Arif et al. report that phosphorylation of the important metabolism-controlling kinase, S6K1, at two sites near the protein terminus induces its phosphorylation of multiple targets related to lipid metabolism. These insulin-stimulated phosphorylation events in adipocytes (fat cells) might contribute to the known influence of S6K1 on obesity.
Overcoming therapeutic resistance in glioblastoma (GBM) is an essential strategy for improving cancer therapy. However, cancer cells possess various evasion mechanisms, such as metabolic reprogramming, which promote cell survival and limit therapy. The diverse metabolic fuel sources that are produced by autophagy provide tumors with metabolic plasticity and are known to induce drug or radioresistance in GBM. This study determined that autophagy, a common representative cell homeostasis mechanism, was upregulated upon treatment of GBM cells with ionizing radiation (IR). Nuclear receptor binding factor 2 (NRBF2)—a positive regulator of the autophagy initiation step—was found to be upregulated in a GBM orthotopic xenograft mouse model. Furthermore, ATP production and the oxygen consumption rate (OCR) increased upon activation of NRBF2-mediated autophagy. It was also discovered that changes in metabolic state were induced by alterations in metabolite levels caused by autophagy, thereby causing radioresistance. In addition, we found that lidoflazine—a vasodilator agent discovered through drug repositioning—significantly suppressed IR-induced migration, invasion, and proliferation by inhibiting NRBF2, resulting in a reduction in autophagic flux in both in vitro models and in vivo orthotopic xenograft mouse models. In summary, we propose that the upregulation of NRBF2 levels reprograms the metabolic state of GBM cells by activating autophagy, thus establishing NRBF2 as a potential therapeutic target for regulating radioresistance of GBM during radiotherapy.
by
Qiang Wen;
Benjamin Goldenson;
Serena J. Silver;
Monica Schenone;
Vladimir Dancik;
Zan Huang;
Ling-Zhi Wang;
Timothy Lewis;
W. Frank An;
Xiaoyu Li;
Mark-Anthony Bray;
Clarisse Thiollier;
Lauren Diebold;
Laure Gilles;
Martha S. Vokes;
Christopher B. Moore;
Meghan Bliss-Moreau;
Lynn VerPlank;
Nicola J. Tolliday;
William G Woods
The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.
Ca2+-activated Cl− channels (CaCCs) perform a multitude of functions including the control of cell excitability, regulation of cell volume and ionic homeostasis, exocrine and endocrine secretion, fertilization, amplification of olfactory sensory function, and control of smooth muscle cell contractility. CaCCs are the translated products of two members (ANO1 and ANO2, also known as TMEM16A and TMEM16B) of the Anoctamin family of genes comprising ten paralogs. This review focuses on recent progress in understanding the molecular mechanisms involved in the regulation of ANO1 by cytoplasmic Ca2+, post-translational modifications, and how the channel protein interacts with membrane lipids and protein partners. After first reviewing the basic properties of native CaCCs, we then present a brief historical perspective highlighting controversies about their molecular identity in native cells. This is followed by a summary of the fundamental biophysical and structural properties of ANO1. We specifically address whether the channel is directly activated by internal Ca2+ or indirectly through the intervention of the Ca2+-binding protein Calmodulin (CaM), and the structural domains responsible for Ca2+- and voltage-dependent gating. We then review the regulation of ANO1 by internal ATP, Calmodulin-dependent protein kinase II-(CaMKII)-mediated phosphorylation and phosphatase activity, membrane lipids such as the phospholipid phosphatidyl-(4,5)-bisphosphate (PIP2), free fatty acids and cholesterol, and the cytoskeleton. The article ends with a survey of physical and functional interactions of ANO1 with other membrane proteins such as CLCA1/2, inositol trisphosphate and ryanodine receptors in the endoplasmic reticulum, several members of the TRP channel family, and the ancillary Κ+ channel β subunits KCNE1/5.
Sepsis, a pathology resulting from excessive inflammatory response that leads to multiple organ failure, is a major cause of mortality in intensive care units. Macrophages play an important role in the pathophysiology of sepsis. Accumulating evidence has suggested an upregulated rate of aerobic glycolysis as a key common feature of activated proinflammatory macrophages. Here, we identified a crucial role of myeloid 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (Pfkfb3), a glycolytic activator in lipopolysaccharide (LPS)-induced endotoxemia in mice. Pfkfb3 expression is substantially increased in bone marrow derived macrophages (BMDMs) treated with LPS in vitro and in lung macrophages of mice challenged with LPS in vivo. Myeloid-specific knockout of Pfkfb3 in mice protects against LPS-induced lung edema, cardiac dysfunction and hypotension, which were associated with decreased expression of interleukin 1 beta (Il1b), interleukin 6 (Il6) and nitric oxide synthase 2 (Nos2), as well as reduced infiltration of neutrophils and macrophages in lung tissue. Pfkfb3 ablation in cultured macrophages attenuated LPS-induced glycolytic flux, resulting in a decrease in proinflammatory gene expression. Mechanistically, Pfkfb3 ablation or inhibition with a Pfkfb3 inhibitor AZ26 suppresses LPS-induced proinflammatory gene expression via the NF-κB signaling pathway. In summary, our study reveals the critical role of Pfkfb3 in LPS-induced sepsis via reprogramming macrophage metabolism and regulating proinflammatory gene expression. Therefore, PFKFB3 is a potential target for the prevention and treatment of inflammatory diseases such as sepsis.
