DOCSLIB.ORG

Role of Unc51.1 and Its Binding Partners in CNS Axon Outgrowth

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Role of Unc51.1 and Its Binding Partners in CNS Axon Outgrowth Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Role of Unc51.1 and its binding partners in CNS axon outgrowth Toshifumi Tomoda, Jee Hae Kim, Caixin Zhan, and Mary E. Hatten1 Laboratory of Developmental Neurobiology, The Rockefeller University, New York, New York 10021-6399, USA Previous studies showed that the serine/threonine kinase Unc51.1 is one of the earliest genes in neuronal differentiation and is required for granule cell axon formation. To examine the mechanism of Unc51.1 regulation of axon extension, we have identified two direct binding partners. The first, SynGAP, a negative regulator of Ras, is expressed within axons and growth cones of developing granule cells. Overexpression of SynGAP blocks neurite outgrowth by a mechanism that involves Ras-like GTPase cascade. The second binding partner is a PDZ domain-containing scaffolding protein, Syntenin, that binds Rab5 GTPase, the activity of which is attenuated by SynGAP. Thus, our results demonstrate that the Unc51.1-containing protein complex governs axon formation via Ras-like GTPase signaling and through regulation of the Rab5-mediated endocytic pathways within developing axons. [Keywords: Unc51.1; SynGAP; Syntenin; axon formation; vesicular membrane.] Received September 5, 2003; revised version accepted February 5, 2004. The synaptic circuitry of the cerebellar cortex develops calized to both axonal shafts and growth cones of extend- through the coordinated outgrowth of the axons and den- ing axons. Retroviral infection of granule cell precursors drites of the two principal classes of neurons, the granule with a kinase-deficient form of Unc51.1 demonstrates cell and the Purkinje cell, and the subsequent formation that Unc51.1 is essential for neurite extension/parallel of synaptic junctions among them. Parallel fibers of the fiber formation in cerebellar granule neurons (Tomoda et granule cells form just after the granule cell progenitors al. 1999). Others have recently reported that ULK1, a exit the cell cycle, as an early step in the program of human ortholog of Unc51.1, interacts with the Golgi- neuronal differentiation (Ramon y Cajal 1995). The cer- associated ATPase enhancer of 16 kD (GATE-16) and ebellar cortex provides an unique system for studying with the ␥2subunit of GABA-A receptor associated pro- neurite outgrowth and connectivity. The granule cell, tein (GABARAP; Okazaki et al. 2000). The GATE-16 is the most abundant neuron of the central nervous system an essential factor for intra-Golgi transport (Sagiv et al. (CNS), has facilitated analyses of the molecular and cel- 2000), and the GABARAP has a possible role in the regu- lular mechanisms that underlie the basic steps in neu- lation of receptor trafficking (Wang et al. 1999). How- ronal differentiation. The development of the cerebellar ever, it is not yet known how these Unc51.1 binding granule cell has been studied extensively, including partners function with Unc51.1 to regulate neurite mechanisms of proliferation, neuronal migration, axon formation. formation, and synaptogenesis in vivo and in vitro. To further investigate the molecular basis of Unc51.1/ To gain insight into initial steps of parallel fiber Unc51.2function in neurite extension, we searched for formation/axon outgrowth, we previously identified additional proteins that bind Unc51.1/Unc51.2in yeast Unc51.1/Unc51.2, two murine homologs of the Cae- two-hybrid screens. Interacting molecules identified in- norhabditis elegans unc-51 gene (Tomoda et al. 1999). In cluded SynGAP, a synaptic GTPase-activating protein