKs and LEPR. Importantly, leptin-induced breast cancer cell proliferation was abrogated by SK1-specific small interfering RNA (siRNA). Conclusions: Overall, our findings demonstrate a novel SFK/ERK1/2-mediated pathway that links leptin signalling and expression of oncogenic enzyme SK1 in breast tumours and suggest the potential significance of this pathway in ER-negative breast cancer.
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Leptin induces upregulation of sphingosine Kinase 1 in oestrogen receptor-negative breast cancer via Src family Kinase-mediated, janus Kinase 2-independent pathway
Breast Cancer Research, 2014Co-Authors: Heba Alshaker, Jonathan Krell, Adam E Frampton, Jonathan Waxman, Oleg Blyuss, Alexey Zaikin, Mathias Winkler, Justin Stebbing, Ernesto Yague, Dmitri PchejetskiAbstract:Introduction Obesity is a known risk factor for breast cancer. Sphingosine Kinase 1 (SK1) is an oncogenic lipid Kinase that is overexpressed in breast tumours and linked with poor prognosis, however, its role in obesity-driven breast cancer was never elucidated. Methods Human primary and secondary breast cancer tissues were analysed for SK1 and leptin receptor expression using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Leptin-induced signalling was analysed in human oestrogen receptor (ER)-positive and negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Results Our findings show for the first time that human primary breast tumours and associated lymph node metastases exhibit a strong correlation between SK1 and leptin receptor expression (Pearson R = 0.78 and R = 0.77, respectively, P
Thomas E. Smithgall - One of the best experts on this subject based on the ideXlab platform.
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Remodeling of HIV-1 Nef Structure by SRC-Family Kinase Binding.
Journal of Molecular Biology, 2017Co-Authors: Jamie A Moroco, Thomas E. Smithgall, Thomas E Wales, John Jeff Alvarado, Ryan P. Staudt, John R EngenAbstract:Abstract The HIV-1 accessory protein Nef controls multiple aspects of the viral life cycle and host immune response, making it an attractive therapeutic target. Previous X-ray crystal structures of Nef in complex with key host cell binding partners have shed light on protein–protein interactions critical to Nef function. Crystal structures of Nef in complex with either the SH3 or tandem SH3–SH2 domains of SRC-Family Kinases reveal distinct dimer conformations of Nef. However, the existence of these Nef dimer complexes in solution has not been established. Here we used hydrogen exchange mass spectrometry (HX MS) to compare the solution conformation of Nef alone and in complexes with the SH3 or the SH3–SH2 domains of the SRC-Family Kinase Hck. HX MS revealed that interaction with the Hck SH3 or tandem SH3–SH2 domains induces protection of the Nef αB-helix from deuterium uptake, consistent with a role for αB in dimer formation. HX MS analysis of a Nef mutant (position Asp123, a site buried in the Nef:SH3 dimer but surface exposed in the Nef:SH3–SH2 complex), showed a Hck-induced conformational change in Nef relative to wild-type Nef. These results support a model in which SRC-Family Kinase binding induces conformational changes in Nef to expose residues critical for interaction with the μ1 subunit of adaptor protein 1 and the major histocompatibility complex-1 tail, and subsequent major histocompatibility complex-1 downregulation and immune escape of HIV-infected cells required for functional interactions with downstream binding partners.
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HIV-1 Nef interaction influences the ATP-binding site of the SRC-Family Kinase, Hck
BMC Chemical Biology, 2012Co-Authors: Teodora Pene-dumitrescu, Sherry T Shu, John J Alvarado, Purushottam Narute, Jamie A Moroco, Joanne I Yeh, Haibin Shi, Thomas E Wales, John R Engen, Thomas E. SmithgallAbstract:BackgroundNef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src Kinase family. Nef preferentially activates Hck, a SRC-Family Kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of Kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the Kinase active site to favor inhibitor association.ResultsTo test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine Kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the Kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based Kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the K_m for ATP as well as enhanced inhibitor potency.ConclusionsThese observations suggest that stable interaction with Nef may induce SRC-Family Kinase active site conformations amenable to selective inhibitor targeting.
