The mitochondrial enzyme glutaminase (GLS) is frequently up-regulated during tumorigenesis and being evaluated as a target for cancer therapy. GLS catalyzes the hydrolysis of glutamine to glutamate, which then supplies diverse metabolic pathways with carbon and/or nitrogen. Here, we report that SIRT5, NAD+-dependent lysine deacylase, plays key role in stabilizing GLS. In transformed cells, SIRT5 regulates metabolism by desuccinylating thereby protecting it from ubiquitin-mediated...

Efforts to target glutamine metabolism for cancer therapy have focused on the glutaminase isozyme GLS. The importance of other isozyme, GLS2, in has remained unclear, and it been described as a tumor suppressor some contexts. Here, we report that GLS2 is upregulated essential luminal-subtype breast tumors, which account >70% incidence. We show expression elevated by GATA3 cells but suppressed promoter methylation basal-subtype cells. Although luminal cancers resist GLS-selective inhibitors,...

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation bromide into tyrosine residues. In this work, we sought to identify major target proteins for bromination HOBr or PXDN-mediated oxidation in mouse...

Overexpression of the receptor tyrosine kinase HER2/ErbB2 (ERBB2) has been linked to a poor prognosis for patients with breast cancer; thus, its activity is central target cancer therapy. Likewise, overexpression heregulin (HRG/NRG1), growth factor responsible ErbB2 activation, also shown be driver progression. Although inhibitors offer major advancement in treatment ErbB2-dependent cancers, are highly susceptible developing clinical resistance these drugs. Therefore, detailed understanding...

Abstract Lung cancers are frequently characterized by inappropriate activation of epidermal growth factor receptor (EGFR)-dependent signaling and epigenetic silencing the NADPH oxidase (NOX) enzyme DUOX1, both potentially contributing to worse prognosis. Based on previous findings linking DUOX1 with redox-dependent EGFR activation, present studies were designed evaluate whether in lung may be responsible for altered regulation. In contrast normal epithelial cells, EGF stimulation cancer cell...

Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin (PH) domains more recently described Dock180 little sequence with are characterized Dock regions 1 2 (DHR-1 -2, respectively). While extensive characterization has performed, less is known about how act as GEFs,...

More than 50% of people with asthma in the United States are obese, and obesity often worsens symptoms allergic impairs response to treatment. Based on previously established roles epithelial NADPH oxidase DUOX1 airway inflammation, we addressed potential involvement altered inflammation context obesity. Intranasal house dust mite (HDM) allergen challenge subjects induced rapid secretion IL-33, then IL-13, into nasal lumen, responses that were significantly enhanced obese asthmatic (BMI...

Abstract The respiratory epithelium forms the first line of defense against inhaled pathogens and acts as an important source innate cytokine responses to environmental insults. One critical mediator these is IL-1 family IL-33, which rapidly secreted upon acute epithelial injury alarmin induces type 2 immune responses. Our recent work highlighted importance NADPH oxidase dual 1 (DUOX1) in airway IL-33 secretion by various airborne allergens associated with H2O2 production...

The ability of cancer cells to survive microenvironmental stresses is critical for tumor progression and metastasis; however, how they these challenges not fully understood. Here, we describe a novel multiprotein complex (DockTOR) essential the survival under stress, triggered by GTPase Cdc42 signaling partner Dock7, which includes AKT, mTOR, mTOR regulators TSC1, TSC2, Rheb. DockTOR enables maintain low but mTORC2-dependent phosphorylation AKT during serum deprivation preventing...

Abstract Akt is a well-known target of mitogenic signaling, which activated by phosphoinositide 3-kinase, PDK1 and mTORC2, commonly regarded as master regulator cell survival. However, there still good deal to learn regarding how can promote survival when cancer cells are faced the multitude challenges they need overcome during process malignant transformation. While investigating role Dock7, Cdc42 Rac guanine nucleotide exchange factor Dock180 family, in tumorigenesis, we discovered an...

<p>PDF file - 42K, Attenuation of HRG-stimulated AKT phosphorylation by the knock-down Rictor can be rescued expression an shRNA-insensitive construct. SKBR3 cells were infected either with control or shRNA-containing virus twice, one day apart, and selected 2 microg/mL puromycin for 48 h. Cells then subjected to transfection mock (no DNA) 5 microg plasmid 3 hours, followed serum-starvation On harvest, treated without 1 nM HRG. Whole cell lysates collected Western blotting probing...