the worm, unc-51 is important for axon elongation (GAP; Chen et al. 1998; Kim et al. 1998), and Syntenin, (Hedgecock et al. 1985; Desai et al. 1988; Siddiqui 1990; an endocytic vesicular membrane protein with PDZ do- McIntire et al. 1992; Ogura et al. 1994). The unc-51/ mains (Grootjans et al. 1997; Fialka et al. 1999). SynGAP Unc51.1/Unc51.2 genes belong to a subfamily of protein interacts with the PDZ domains of the PSD-95/SAP90 serine/threonine kinases. Unc51.1 is expressed in a family of proteins via its CЈ-terminal amino acids, number of neuronal populations, including the cerebel- T/SXV (Kim et al. 1998), and is a major component of lar granule cell, during development. The protein is lo- postsynaptic density (PSD), a dense cytoskeletal matrix found beneath the postsynaptic membrane that is nota- bly prominent in excitatory synapses (Chen et al. 1998). SynGAP functions as a brain-specific Ras GTPase-acti- 1Corresponding author. vating protein and is associated in a large complex with E-MAIL [email protected]; FAX (212) 327-7145. Article published online ahead of print. Article and publication date are PSD-95/SAP90, SAP102, and the NMDA receptors at ex- at http://www.genesdev.org/cgi/doi/10.1101/gad.1151204. citatory synapses in cortex and hippocampus. Recent GENES & DEVELOPMENT 18:541–558 © 2004 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/04; www.genesdev.org 541 Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Tomoda et al. studies have shown that SynGAP contributes to neural The SynGAP clone isolated in our screen corresponded plasticity by modulating Ras activity in brain (Komi- to the ␣2isoform that encodes a protein 35 amino acids yama et al. 2002; Kim et al. 2003). Syntenin contains two longer than that encoded by the ␣1 isoform, an isoform PDZ domains that bind cytoplasmic domains of various originally reported (Kim et al. 1998; Chen et al. 1998). proteins, including syndecan, neurofascin, glutamate re- The ␣1 isoform ends with TRV, through which it binds ceptors, and ephrin-B2(Grootjans et al. 1997; Lin et al. a PDZ domain of PSD-95. The ␣2isoform has one base 1999; Koroll et al. 2001; Hirbec et al. 2003). Syntenin insertion within the CЈ-terminal tail region of the pro- often serves as a scaffolding molecule for synaptic pro- tein compared with the ␣1 isoform (Li et al. 2001), ends teins (Hirbec et al. 2003). with ADH, and does not bind PSD-95 (data not shown). In this report, we present evidence that SynGAP is Both the ␣1 and ␣2isoforms were expressed in P6 cer- expressed during early stages of granule neuron develop- ebellum as confirmed by RT–PCR, and both bound the ment and that Unc51.1 and SynGAP function in concert Unc51.1/Unc51.2baits, although the ␣2isoform consis- to regulate axon outgrowth of developing granule neu- tently displayed slightly higher affinity to the baits than rons. By using bioassays of primary granule neurons ex- did the ␣1 isoform in a yeast two-hybrid assay (data not pressing either Unc51.1 or SynGAP or both, we show shown). We reasoned that the CЈ-terminal sequence of that Unc51.1 and SynGAP function cooperatively in SynGAP functions as a tag that determines subcellular axon formation during brain development. We also show localizations. We therefore generated GFP fusion pro- that Unc51.1 affects the extent of SynGAP modulation teins with the CЈ-tail domain of SynGAP ␣1or␣2, in- of Ras activity, suggesting a functional link between troduced them retrovirally into granule cells in culture, Unc51.1 and SynGAP that could lead to long-term and found that the GFP-CЈ-tail(␣2) was localized to ex- changes in gene expression responsible for axon forma- tending axons but the GFP-CЈ-tail(␣1) was only localized tion. Syntenin provides a scaffold for Unc51.1 