Melanie H Cobb

University of Oxford, Oxford, England, United Kingdom

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Publications (221)1797.86 Total impact

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    ABSTRACT: In this issue of Cancer Cell, Herrero and colleagues identify an anti-tumorigenic small molecule that blocks ERK dimerization, but neither its catalytic activity nor its phosphorylation by MEK. These findings demonstrate that targeting protein dimerization could be a therapeutic avenue for inhibiting kinase signaling pathways associated with lower drug resistance. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cancer cell 08/2015; 28(2):145-7. DOI:10.1016/j.ccell.2015.07.008 · 23.89 Impact Factor
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    ABSTRACT: The related protein kinases SPAK and OSR1 regulate ion homeostasis in part by phosphorylating cation cotransporter family members. The structure of the kinase domain of OSR1 was solved in the unphosphorylated inactive form, and like some other Ste20 kinases, exhibited a domain-swapped activation loop. To further probe the role of domain swapping in SPAK/OSR1, we have determined the crystal structures of SPAK 63-403 at 3.1 Å and SPAK 63-390 T243D at 2.5 Å resolutions. These structures encompass the kinase domain and different portions of the C-terminal tail, the longer without, and the shorter with an activating point mutation T243D. The structure of the T243D protein reveals significant conformational differences relative to unphosphorylated SPAK and OSR1, but also has some features of an inactive kinase. Both structures are domain-swapped dimers. Sequences involved in domain swapping were identified and mutated to create a SPAK monomeric mutant with kinase activity, indicating that monomeric forms are active. The monomeric mutant is activated by WNK1, but has reduced activity toward its substrate NKCC2, suggesting regulatory roles for domain swapping. The structure of the partially active SPAK T243D is consistent with a multi-stage activation process in which phosphorylation induces a SPAK conformation that requires further remodeling to build the active structure.
    Biochemistry 07/2015; DOI:10.1021/acs.biochem.5b00593 · 3.01 Impact Factor
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    ABSTRACT: The mitogen-activated protein kinases ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic β cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic β cell-derived line by extracellular amino acids is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor (GPCR). We show here that amino acids differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent activation of either ERK1/2 or mTORC1 by amino acids, indicating that Gi is not central to either pathway. Although glucagon-like peptide 1 (GLP-1), an agonist for a Gs-coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, amino acids had no effect on cytosolic cyclic AMP accumulation. Ca(2+) entry is required for ERK1/2 activation by amino acids but is dispensable for amino acid activation of mTORC1. Pretreatment with UBO-QIC, a selective Gq inhibitor, reduced activation of ERK1/2 but had little effect on activation of mTORC1 by amino acids, suggesting a differential requirement for Gq. Inhibition of G12/13 by overexpression of the RGS domain of p115 RhoGEF had no effect on mTORC1 activation by amino acids, suggesting that these G proteins are also not involved. We conclude that amino acids regulate ERK1/2 and mTORC1 through distinct signaling pathways.
    Molecular Endocrinology 07/2015; DOI:10.1210/ME.2014-1181 · 4.20 Impact Factor
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    ABSTRACT: The with no lysine (K) (WNK) family of enzymes is best known for control of blood pressure through regulation of the function and membrane localization of ion cotransporters. In mice, global as well as endothelial-specific WNK1 gene disruption results in embryonic lethality due to angiogenic and cardiovascular defects. WNK1(-/-) embryos can be rescued by endothelial-specific expression of a constitutively active form of the WNK1 substrate protein kinase OSR1 (oxidative stress responsive 1). Using human umbilical vein endothelial cells (HUVECs), we explored mechanisms underlying the requirement of WNK1-OSR1 signaling for vascular development. WNK1 is required for cord formation in HUVECs, but the actions of the two major WNK1 effectors, OSR1 and its close relative SPAK (STE20/SPS1-related proline-, alanine-rich kinase), are distinct. SPAK is important for endothelial cell proliferation, whereas OSR1 is required for HUVEC chemotaxis and invasion. We also identified the zinc-finger transcription factor Slug in WNK1-mediated control of endothelial functions. Our study identifies a separation of functions for the WNK1-activated protein kinases OSR1 and SPAK in mediating proliferation, invasion, and gene expression in endothelial cells and an unanticipated link between WNK1 and Slug that is important for angiogenesis.
    Proceedings of the National Academy of Sciences 10/2014; 111(45). DOI:10.1073/pnas.1419057111 · 9.81 Impact Factor