Ras-related C3 botulinum toxin substrate 1 (Rac1) is a small GTPase that is well known for its sensitivity to the environmental stress of a cell or an organism. It senses the external signals which are transmitted from membrane-bound receptors and induces downstream signaling cascades to exert its physiological functions. Rac1 is an important regulator of a variety of cellular processes, such as cytoskeletal organization, generation of oxidative products, and gene expression. In particular, Rac1 has a significant influence on certain brain functions like neuronal migration, synaptic plasticity, and memory formation via regulation of actin dynamics in neurons. Abnormal Rac1 expression and activity have been observed in multiple neurological diseases. Here, we review recent findings to delineate the role of Rac1 signaling in neurodevelopmental disorders associated with abnormal spine morphology, synaptogenesis, and synaptic plasticity. Moreover, certain novel inhibitors of Rac1 and related pathways are discussed as potential avenues toward future treatment for these diseases.
by
Shuo Zhang;
Paul Lyuboslavsky;
Jendayi Azeezah Dixon;
Micah A. Chrenek;
Jana T. Sellers;
Jessica M. Hamm;
P Michael Iuvone;
Christophe P. Ribelayga;
Zhijing Zhang;
Yun Z. Le
PURPOSE. The present study tested the hypothesis that connexin-36 (Cx36) and gap junctions between photoreceptor cells contribute to the circadian rhythm of the photopic electroretinogram (ERG) b-wave amplitude. METHODS. Cone-specific disruption of Cx36 was obtained in mice with a floxed Gjd2 gene and human red/green pigment promoter (HRGP)-driven Cre recombinase. Standard ERG, spectral-domain optical coherence tomography (SD-OCT) and histochemical methods were used. RESULTS. HRGPcreGjd2fl/fl mice had a selective reduction in Cx36 protein in the outer plexiform layer; no reduction in Cx36 was observed in the inner plexiform layer. Cx36 disruption had no effect on the number of cones, the thickness of the photoreceptor layer, or the scotopic ERG responses. However, there was a reduction of the photopic ERG circadian rhythm, with b-wave amplitudes in the day and the night locked in the daytime, light-adapted state. In HRGPcreGjd2+/+ and Gjd2fl/fl controls, the circadian rhythm of light-adapted ERG persisted, similar to that in wild type mice. CONCLUSIONS. Cx36 regulation contributes to the circadian rhythm of light-adapted ERG; in the absence of photoreceptor gap junctions, mice appear to be in a fully light-adapted state regardless of the time of day. The higher amplitudes and reduced circadian regulation of the b-wave of HRGPcreGjd2fl/fl mice may be due to increased synaptic strength at the cone to ON bipolar cell synapse due to electrotonic isolation of the terminals lacking gap junctions.
by
PamelaSara E. Head;
Priya Kapoor-Vazirani;
Purnachandra Ganji;
Hui Zhang;
Sandip K. Rath;
Nho C. Luong;
Ramona Haji-Seyed-Javadi;
Fatmata Sesay;
Shi-Ya Wang;
Duc M. Duong;
Waago Daddacha;
Elizabeth V. Minten;
Boying Song;
Diana Danelia;
Xu Liu;
Shuyi Li;
Eric Ortlund;
Nicholas Seyfried;
David M. Smalley;
Ya Wang;
Xingming Deng;
William Dynan;
Bassel El-Rayes;
Anthony J. Davis;
David Yu
DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.
by
Zhaohui Cai;
Julie A. Saugstad;
Scott D. Sorensen;
Kelly J. Ciombor;
Congxiao Zhang;
Herve Schaffhauser;
Frantisek Hubalek;
Jan Pohl;
Robert M. Duvoisin;
P. Jeffrey Conn
Recent evidence suggests that the functions of presynaptic metabotropic glutamate receptors (mGluRs) are tightly regulated by protein kinases. We previously reported that cAMP-dependent protein kinase (PKA) directly phosphorylates mGluR2 at a single serine residue (Ser843) on the C-terminal tail region of the receptor, and that phosphorylation of this site inhibits coupling of mGluR2 to GTP-binding proteins. This may be the mechanism by which the adenylyl cyclase activator forskolin inhibits presynaptic mGluR2 function at the medial perforant path-dentate gyrus synapse. We now report that PKA also directly phosphorylates several group III mGluRs (mGluR4a, mGluR7a, and mGluR8a), as well as mGluR3 at single conserved serine residues on their C-terminal tails. Furthermore, activation of PKA by forskolin inhibits group III mGluR-mediated responses at glutamatergic synapses in the hippocampus. Interestingly, β-adrenergic receptor activation was found to mimic the inhibitory effect of forskolin on both group II and III mGluRs. These data suggest that a common PKA-dependent mechanism may be involved in regulating the function of multiple presynaptic group II and group III mGluRs. Such regulation is not limited to the pharmacological activation of adenylyl cyclase but can also be elicited by the stimulation of endogenous Gs-coupled receptors, such as β-adrenergic receptors.