and for to cell soma (data not shown). This demonstrates that Rab5 GTPase, an endocytic membrane regulator, and the ␣1 isoform with TRV motif could be the isoform that SynGAP stimulates Rab5 GTPase activity in vitro. In partitions into postsynaptic compartments, whereas the addition, we show that the SynGAP-induced axon ␣2isoform is the one that preferentially goes to axons. truncation is restored by overexpressing Ras or Rab5 Therefore, we used only the ␣2isoform in subsequent GTPases, and we provide evidence that the dominant- experiments. negative Unc51.1 and SynGAP expression disorganizes To determine the minimum region of Unc51.1 that vesicular membranes within extending axons of primary binds SynGAP, we generated a series of deletion con- granule neuron culture. These studies suggest that structs of Unc51.1 and tested them with SynGAP (amino Unc51.1 and its binding partners influence axon forma- acids 829–1328) in a ␤-galactosidase assay in yeast (Fig. tion via the neuronal endocytic pathway. 1A). Although the CЈ-terminal 399 amino acids of Unc51.1 (amino acids 653–1051), as well as the shorter CЈ-terminal domain of Unc51.1 (amino acids 829–1051), Results strongly bound SynGAP, the spacer region (amino acids 653–828) and subregions of the CЈ-terminal domain Unc51.1/Unc51.2 binds SynGAP (amino acids 829–1001 and amino acids 913–1051) did Previous studies suggested the importance of the CЈ-ter- not bind SynGAP. This result demonstrates that the CЈ- minal domains of unc-51/Unc51.1/Unc51.2 in their bio- terminal domain of Unc51.1 (amino acids 829–1051) is logical function (Ogura et al. 1994; Tomoda et al. 1999). the minimum requirement for the binding to SynGAP, Thus, we used the CЈ-terminal region of Unc51.1 (amino and suggests an important role of this domain as a func- acids 653–1051) and Unc51.2(amino acids 531–1037) as tional unit. It is noteworthy that this domain is highly baits to screen a yeast two-hybrid library prepared from conserved between the C. elegans unc-51 and the two postnatal day 6 (P6) mouse cerebellum. Screening of 3 murine homologs, Unc51.1 and Unc51.2 (Tomoda et al. million clones with Unc51.2(amino acids 531–1037) and 1999). To further confirm the binding of Unc51.1 to Syn- subsequent sequencing analyses revealed that one clone GAP, a glutathione S-transferase (GST) pull-down assay spanning ∼1.7 kb encoded the CЈ-terminal 500 amino was performed.
Load more

Recommended publications
  • Sensing and Relayaing of the Autophagic Signal by the ULK1
    SENSING AND RELAYING THE AUTOPHAGIC SIGNAL BY THE ULK1 KINASE COMPLEX by Cindy Puente A Dissertation Presented to the Faculty of the Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy New York, NY May, 2016 __________________________ __________________________ Xuejun Jiang, PhD Date Dissertation Mentor Copyright © 2016 by Cindy Puente To my husband, son, family and friends for their indefinite love and support iii ABSTRACT Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. Autophagy plays a vital role in maintaining cellular homeostasis, especially under stressful conditions, such as nutrient starvation. As such, it is implicated in a plethora of human diseases, particularly age-related conditions such as neurodegenerative disorders. The long-term goals of this proposal were to understand the upstream molecular mechanisms that regulate the induction of mammalian autophagy and understand how this signal is transduced to the downstream, core machinery of the pathway. In yeast, the upstream signals that modulate the induction of starvation- induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyper-phosphorylation of Atg13. However, in mammals, the homologous complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown.