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An inhibitor-resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family Kinase inhibitor A-419259
Oncogene, 2008Co-Authors: Teodora Pene-dumitrescu, L F Peterson, N J Donato, Thomas E. SmithgallAbstract:Chronic myelogenous leukemia (CML) is driven by Bcr-Abl, a constitutively active protein-tyrosine Kinase that stimulates proliferation and survival of myeloid progenitors. Global inhibition of myeloid Src family Kinase (SFK) activity with the broad-spectrum pyrrolo-pyrimidine inhibitor, A-419259, blocks proliferation and induces apoptosis in CML cell lines, suggesting that transformation by Bcr-Abl requires SFK activity. However, the contribution of Hck and other individual SFKs to Bcr-Abl signaling is less clear. Here, we developed an A-419259-resistant mutant of Hck by replacing the gatekeeper residue (Thr-338; c-Src numbering) in the inhibitor-binding site with a bulkier methionine residue (Hck-T338M). This substitution reduced Hck sensitivity to A-419259 by more than 30-fold without significantly affecting Kinase activity in vitro . Expression of Hck-T338M protected K-562 CML cells and Bcr-Abl-transformed TF-1 myeloid cells from the apoptotic and antiproliferative effects of A-419259. These effects correlated with persistence of Hck-T338M Kinase activity in the presence of the compound, and were accompanied by sustained Erk and Stat5 activation. In contrast, control cells expressing equivalent levels of wild-type Hck retained sensitivity to the inhibitor. We also show for the first time that A-419259 induces cell-cycle arrest and apoptosis in primary CD34^+ CML cells with equal potency to imatinib. These data suggest that Hck has a nonredundant function as a key downstream signaling partner for Bcr-Abl and may represent a potential drug target in CML.
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Src Family Kinases Phosphorylate the Bcr-Abl SH3-SH2 Region and Modulate Bcr-Abl Transforming Activity
Journal of Biological Chemistry, 2006Co-Authors: Malcolm A. Meyn, John R Engen, Anthony P. Schiavone, Matthew B. Wilson, Fadi A. Abdi, Nathalie Fahey, Jiong Wu, James M. Hochrein, Thomas E. SmithgallAbstract:Abstract Bcr-Abl is the oncogenic protein-tyrosine Kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family Kinase activity (e.g. Hck, Lyn, and Fyn) induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family Kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family Kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family Kinases impacts Bcr-Abl protein conformation and signaling.
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Activation of STAT3 by the Src family Kinase Hck requires a functional SH3 domain.
Journal of Biological Chemistry, 2002Co-Authors: Steven J. Schreiner, Anthony P. Schiavone, Thomas E. SmithgallAbstract:STAT3 is a member of a family of transcription factors with Src homology 2 (SH2) domains that are activated by tyrosine phosphorylation in response to a wide variety of cytokines and growth factors. In this study, we investigated the mechanism of STAT3 activation by the Src family of nonreceptor tyrosine Kinases, which have been linked to STAT activation in both normal and transformed cell types. Using Sf-9 insect cells, we demonstrate direct STAT3 tyrosine phosphorylation and stimulation of DNA binding activity by five members of the Src Kinase family (Src, Hck, Lyn, Fyn, and Fgr). We also observed stable STAT3.Src family Kinase complex formation in this system. Recombinant Src family Kinase SH3 domains were sufficient for interaction with STAT3, suggesting a mechanistic basis for the Src Kinase-STAT3 interaction. To test the contribution of Src family Kinase SH3 domains to the recruitment and activation of STAT3 in vivo, we used Rat-2 fibroblasts expressing activated mutants of the myeloid Src family member Hck. Transformation of fibroblasts by an activated Hck mutant lacking the negative regulatory tail tyrosine residue (Hck-YF) induced strong DNA binding activity of endogenous STAT3. Inactivation of Hck SH3 function by Ala replacement of a conserved Trp residue (W93A mutant) completely abolished STAT3 activation by Hck-YF and reduced transforming activity by 50% without affecting Hck Kinase activity. Finally, overexpression of STAT3 in Rat-2 cells transiently stimulated Hck and c-Src Kinase activity in the absence of extracellular signals, an effect that was dependent upon a putative SH3 binding motif in STAT3. These results support a model in which Src family Kinases recruit STAT3 through an SH3-dependent mechanism, resulting in transient Kinase activation and STAT3 phosphorylation.
Dmitri Pchejetski - One of the best experts on this subject based on the ideXlab platform.
