Examining novel roles for the IκB kinase in coordinating the cellular response to metabolic stress

The induction of mammalian autophagy, a conserved cellular bulk-degradation process, was recently shown to require Inhibitor of κB (IκB) Kinase (IKK), the upstream regulator of nuclear factor (NF)-κB transcription factors. In response to cell stress IKK has been exclusively studied for its ability to activate NF-κB-dependent pro-inflammatory gene expression; surprisingly this activity is not required for starvation-induced autophagy and the mechanism by which IKK promotes this activity are largely unknown. Here we investigate the role of IKK/NF-κB pathway in response to both acute and prolonged nutrient deprivation, a classic autophagy- and novel NF-κB-inducing stimulus. We demonstrate that classic IKK-dependent NF-κB activation and gene expression occurs in response to cell starvation. Independently, IKK controls expression of genes necessary for autophagic machinery in response to prolonged starvation. The work presented in Chapter 2 will demonstrate that IKK is important molecule upstream of changes in gene expression induced by cellular starvation. IKK activity in response to acute starvation is also explored within and we identify that this kinase is important for transducing signals that inhibit growth and metabolic pathways. We find that IKK is required for inhibition of growth factor-dependent signaling through phosphorylation of the novel IKK substrate Phosphoinonsitide 3-Kinase (PI3K). The work outlined in Chapter 3 identifies a novel cross-talk mechanism between growth and stress responsive signal transduction pathways important for coordinating the cellular response to nutrient availability. In summary, the following manuscript will identify two novel functions for the IKK complex in regulating nutrient sensitive pathways, establishing the critical role of this kinase in cellular adaptation to metabolic stress

Nutrient-sensing mechanisms and pathways

The ability to sense and respond to fluctuations in environmental nutrient levels is a requisite for life. Nutrient scarcity is a selective pressure that has shaped the evolution of most cellular processes. Different pathways that detect intracellular and extracellular levels of sugars, amino acids, lipids and surrogate metabolites are integrated and coordinated at the organismal level through hormonal signals. During food abundance, nutrient-sensing pathways engage anabolism and storage, whereas scarcity triggers homeostatic mechanisms, such as the mobilization of internal stores through autophagy. Nutrient-sensing pathways are commonly deregulated in human metabolic diseases.National Institutes of Health (U.S.) (Grant R01 CA129105)National Institutes of Health (U.S.) (Grant R01 CA103866)National Institutes of Health (U.S.) (Grant R01 AI047389)National Institutes of Health (U.S.) (Grant R21 AG042876)American Federation for Aging ResearchStarr FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT (Frontier Research Program)Ellison Medical FoundationCharles A. King TrustAmerican Cancer Society (Ellison Medical Foundation Postdoctoral Fellowship PF-13-356-01-TBE

Quantitative modelling of amino acid transport and homeostasis in mammalian cells

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signaling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell’s endowment with amino acid transporters can be deconvoluted experimentally and used this data to computationally simulate amino acid translocation across the plasma membrane. Transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system are replicated in silico and can be applied to systems where only transcriptomic data are available. This work explains amino acid homeostasis at the systems-level, through a combination of secondary active transporters, functionally acting as loaders, harmonizers and controller transporters to generate a stable equilibrium of all amino acid concentrations

Dihydropyrimidine Accumulation Is Required for the Epithelial-Mesenchymal Transition

It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the “mesenchymal metabolic signature” (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits.United States. National Institutes of Health (RO1 CA103866)United States. National Institutes of Health (AI047389)United States. National Institutes of Health (K99 CA168940)American Cancer Society (PF-12-099-01-TGB)American Cancer Society (PF-13-356-01-TBE)United States. Department of Defense (BC123066)United States. National Institutes of Health (CA112967)United States. National Institutes of Health (ES015339

Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB ▿

Glutamate is a critical neurotransmitter of the central nervous system (CNS) and also an important regulator of cell survival and proliferation. The binding of glutamate to metabotropic glutamate receptors induces signal transduction cascades that lead to gene-specific transcription. The transcription factor NF-κB, which regulates cell proliferation and survival, is activated by glutamate; however, the glutamate receptor-induced signaling pathways that lead to this activation are not clearly defined. Here we investigate the glutamate-induced activation of NF-κB in glial cells of the CNS, including primary astrocytes. We show that glutamate induces phosphorylation, nuclear accumulation, DNA binding, and transcriptional activation function of glial p65. The glutamate-induced activation of NF-κB requires calcium-dependent IκB kinase α (IKKα) and IKKβ activation and induces p65-IκBα dissociation in the absence of IκBα phosphorylation or degradation. Moreover, glutamate-induced IKK preferentially targets the phosphorylation of p65 but not IκBα. Finally, we show that the ability of glutamate to activate NF-κB requires cross-coupled signaling with the epidermal growth factor receptor. Our results provide insight into a glutamate-induced regulatory pathway distinct from that described for cytokine-induced NF-κB activation and have important implications with regard to both normal glial cell physiology and pathogenesis

Disruption of the Rag-Ragulator Complex by c17orf59 Inhibits mTORC1

mTORC1 controls key processes that regulate cell growth, including mRNA translation, ribosome biogenesis, and autophagy. Environmental amino acids activate mTORC1 by promoting its recruitment to the cytosolic surface of the lysosome, where its kinase is activated downstream of growth factor signaling. mTORC1 is brought to the lysosome by the Rag GTPases, which are tethered to the lysosomal membrane by Ragulator, a lysosome-bound scaffold. Here, we identify c17orf59 as a Ragulator-interacting protein that regulates mTORC1 activity through its interaction with Ragulator at the lysosome. The binding of c17orf59 to Ragulator prevents Ragulator interaction with the Rag GTPases, both in cells and in vitro, and decreases Rag GTPase lysosomal localization. Disruption of the Rag-Ragulator interaction by c17orf59 impairs mTORC1 activation by amino acids by preventing mTOR from reaching the lysosome. By disrupting the Rag-Ragulator interaction to inhibit mTORC1, c17orf59 expression may represent another mechanism to modulate nutrient sensing by mTORC1

p85α SH2 Domain Phosphorylation by IKK Promotes Feedback Inhibition of PI3K and Akt in Response to Cellular Starvation

The IκB kinase (IKK) pathway is an essential mediator of inflammatory, oncogenic, and cell stress pathways. Recently IKK was shown to be essential for autophagy induction in mammalian cells independent of its ability regulate NF-κB, but the mechanism by which this occurs is unclear. Here we demonstrate that the p85 regulatory subunit of PI3K is an IKK substrate, phosphorylated at S690 in vitro and in vivo in response to cellular starvation. Cells expressing p85 S690A or inhibited for IKK activity exhibit increased Akt activity following cell starvation, demonstrating that p85 phosphorylation is required for starvation-induced PI3K feedback inhibition. S690 is in a conserved region of the p85 cSH2 domain, and IKK-mediated phosphorylation of this site results in decreased affinity for tyrosine-phosphorylated proteins and decreased PI3K membrane localization. Finally, leucine deprivation is shown to be necessary and sufficient for starvation-induced, IKK-mediated p85 phosphorylation and PI3K feedback inhibition

Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1

The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase–dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid–sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.National Institutes of Health (U.S.) (Grant R01 CA103866)National Institutes of Health (U.S.) (Grant AI47389)United States. Dept. of Defense (W81XWH-07-0448)National Institutes of Health (U.S.) (Fellowship F30CA180754)National Institutes of Health (U.S.) (Fellowship T32 GM007753)National Institutes of Health (U.S.) (Fellowship F31 AG044064)National Institutes of Health (U.S.) (Fellowship F31CA180271)United States. Dept. of Defense (National Defense Science and Engineering Graduate Fellowship)National Science Foundation (U.S.). Graduate Research Fellowship ProgramAmerican Cancer Society (Ellison Medical Foundation. Postdoctoral Fellowship PF-13-356-01-TBE)Howard Hughes Medical Institut