    [Show full text]
  • Coxsackievirus Infection Induces a Non-Canonical Autophagy Independent of the ULK and PI3K Complexes
    www.nature.com/scientificreports OPEN Coxsackievirus infection induces a non‑canonical autophagy independent of the ULK and PI3K complexes Yasir Mohamud1,2, Junyan Shi1,2, Hui Tang1,3, Pinhao Xiang1,2, Yuan Chao Xue1,2, Huitao Liu1,2, Chen Seng Ng1,2 & Honglin Luo1,2* Coxsackievirus B3 (CVB3) is a single‑stranded positive RNA virus that usurps cellular machinery, including the evolutionarily anti‑viral autophagy pathway, for productive infections. Despite the emergence of double‑membraned autophagosome‑like vesicles during CVB3 infection, very little is known about the mechanism of autophagy initiation. In this study, we investigated the role of established autophagy factors in the initiation of CVB3‑induced autophagy. Using siRNA‑mediated gene‑silencing and CRISPR‑Cas9‑based gene‑editing in culture cells, we discovered that CVB3 bypasses the ULK1/2 and PI3K complexes to trigger autophagy. Moreover, we found that CVB3‑ induced LC3 lipidation occurred independent of WIPI2 and the transmembrane protein ATG9 but required components of the late‑stage ubiquitin‑like ATG conjugation system including ATG5 and ATG16L1. Remarkably, we showed the canonical autophagy factor ULK1 was cleaved through the catalytic activity of the viral proteinase 3C. Mutagenesis experiments identifed the cleavage site of ULK1 after Q524, which separates its N‑terminal kinase domain from C‑terminal substrate binding domain. Finally, we uncovered PI4KIIIβ (a PI4P kinase), but not PI3P or PI5P kinases as requisites for CVB3‑induced LC3 lipidation. Taken together, our studies reveal that CVB3 initiates a non‑canonical form of autophagy that bypasses ULK1/2 and PI3K signaling pathways to ultimately converge on PI4KIIIβ‑ and ATG5–ATG12–ATG16L1 machinery.
    [Show full text]
  • 12Q Deletions FTNW
    12q deletions rarechromo.org What is a 12q deletion? A deletion from chromosome 12q is a rare genetic condition in which a part of one of the body’s 46 chromosomes is missing. When material is missing from a chromosome, it is called a deletion. What are chromosomes? Chromosomes are the structures in each of the body’s cells that carry genetic information telling the body how to develop and function. They come in pairs, one from each parent, and are numbered 1 to 22 approximately from largest to smallest. Additionally there is a pair of sex chromosomes, two named X in females, and one X and another named Y in males. Each chromosome has a short (p) arm and a long (q) arm. Looking at chromosome 12 Chromosome analysis You can’t see chromosomes with the naked eye, but if you stain and magnify them many hundreds of times under a microscope, you can see that each one has a distinctive pattern of light and dark bands. In the diagram of the long arm of chromosome 12 on page 3 you can see the bands are numbered outwards starting from the point at the top of the diagram where the short and long arms meet (the centromere). Molecular techniques If you magnify chromosome 12 about 850 times, a small piece may be visibly missing. But sometimes the missing piece is so tiny that the chromosome looks normal through a microscope. The missing section can then only be found using more sensitive molecular techniques such as FISH (fluorescence in situ hybridisation, a technique that reveals the chromosomes in fluorescent colour), MLPA (multiplex ligation-dependent probe amplification) and/or array-CGH (microarrays), a technique that shows gains and losses of tiny amounts of DNA throughout all the chromosomes.
    [Show full text]
  • Regulation of ULK1 in Autophagy 2012 Stefan Ludwik Loska
    Regulation of ULK1 in autophagy A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2012 Stefan Ludwik Loska Table of Contents List of Figures...................................................................................................................6 Abstract.............................................................................................................................8 Declaration........................................................................................................................9 Copyright statement........................................................................................................10 Acknowledgements.........................................................................................................11 Autobiographical statement............................................................................................12 Abbreviations..................................................................................................................13 1. Introduction................................................................................................................19 1.1. Autophagy...........................................................................................................19 1.1.1. Role.............................................................................................................19 1.1.2. Mechanism..................................................................................................23
    [Show full text]
  • ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases
    cells Article ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases Tahira Anwar 1, Xiaonan Liu 2 , Taina Suntio 3, Annika Marjamäki 1, Joanna Biazik 1, Edmond Y. W. Chan 4,5, Markku Varjosalo 2 and Eeva-Liisa Eskelinen 1,6,* 1 Molecular and Integrative Biosciences Research Programme, University of Helsinki, 00014 Helsinki, Finland; tahira.anwar@helsinki.fi (T.A.); [email protected] (A.M.); [email protected] (J.B.) 2 Institute of Biotechnology & HiLIFE, University of Helsinki, 00014 Helsinki, Finland; xiaonan.liu@helsinki.fi (X.L.); markku.varjosalo@helsinki.fi (M.V.) 3 Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland; taina.suntio@helsinki.fi 4 Department of Biomedical and Molecular Sciences and Department of Pathology and Molecular Medicine, Queen’s University, Kingston, ON K7L 3N6, Canada; [email protected] 5 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, UK 6 Institute of Biomedicine, University of Turku, 20520 Turku, Finland * Correspondence: eeva-liisa.eskelinen@utu.fi; Tel.: +358-505115631 Received: 24 April 2019; Accepted: 15 May 2019; Published: 17 May 2019 Abstract: Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes.