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Leptin induces upregulation of sphingosine Kinase 1 in oestrogen receptor-negative breast cancer via Src family Kinase-mediated, janus Kinase 2-independent pathway
Breast Cancer Research, 2014Co-Authors: Heba Alshaker, Jonathan Krell, Adam E Frampton, Jonathan Waxman, Oleg Blyuss, Alexey Zaikin, Mathias Winkler, Justin Stebbing, Ernesto Yague, Dmitri PchejetskiAbstract:Introduction Obesity is a known risk factor for breast cancer. Sphingosine Kinase 1 (SK1) is an oncogenic lipid Kinase that is overexpressed in breast tumours and linked with poor prognosis, however, its role in obesity-driven breast cancer was never elucidated. Methods Human primary and secondary breast cancer tissues were analysed for SK1 and leptin receptor expression using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Leptin-induced signalling was analysed in human oestrogen receptor (ER)-positive and negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Results Our findings show for the first time that human primary breast tumours and associated lymph node metastases exhibit a strong correlation between SK1 and leptin receptor expression (Pearson R = 0.78 and R = 0.77, respectively, P
Mathias Winkler - One of the best experts on this subject based on the ideXlab platform.
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leptin induces upregulation of sphingosine Kinase 1 in oestrogen receptor negative breast cancer via src family Kinase mediated janus Kinase 2 independent pathway
Breast Cancer Research, 2014Co-Authors: Heba Alshaker, Jonathan Krell, Adam E Frampton, Jonathan Waxman, Oleg Blyuss, Alexey Zaikin, Mathias Winkler, Justin Stebbing, Ernesto YagueAbstract:Introduction: Obesity is a known risk factor for breast cancer. Sphingosine Kinase 1 (SK1) is an oncogenic lipid Kinase that is overexpressed in breast tumours and linked with poor prognosis, however, its role in obesity-driven breast cancer was never elucidated. Methods: Human primary and secondary breast cancer tissues were analysed for SK1 and leptin receptor expression using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Leptin-induced signalling was analysed in human oestrogen receptor (ER)-positive and negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Results: Our findings show for the first time that human primary breast tumours and associated lymph node metastases exhibit a strong correlation between SK1 and leptin receptor expression (Pearson R= 0.78 and R = 0.77, respectively, P <0.001). Both these genes are elevated in metastases of ER-negative patients and show a significant increase in patients with higher body mass index (BMI). Leptin induces SK1 expression and activation in ER-negative breast cancer cell lines MDAMB-231 and BT-549, but not in ER-positive cell lines. Pharmacological inhibition and gene knockdown showed that leptin-induced SK1 activity and expression are mediated by activation of extracellular signal-regulated Kinases 1/2 (ERK1/2) and Src family Kinase (SFK) pathways, but not by the major pathways downstream of leptin receptor (LEPR) - janus Kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Src-homology 2 domain-containing phosphatase 2 (SHP2) appeared to be key to SK1 activation, and may function as an adaptor protein between SFKs and LEPR. Importantly, leptin-induced breast cancer cell proliferation was abrogated by SK1-specific small interfering RNA (siRNA). Conclusions: Overall, our findings demonstrate a novel SFK/ERK1/2-mediated pathway that links leptin signalling and expression of oncogenic enzyme SK1 in breast tumours and suggest the potential significance of this pathway in ER-negative breast cancer.
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Leptin induces upregulation of sphingosine Kinase 1 in oestrogen receptor-negative breast cancer via Src family Kinase-mediated, janus Kinase 2-independent pathway
Breast Cancer Research, 2014Co-Authors: Heba Alshaker, Jonathan Krell, Adam E Frampton, Jonathan Waxman, Oleg Blyuss, Alexey Zaikin, Mathias Winkler, Justin Stebbing, Ernesto Yague, Dmitri PchejetskiAbstract:Introduction Obesity is a known risk factor for breast cancer. Sphingosine Kinase 1 (SK1) is an oncogenic lipid Kinase that is overexpressed in breast tumours and linked with poor prognosis, however, its role in obesity-driven breast cancer was never elucidated. Methods Human primary and secondary breast cancer tissues were analysed for SK1 and leptin receptor expression using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Leptin-induced signalling was analysed in human oestrogen receptor (ER)-positive and negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays. Results Our findings show for the first time that human primary breast tumours and associated lymph node metastases exhibit a strong correlation between SK1 and leptin receptor expression (Pearson R = 0.78 and R = 0.77, respectively, P
