CUL3 Gene Cullin 3
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Ubiquitin Ligase Trim32 and Chloride-Sensitive WNK1 As Regulators of Potassium Channels in the Brain Eugene Miler Cilento University of Vermont
University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2015 Ubiquitin Ligase Trim32 and Chloride-sensitive WNK1 as Regulators of Potassium Channels in the Brain Eugene Miler Cilento University of Vermont Follow this and additional works at: http://scholarworks.uvm.edu/graddis Part of the Neurosciences Commons, and the Pharmacology Commons Recommended Citation Cilento, Eugene Miler, "Ubiquitin Ligase Trim32 and Chloride-sensitive WNK1 as Regulators of Potassium Channels in the Brain" (2015). Graduate College Dissertations and Theses. Paper 431. This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. UBIQUITIN LIGASE TRIM32 AND CHLORIDE-SENSITIVE WNK1 AS REGULATORS OF POTASSIUM CHANNELS IN THE BRAIN A Dissertation Presented by Eugene Miler Cilento to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Neuroscience October, 2015 Defense Date: August 04, 2014 Dissertation Examination Committee: Anthony Morielli, Ph.D., Advisor John Green, Ph.D., Chairperson Bryan Ballif, Ph.D. Wolfgang Dostmann Ph.D. George Wellman, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT The voltage-gated potassium channel Kv1.2 impacts membrane potential and therefore excitability of neurons. Expression of Kv1.2 at the plasma membrane (PM) is critical for channel function, and altering Kv1.2 at the PM is one way to affect membrane excitability. -
The Human Dcn1-Like Protein DCNL3 Promotes Cul3 Neddylation at Membranes
The human Dcn1-like protein DCNL3 promotes Cul3 neddylation at membranes Nathalie Meyer-Schallera, Yang-Chieh Choub,c, Izabela Sumaraa, Dale D. O. Martind, Thimo Kurza, Nadja Kathedera, Kay Hofmanne, Luc G. Berthiaumed, Frank Sicherib,c, and Matthias Petera,1 aInstitute of Biochemistry, Eidgeno¨ssiche Technische Hochschule, 8093 Zurich, Switzerland; bCenter for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, ON, Canada M5G 1X5; cDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; dDepartment of Cell Biology, University of Alberta, Edmonton, AB, Canada T6G 2H7; and eBioinformatics Group, Miltenyi Biotec, 51429 Bergisch-Gladbach, Germany Edited by Michael Rape, University of California, Berkeley, CA, and accepted by the Editorial Board June 9, 2009 (received for review December 9, 2008) Cullin (Cul)-based E3 ubiquitin ligases are activated through the enzyme and promotes Nedd8 conjugation through formation of attachment of Nedd8 to the Cul protein. In yeast, Dcn1 (defective this complex (14, 15). Human cells harbor 5 Dcn1-like proteins in Cul neddylation 1 protein) functions as a scaffold-like Nedd8 termed DCNL1–DCNL5 (also named DCUN1D 1–5 for defec- E3-ligase by interacting with its Cul substrates and the Nedd8 E2 tive in Cul neddylation 1 domain-containing protein 1–5) (Fig. Ubc12. Human cells express 5 Dcn1-like (DCNL) proteins each S1). These DCNLs have distinct amino-terminal domains, but containing a C-terminal potentiating neddylation domain but dis- share a conserved C-terminal potentiating neddylation (PONY) tinct amino-terminal extensions. Although the UBA-containing domain, which in yeast Dcn1 is necessary and sufficient for Cul DCNL1 and DCNL2 are likely functional homologues of yeast Dcn1, neddylation in vivo and in vitro (14). -
And CAND1-Dependent Remodelling of the Budding Yeast SCF Complex
ARTICLE Received 31 Jan 2013 | Accepted 20 Feb 2013 | Published 27 Mar 2013 DOI: 10.1038/ncomms2628 OPEN CSN- and CAND1-dependent remodelling of the budding yeast SCF complex Aleksandra Zemla1, Yann Thomas1, Sylwia Kedziora1, Axel Knebel1, Nicola T Wood1, Gwenae¨l Rabut2 & Thimo Kurz1 Cullin–RING ligases (CRLs) are ubiquitin E3 enzymes with variable substrate-adaptor and -receptor subunits. All CRLs are activated by modification of the cullin subunit with the ubiquitin-like protein Nedd8 (neddylation). The protein CAND1 (Cullin-associated-Nedd8- dissociated-1) also promotes CRL activity, even though it only interacts with inactive ligase complexes. The molecular mechanism underlying this behaviour remains largely unclear. Here, we find that yeast SCF (Skp1–Cdc53–F-box) Cullin–RING complexes are remodelled in a CAND1-dependent manner, when cells are switched from growth in fermentable to non-fermentable carbon sources. Mechanistically, CAND1 promotes substrate adaptor release following SCF deneddylation by the COP9 signalosome (CSN). CSN- or CAND1- mutant cells fail to release substrate adaptors. This delays the formation of new complexes during SCF reactivation and results in substrate degradation defects. Our results shed light on how CAND1 regulates CRL activity and demonstrate that the cullin neddylation– deneddylation cycle is not only required to activate CRLs, but also to regulate substrate specificity through dynamic substrate adaptor exchange. 1 Scottish Institute for Cell Signalling, Protein Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. 2 CNRS, Universite´ Rennes 1, Institut de Ge´ne´tique et De´veloppement de Rennes, 2 avenue du Professeur Le´on Bernard, CS 34317, Rennes Cedex 35043, France. -
Kelch-Like Protein 2 Mediates Angiotensin II–With No Lysine 3 Signaling in the Regulation of Vascular Tonus