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    ABSTRACT: Aggressive neuroendocrine lung cancers, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), represent an understudied tumor subset that accounts for approximately 40,000 new lung cancer cases per year in the United States. No targeted therapy exists for these tumors. We determined that achaete-scute homolog 1 (ASCL1), a transcription factor required for proper development of pulmonary neuroendocrine cells, is essential for the survival of a majority of lung cancers (both SCLC and NSCLC) with neuroendocrine features. By combining whole-genome microarray expression analysis performed on lung cancer cell lines with ChIP-Seq data designed to identify conserved transcriptional targets of ASCL1, we discovered an ASCL1 target 72-gene expression signature that (i) identifies neuroendocrine differentiation in NSCLC cell lines, (ii) is predictive of poor prognosis in resected NSCLC specimens from three datasets, and (iii) represents novel "druggable" targets. Among these druggable targets is B-cell CLL/lymphoma 2, which when pharmacologically inhibited stops ASCL1-dependent tumor growth in vitro and in vivo and represents a proof-of-principle ASCL1 downstream target gene. Analysis of downstream targets of ASCL1 represents an important advance in the development of targeted therapy for the neuroendocrine class of lung cancers, providing a significant step forward in the understanding and therapeutic targeting of the molecular vulnerabilities of neuroendocrine lung cancer.
    Proceedings of the National Academy of Sciences 09/2014; 111(41). DOI:10.1073/pnas.1410419111 · 9.81 Impact Factor
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    ABSTRACT: Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors.
    Journal of Clinical Investigation 08/2014; 124(9). DOI:10.1172/JCI69004 · 13.77 Impact Factor
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    ABSTRACT: The kinesin family members (KIFs) KIF2A and KIF2C depolymerize microtubules, unlike the majority of other kinesins, which transport cargo along microtubules. KIF2A regulates the localization of lysosomes in the cytoplasm, which assists in activation of the mechanistic target of rapamycin complex 1 (mTORC1) on the lysosomal surface. We find that the closely related kinesin KIF2C also influences lysosomal organization in immortalized human bronchial epithelial cells (HBECs). Expression of KIF2C and, to a lesser extent, KIF2A in untransformed and mutant K-Ras-transformed cells is regulated by ERK1/2. Prolonged inhibition of ERK1/2 activation with PD0325901 mimics nutrient deprivation by disrupting lysosome organization and decreasing mTORC1 activity in HBEC, suggesting a long-term mechanism for optimization of mTORC1 activity by ERK1/2. We tested the hypothesis that up-regulation of KIF2C and KIF2A by ERK1/2 caused aberrant lysosomal positioning and mTORC1 activity in a mutant K-Ras-dependent cancer and cancer model. In Ras-transformed cells, however, mTORC1 activity and lysosome organization appear independent of ERK1/2 and these kinesins although ERK1/2 activity and the kinesins are required for Ras-dependent proliferation and migration. We conclude that mutant K-Ras repurposes these signaling and regulatory proteins to support the transformed phenotype.
    Proceedings of the National Academy of Sciences 07/2014; 111(29). DOI:10.1073/pnas.1411016111 · 9.81 Impact Factor
  • Aileen M Klein · Melanie H Cobb
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    ABSTRACT: Mitogen-activated protein kinase (MAPK) cascades are tightly controlled through a series of well-characterized phospho-regulatory events. In this issue, Takeda et al (2014) identify the inhibitor of apoptosis protein, XIAP, as a key regulator of ERK5 activation via uncoupling of upstream kinase activity by non-degradative ubiquitination.
    The EMBO Journal 07/2014; 33(16). DOI:10.15252/embj.201489205 · 10.75 Impact Factor
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    ABSTRACT: WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade, leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. We found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride-binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation.
    Science Signaling 05/2014; 7(324):ra41. DOI:10.1126/scisignal.2005050 · 7.65 Impact Factor
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    ABSTRACT: Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. Although autophagy is generally accepted to be regulated in a cell-autonomous fashion, recent studies suggest that nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets.
    Trends in Endocrinology and Metabolism 04/2014; 25(5). · 8.87 Impact Factor
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    Eric M Wauson · Hashem A Dbouk · Anwesha B Ghosh · Melanie H Cobb
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    ABSTRACT: Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. Although autophagy is generally accepted to be regulated in a cell-autonomous fashion, recent studies suggest that nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets.
    Trends in Endocrinology and Metabolism 04/2014; 25(5). DOI:10.1016/j.tem.2014.03.006 · 8.87 Impact Factor