    [Show full text]
  • Selective Reversible Inhibition of Autophagy in Hypoxic Breast Cancer Cells Promotes
    Author Manuscript Published OnlineFirst on November 15, 2016; DOI: 10.1158/0008-5472.CAN-15-3458 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Selective reversible inhibition of autophagy in hypoxic breast cancer cells promotes pulmonary metastasis Christopher M. Dower, Neema Bhat, Edward W. Wang and Hong-Gang Wang* Department of Pediatrics The Pennsylvania State University College of Medicine, Milton Hershey Medical Center 500 University Drive, Hershey, PA 17033, USA Running Title: Hypoxia-regulated autophagy in cancer metastasis Key words: Autophagy, Hypoxia, Tumor Microenvironment, Metastasis, Breast Cancer Grant Support: This work was supported by National Institutes of Health Grant CA171501, and by the Lois High Berstler Endowment Fund and the Four Diamonds Fund of the Pennsylvania State University College of Medicine *Corresponding author: Mailing address: 500 University Drive, Hershey, PA 17033, USA; Phone: (717) 531-4574; email: [email protected] Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed. Word Count: 5759 Figures: 7 + 4 Supplementary Figures Page 1 of 21 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 15, 2016; DOI: 10.1158/0008-5472.CAN-15-3458 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Autophagy influences how cancer cells respond to nutrient deprivation and hypoxic stress, two hallmarks of the tumor microenvironment (TME). In this study, we explored the impact of autophagy on the pathophysiology of breast cancer cells, using a novel hypoxia-dependent, reversible dominant negative strategy to regulate autophagy at the cellular level within the TME.
    [Show full text]
  • The Autophagy Initiating Kinase ULK1 Is Required for Pancreatic Cancer Cell Growth and Survival
    bioRxiv preprint doi: https://doi.org/10.1101/2021.05.15.444304; this version posted May 17, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The autophagy initiating kinase ULK1 is required for pancreatic cancer cell growth and survival Sonja N. Brun1, Gencer Sancar2, Jan Lumibao3, Allison S. Limpert5, Huiyu Ren5, Angela Ianniciello1, Herve Tiriac4, Michael Downes2, Danielle D. Engle3, Ronald M. Evans2, Nicholas D.P. Cosford5, and Reuben J. Shaw1* 1Molecular and Cell Biology Laboratory, 2Gene Expression Laboratory, 3Regulatory Biology Laboratory, Salk Cancer Center, The Salk Institute for Biological Studies, La Jolla, California 92037 4Division of Surgical Oncology, Department of Surgery, Moores Cancer Center, University of California San Diego, La Jolla, California. 5Cancer Molecules & Structures Program, NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037 *correspondence: [email protected] Abstract Amongst cancer subtypes, pancreatic ductal adenocarcinoma (PDA) has been demonstrated to be most sensitive to autophagy inhibition, which may be due to unique metabolic rewiring in these cells. The serine/threonine kinase ULK1 forms the catalytic center of a complex mediating the first biochemical step of autophagy. ULK1 directly recieves signals from mTORC1 and AMPK to trigger autophagy under stress and nutrient poor conditions. Studies in genetic engineered mouse models of cancer have revealed that deletion of core downstream autophagy genes (ATG5, ATG7) at the time of tumor iniation leads to a profound block in tumor progression leading to the development of autophagy inhibitors as cancer therapeutics.