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<p>PDF file - 62K, Ectopically-expressed components of both mTORC1 and mTORC2 in HEK 293T cells can interact with TSC2 as well mTOR. were transfected either Myc-Raptor or Myc-Rictor. Cells then lysed lysis buffer containing 0.3% CHAPS subjected to immunoprecipitation using anti-Myc antibody precipitate Raptor Rictor. The precipitated samples Western blotting probing for mTOR, TSC2, Myc.</p>

<p>PDF file - 128K, Two mechanistically-distinct mTOR inhibitors, rapamycin and INK-128, show differential abilities to inhibit signaling components upstream of mTORC1. SKBR3 cells were serum-starved for 40-48 h. Rapamycin (50 nM) or INK was added the 30 minutes prior additional treatment with without 1 nM HRG min. Cells then harvested, lysed, lysates analyzed by Western blotting relative levels phospho-mTOR (S2448), phospho-TSC2 (T1462), phopsho-AKT (T308), phospho-AKT (S473)...

<p>PDF file - 201K, HRGalpha, as well HRGbeta, is capable of stimulating the ability SKBR3 cells to form colonies and activate components mTORC1 signaling pathway. A, were seeded in 0.3% agarose-containing complete medium with addition 1 nM HRGalpha or HRGbeta. Cells fed every three days growth factor-containing counted on day 13. The experiment was performed triplicate results averaged graphed. B, serum-starved for 40-48 h, then challenged indicated times. Cell lysates collected...

<p>PDF file - 136K, HRG-dependent stimulation of mTORC1-related signaling activities in SKBR3 cells is blocked by pre-treatment with the ErbB2-specific kinase inhibitor, CP-724,714. were serum-starved followed treatment or without 5 microM CP-724,714 for 30 minutes prior to 1 nM HRG an additional minutes. Cells collected and lysed, lysates analyzed Western blotting using specific antibodies probe phospho-ErbB2 (Y1248), phospho-mTOR (S2448), phospho-TSC2 (T1462), phospho-AKT (T308),...

<p>PDF file - 128K, Two mechanistically-distinct mTOR inhibitors, rapamycin and INK-128, show differential abilities to inhibit signaling components upstream of mTORC1. SKBR3 cells were serum-starved for 40-48 h. Rapamycin (50 nM) or INK was added the 30 minutes prior additional treatment with without 1 nM HRG min. Cells then harvested, lysed, lysates analyzed by Western blotting relative levels phospho-mTOR (S2448), phospho-TSC2 (T1462), phopsho-AKT (T308), phospho-AKT (S473)...

<p>PDF file - 136K, HRG-dependent stimulation of mTORC1-related signaling activities in SKBR3 cells is blocked by pre-treatment with the ErbB2-specific kinase inhibitor, CP-724,714. were serum-starved followed treatment or without 5 microM CP-724,714 for 30 minutes prior to 1 nM HRG an additional minutes. Cells collected and lysed, lysates analyzed Western blotting using specific antibodies probe phospho-ErbB2 (Y1248), phospho-mTOR (S2448), phospho-TSC2 (T1462), phospho-AKT (T308),...

<p>PDF file - 201K, HRGalpha, as well HRGbeta, is capable of stimulating the ability SKBR3 cells to form colonies and activate components mTORC1 signaling pathway. A, were seeded in 0.3% agarose-containing complete medium with addition 1 nM HRGalpha or HRGbeta. Cells fed every three days growth factor-containing counted on day 13. The experiment was performed triplicate results averaged graphed. B, serum-starved for 40-48 h, then challenged indicated times. Cell lysates collected...

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<p>PDF file - 42K, Attenuation of HRG-stimulated AKT phosphorylation by the knock-down Rictor can be rescued expression an shRNA-insensitive construct. SKBR3 cells were infected either with control or shRNA-containing virus twice, one day apart, and selected 2 microg/mL puromycin for 48 h. Cells then subjected to transfection mock (no DNA) 5 microg plasmid 3 hours, followed serum-starvation On harvest, treated without 1 nM HRG. Whole cell lysates collected Western blotting probing...