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    ABSTRACT: Cigarette smoking is a major risk factor for acquisition of small cell lung cancer (SCLC). We and others have demonstrated a role for the basic helix-loop-helix (bHLH) transcription factor NeuroD1 in the pathogenesis of neural and neuroendocrine lung cancer including SCLC. In the current study we investigated the possible function of NeuroD1 in established tumors as well as actions early on in pathogenesis in response to nicotine. We demonstrate that nicotine up-regulates NeuroD1 in immortalized normal bronchial epithelial cells (HBECs) and a subset of undifferentiated carcinomas. Increased expression of NeuroD1 subsequently leads to regulation of expression and function of the nicotinic acetylcholine receptor (nAChR) subunit cluster of α3, α5, and β4. Additionally, we find that coordinated expression of these subunits by NeuroD1 leads to enhanced nicotine-induced migration and invasion likely through changes in intracellular calcium. These findings suggest that aspects of the pathogenesis of neural and neuroendocrine lung cancers may be affected by a nicotine- and NeuroD1-induced positive feedback loop.
    Molecular biology of the cell 04/2014; 25(11). DOI:10.1091/mbc.E13-06-0316 · 5.98 Impact Factor
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    ABSTRACT: We have recently shown that the class C GPCR T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic β cells. Amino acids are unable to activate ERK1/2 in β cells in which T1R3 has been depleted. The muscarinic receptor agonist, carbachol, activated ERK1/2 better in T1R3-depleted than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic β cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in β cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and calcium responses were altered. To determine if these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased and some but not all changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in β cells as a mechanism to compensate for amino acid deficiency.
    Journal of Biological Chemistry 04/2014; 289(20). DOI:10.1074/jbc.M114.565069 · 4.57 Impact Factor
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    ABSTRACT: We show that expression of the microtubule depolymerizing kinesin KIF2C is induced by transformation of immortalized human bronchial epithelial cells (HBEC) by expression of K-Ras(G12V) and knockdown of p53. Further investigation demonstrates that this is due to the K-Ras/ERK1/2 MAPK pathway, as loss of p53 had little effect on KIF2C expression. In addition to KIF2C, we also found that the related kinesin KIF2A is modestly upregulated in this model system; both proteins are expressed more highly in many lung cancer cell lines compared to normal tissue. As a consequence of their depolymerizing activity, these kinesins increase dynamic instability of microtubules. Depletion of either of these kinesins impairs the ability of cells transformed with mutant K-Ras to migrate and invade matrigel. However, depletion of these kinesins does not reverse the epithelial to mesenchymal transition (EMT) caused by mutant K-Ras. Our studies indicate that increased expression of microtubule destabilizing factors can occur during oncogenesis to support enhanced migration and invasion of tumor cells.Oncogene advance online publication, 18 November 2013; doi:10.1038/onc.2013.486.
    Oncogene 11/2013; 33(47). DOI:10.1038/onc.2013.486 · 8.56 Impact Factor
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    ABSTRACT: The Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase directly regulate the solute carrier 12 family of cation-chloride cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. OSR1 and STE20/SPS1-related proline-, alanine-rich kinase are activated by with no lysine [K] protein kinases that phosphorylate the essential activation loop regulatory site on these kinases. We found that inhibition of phosphoinositide 3-kinase (PI3K) reduced OSR1 activation by osmotic stress. Inhibition of the PI3K target pathway, the mammalian target of rapamycin complex 2 (mTORC2), by depletion of Sin1, one of its components, decreased activation of OSR1 by sorbitol and reduced activity of the OSR1 substrate, the sodium, potassium, two chloride cotransporter, in HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2 phosphorylated OSR1 on S339 in vitro, and mutation of this residue eliminated OSR1 phosphorylation by mTORC2. Thus, we identify a previously unrecognized connection of the PI3K pathway through mTORC2 to a Ste20 protein kinase and ion homeostasis.
    Proceedings of the National Academy of Sciences 11/2013; 110(47). DOI:10.1073/pnas.1318676110 · 9.81 Impact Factor
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    ABSTRACT: The developmental transcription factor NeuroD1 is anomalously expressed in a subset of aggressive neuroendocrine tumors. Previously, we demonstrated that TrkB and neural cell adhesion molecule (NCAM) are downstream targets of NeuroD1 that contribute to the actions of neurogenic differentiation 1 (NeuroD1) in neuroendocrine lung. We found that several malignant melanoma and prostate cell lines express NeuroD1 and TrkB. Inhibition of TrkB activity decreased invasion in several neuroendocrine pigmented melanoma but not in prostate cell lines. We also found that loss of the tumor suppressor p53 increased NeuroD1 expression in normal human bronchial epithelial cells and cancer cells with neuroendocrine features. Although we found that a major mechanism of action of NeuroD1 is by the regulation of TrkB, effective targeting of TrkB to inhibit invasion may depend on the cell of origin. These findings suggest that NeuroD1 is a lineage-dependent oncogene acting through its downstream target, TrkB, across multiple cancer types, which may provide new insights into the pathogenesis of neuroendocrine cancers.