    [Show full text]
  • ULK1 Forms Distinct Oligomeric States and Nanoscopic Morphologies During Autophagy Initiation
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.187336; this version posted December 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ULK1 forms distinct oligomeric states and nanoscopic morphologies during autophagy initiation Chiranjib Banerjee1, Dushyant Mehra1,3, Daihyun Song2, Angel Mancebo1, Do-Hyung Kim2,* and Elias M. Puchner1,* 1. School of Physics and Astronomy, University of Minnesota, Twin Cities 2. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Twin Cities 3. Department of Biomedical Engineering and Physiology, Mayo Clinic, Rochester, MN * Corresponding authors: [email protected] and [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.187336; this version posted December 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Autophagy is an evolutionarily conserved process for the degradation and recycling of intracellular components. Although autophagy has been extensively studied, it still remains unclear how autophagosome formation occurs in response to starvation. Here we combined CRISPR-cas9- assisted genome-editing with quantitative Photoactivated Localization Microscopy (qPALM) to analyze the nanoscopic spatial distribution and oligomeric states of endogenous ULK1, the central autophagy induction regulator with single molecule sensitivity. Amino acid starvation induced a small fraction of ULK1 molecules to localize to arc-shaped and spherical structures with radii up to 300 nm and with more than 30 ULK1 molecules. These starvation-induced structures with high ULK1 content occurred only when ULK1 was colocalized with Atg13 and within 100 nm distance to the endoplasmic reticulum.
    [Show full text]
  • Tiratha Raj Singh.Pdf
    GENE-40307; No. of pages: 10; 4C: Gene xxx (2015) xxx–xxx Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene 1Q1 Unc-51 like kinase 1 (ULK1) in silico analysis for biomarker 2 identification: A vital component of autophagy 3Q2 Rohit Randhawa a, Manika Sehgal a, Tiratha Raj Singh a, Ajay Duseja b, Harish Changotra a,⁎ 4 a Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan 1732 34 Himachal Pradesh, India 5 b Department of Hepatology, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India 6 article info abstract 7 Article history: Autophagy is a degradation pathway involving lysosomal machinery for degradation of damaged organelles like 19 8 Received 19 October 2014 the endoplasmic reticulum and mitochondria into their building blocks to maintain homeostasis within the cell. 20 9 Received in revised form 3 February 2015 ULK1, a serine/threonine kinase, is conserved across species, from yeasts to mammals, and plays a central role in 21 10 Accepted 5 February 2015 autophagy pathway. It receives signals from upstream modulators such as TIP60, mTOR and AMPK and relays 22 11 Available online xxxx them to its downstream substrates like Ambra1 and ZIP kinase. The activity of this complex is regulated through 23 – fi fi 24 12 Keywords: protein protein interactions and post-translational modi cations. Applying in silico analysis we identi ed 25 13Q3 Autophagy (i) conserved patterns of ULK1 that showed its evolutionary relationship between the species which were closely 14 ULK1 related in a family compared to others.
    [Show full text]
  • The Emerging Roles of Mtorc1 in Macromanaging Autophagy
    cancers Review The Emerging Roles of mTORC1 in Macromanaging Autophagy Akpedje S. Dossou and Alakananda Basu * Department of Microbiology, Immunology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-817-735-2487 Received: 25 August 2019; Accepted: 23 September 2019; Published: 24 September 2019 Abstract: Autophagy is a process of self-degradation that enables the cell to survive when faced with starvation or stressful conditions. The mechanistic target of rapamycin (mTOR), also known as the mammalian target of rapamycin, plays a critical role in maintaining a balance between cellular anabolism and catabolism. mTOR complex 1 (mTORC1) was unveiled as a master regulator of autophagy since inhibition of mTORC1 was required to initiate the autophagy process. Evidence has emerged in recent years to indicate that mTORC1 also directly regulates the subsequent steps of the autophagy process, including the nucleation, autophagosome elongation, autophagosome maturation and termination. By phosphorylating select protein targets of the autophagy core machinery and/or their regulators, mTORC1 can alter their functions, increase their proteasomal degradation or modulate their acetylation status, which is a key switch of the autophagy process. Moreover, it phosphorylates and alters the subcellular localization of transcription factors to suppress the expression of genes needed for autophagosome formation and lysosome biogenesis. The purpose of this review article is to critically analyze current literatures to provide an integrated view of how mTORC1 regulates various steps of the autophagy process. Keywords: macroautophagy; autophagy regulation; mTORC1 substrates; AMPK; ULK1; autophagy initiation; nucleation; elongation; autophagosome maturation; transcriptional regulation 1.