    08/2013; 2(8):e63. DOI:10.1038/oncsis.2013.24
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    ABSTRACT: The mitogen-activated protein kinases (MAPKs) ERK1/2 regulate numerous cellular processes including gene transcription, proliferation, and differentiation. The only known substrates of the MAP2Ks MEK1/2 are ERK1/2; thus, the MEK inhibitors PD98059, U0126, and PD0325901 have been important tools in determining the functions of ERK1/2. By using these inhibitors and genetically manipulating MEK, we find that ERK1/2 activation is neither sufficient nor necessary for regulated insulin secretion from pancreatic beta cells or epinephrine secretion from chromaffin cells. We show that both PD98059 and U0126 reduce agonist-induced calcium entry into cells inde-pendently of their ability to inhibit ERK1/2. Caution should be used when interpreting results from experiments using these compounds.
    Biochemistry 07/2013; 52(31). DOI:10.1021/bi4007644 · 3.01 Impact Factor
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    ABSTRACT: G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are important for cellular decisions are amino acids, required for continued protein synthesis, as metabolic starting materials and energy sources. Amino acids are detected by a number of class C GPCRs. One cluster of amino acid-sensing class C GPCRs includes umami and sweet taste receptors, GPRC6A, and the calcium-sensing receptor. We have recently found that the umami taste receptor heterodimer T1R1/T1R3 is a sensor of amino acid availability that regulates the activity of the mammalian target of rapamycin. This review focuses on an array of findings on sensing amino acids and sweet molecules outside of neurons by this cluster of class C GPCRs and some of the physiologic processes regulated by them.
    Molecular Endocrinology 07/2013; 27(8). DOI:10.1210/me.2013-1100 · 4.20 Impact Factor
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    ABSTRACT: Small-cell lung cancer and other aggressive neuroendocrine cancers are often associated with early dissemination and frequent metastases. We demonstrate that neurogenic differentiation 1 (NeuroD1) is a regulatory hub securing cross talk among survival and migratory-inducing signaling pathways in neuroendocrine lung carcinomas. We find that NeuroD1 promotes tumor cell survival and metastasis in aggressive neuroendocrine lung tumors through regulation of the receptor tyrosine kinase tropomyosin-related kinase B (TrkB). Like TrkB, the prometastatic signaling molecule neural cell adhesion molecule (NCAM) is a downstream target of NeuroD1, whose impaired expression mirrors loss of NeuroD1. TrkB and NCAM may be therapeutic targets for aggressive neuroendocrine cancers that express NeuroD1.
    Proceedings of the National Academy of Sciences 04/2013; 110(16). DOI:10.1073/pnas.1303932110 · 9.81 Impact Factor
  • Aileen M Klein · Elma Zaganjor · Melanie H Cobb
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    ABSTRACT: Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that are essential nodes in many cellular regulatory circuits including those that take place on DNA. Most members of the four MAPK subgroups that exist in canonical three kinase cascades-extracellular signal-regulated kinases 1 and 2 (ERK1/2), ERK5, c-Jun N-terminal kinases (JNK1-3), and p38 (α, β, γ, and δ) families-have been shown to perform regulatory functions on chromatin. This review offers a brief update on the variety of processes that involve MAPKs and available mechanisms garnered in the last two years.
    Current opinion in cell biology 02/2013; 25(2). DOI:10.1016/j.ceb.2013.01.002 · 8.74 Impact Factor

Publication Stats

25k Citations
1,797.86 Total Impact Points

Institutions

  • 2014
    • University of Oxford
      Oxford, England, United Kingdom
  • 1990–2014
    • University of Texas at Dallas
      • Biochemistry
      Richardson, Texas, United States
    • Regeneron
      Terryville, New York, United States
  • 1989–2014
    • University of Texas Southwestern Medical Center
      • • Department of Pharmacology
      • • Department of Biochemistry
      Dallas, Texas, United States
  • 2007
    • Harvard University
      • Department of Molecular and Cell Biology
      Cambridge, Massachusetts, United States
  • 2006
    • Baylor University
      Waco, Texas, United States
  • 2002
    • Institute of Molecular Biology
      Mayence, Rheinland-Pfalz, Germany
  • 1994
    • University of California, San Diego
      • Department of Pharmacology
      San Diego, California, United States
  • 1988–1994
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 1992
    • Louisiana State University Health Sciences Center New Orleans
      • Department of Biochemistry and Molecular Biology
      New Orleans, Louisiana, United States
  • 1986
    • University of Texas Health Science Center at Tyler
      Tyler, Texas, United States