    [Show full text]
  • Mtor Regulation of Autophagy
    FEBS Letters 584 (2010) 1287–1295 journal homepage: www.FEBSLetters.org Review mTOR regulation of autophagy Chang Hwa Jung, Seung-Hyun Ro, Jing Cao, Neil Michael Otto, Do-Hyung Kim * Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA article info abstract Article history: Nutrient starvation induces autophagy in eukaryotic cells through inhibition of TOR (target of rap- Received 1 December 2009 amycin), an evolutionarily-conserved protein kinase. TOR, as a central regulator of cell growth, plays Revised 11 January 2010 a key role at the interface of the pathways that coordinately regulate the balance between cell Accepted 12 January 2010 growth and autophagy in response to nutritional status, growth factor and stress signals. Although Available online 18 January 2010 TOR has been known as a key regulator of autophagy for more than a decade, the underlying regu- Edited by Noboru Mizushima latory mechanisms have not been clearly understood. This review discusses the recent advances in understanding of the mechanism by which TOR regulates autophagy with focus on mammalian TOR (mTOR) and its regulation of the autophagy machinery. Keywords: Mammalian target of rapamycin Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Autophagy-related gene 1 UNC-51-like kinase 1 UNC-51-like kinase 2 Autophagy-related gene 13 1. Introduction the autophagy study field, the mechanism by which TOR regulates autophagy remains not clearly understood. Here, we review recent Nutrient starvation, stress, or reduced availability of growth fac- findings that have made important progress in our understanding tors alarms eukaryotic cells to adjust metabolism to survive.
    [Show full text]
  • VEGF Triggers Transient Induction of Autophagy in Endothelial Cells Via Ampkα1
    cells Article VEGF Triggers Transient Induction of Autophagy in Endothelial Cells via AMPKα1 Katrin Spengler 1, Nderim Kryeziu 1, Silke Große 1, Alexander S. Mosig 2 and Regine Heller 1,* 1 Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, 07743 Jena, Germany; [email protected] (K.S.); [email protected] (N.K.); [email protected] (S.G.) 2 Institute of Biochemistry II and Center for Sepsis Control and Care, Jena University Hospital, 07743 Jena, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-3641-939-5633 Received: 31 January 2020; Accepted: 10 March 2020; Published: 11 March 2020 Abstract: AMP-activated protein kinase (AMPK) is activated by vascular endothelial growth factor (VEGF) in endothelial cells and it is significantly involved in VEGF-induced angiogenesis. This study investigates whether the VEGF/AMPK pathway regulates autophagy in endothelial cells and whether this is linked to its pro-angiogenic role. We show that VEGF leads to AMPKα1-dependent phosphorylation of Unc-51-like kinase 1 (ULK1) at its serine residue 556 and to the subsequent phosphorylation of the ULK1 substrate ATG14. This triggers initiation of autophagy as shown by phosphorylation of ATG16L1 and conjugation of the microtubule-associated protein light chain 3B, which indicates autophagosome formation; this is followed by increased autophagic flux measured in the presence of bafilomycin A1 and by reduced expression of the autophagy substrate p62. VEGF-induced autophagy is transient and probably terminated by mechanistic target of rapamycin (mTOR), which is activated by VEGF in a delayed manner.
    [Show full text]