BASIC RESEARCH www.jasn.org Kelch-Like Protein 2 Mediates Angiotensin II–With No Lysine 3 Signaling in the Regulation of Vascular Tonus Moko Zeniya, Nobuhisa Morimoto, Daiei Takahashi, Yutaro Mori, Takayasu Mori, Fumiaki Ando, Yuya Araki, Yuki Yoshizaki, Yuichi Inoue, Kiyoshi Isobe, Naohiro Nomura, Katsuyuki Oi, Hidenori Nishida, Sei Sasaki, Eisei Sohara, Tatemitsu Rai, and Shinichi Uchida Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan ABSTRACT Recently, the kelch-like protein 3 (KLHL3)–Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3–STE20/SPS1-related proline/alanine-rich kinase–Na/K/Cl cotransporter isoform 1 phosphoryla- tion cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance. -
WNK1 Activates SGK1 to Regulate the Epithelial Sodium Channel
WNK1 activates SGK1 to regulate the epithelial sodium channel Bing-e Xu*, Steve Stippec*, Po-Yin Chu†, Ahmed Lazrak†, Xin-Ji Li†, Byung-Hoon Lee*, Jessie M. English*‡, Bernardo Ortega†, Chou-Long Huang†§, and Melanie H. Cobb*§ Departments of *Pharmacology and †Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041 Communicated by David L. Garbers, University of Texas Southwestern Medical Center, Dallas, TX, May 27, 2005 (received for review April 20, 2005) WNK (with no lysine [K]) kinases are serine–threonine protein (15). ENaC is the rate-limiting step for sodium transport across kinases with an atypical placement of the catalytic lysine. Intronic high-resistance epithelia and is regulated by insulin and mineralo- deletions increase the expression of WNK1 in humans and cause corticoid hormones. The activity of SGK1 depends on phosphati- pseudohypoaldosteronism type II, a form of hypertension. WNKs dylinositol 3-kinase (PI-3 kinase), an essential mediator of insulin have been linked to ion carriers, but the underlying regulatory signaling. PI-3 kinase is also required for SGK1-dependent stimu- mechanisms are unknown. Here, we report a mechanism for the lation of ENaC by mineralocorticoids (16). The impact of SGK1 on control of ion permeability by WNK1. We show that WNK1 acti- ENaC, together with its induction by aldosterone and insulin, places vates the serum- and glucocorticoid-inducible protein kinase SGK1, this protein kinase in a key position to regulate sodium homeostasis leading to activation of the epithelial sodium channel. Increased and blood pressure (14). channel activity induced by WNK1 depends on SGK1 and the E3 Ubiquitinylation controls the cell-surface expression of many ubiquitin ligase Nedd4-2. -
Kelch-Like 3 and Cullin 3 Regulate Electrolyte Homeostasis Via Ubiquitination and Degradation of WNK4
Kelch-like 3 and Cullin 3 regulate electrolyte homeostasis via ubiquitination and degradation of WNK4 Shigeru Shibataa,b, Junhui Zhanga,b, Jeremy Puthumanaa,b, Kathryn L. Stonec, and Richard P. Liftona,b,1 aDepartment of Genetics, bHoward Hughes Medical Institute, and cW. M. Keck Facility, Yale University School of Medicine, New Haven, CT 06510 Contributed by Richard P. Lifton, March 8, 2013 (sent for review February 25, 2013) Pseudohypoaldosteronism type II (PHAII) is a rare Mendelian syn- Mutations in four genes have been identified to cause PHAII drome featuring hypertension and hyperkalemia resulting from (4, 5). Two encode the serine-threonine kinases WNK1 (with no constitutive renal salt reabsorption and impaired K+ secretion. Re- lysine kinase 1) and WNK4 (4). Disease-causing mutations in cently, mutations in Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), compo- WNK4 are missense mutations that cluster in a short, highly acidic nents of an E3 ubiquitin ligase complex, were found to cause PHAII, domain of the protein, whereas mutations in WNK1 are large suggesting that loss of this complex’s ability to target specific sub- deletions of the first intron that increase WNK1 expression. Bio- strates for ubiquitination leads to PHAII. By MS and coimmunopre- chemistry, cell biology, and animal model studies have demon- cipitation, we show that KLHL3 normally binds to WNK1 and WNK4, strated that WNK4 regulates the balance between renal Na-Cl + members of WNK (with no lysine) kinase family that have previously reabsorption and K secretion, with missense mutations found in been found mutated in PHAII. We show that this binding leads to patients with PHAII promoting increased levels of the renal Na-Cl ubiquitination, including polyubiquitination, of at least 15 specific cotransporter NCC and decreased levels of renal outer medullary + + sites in WNK4, resulting in reduced WNK4 levels. -
Antagonistic Regulation of ROMK by Long and Kidney-Specific WNK1 Isoforms
Antagonistic regulation of ROMK by long and kidney-specific WNK1 isoforms Ahmed Lazrak*, Zhen Liu*, and Chou-Long Huang† Department of Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8856 Communicated by Steven C. Hebert, Yale University School of Medicine, New Haven, CT, December 8, 2005 (received for review November 8, 2005) WNK kinases are serine-threonine kinases with an atypical place- of hypertension (7, 8). Others have reported that WNK4 phos- ment of the catalytic lysine. Intronic deletions with increased phorylates claudins 1–4, the tight-junction proteins involved in expression of a ubiquitous long WNK1 transcript cause pseudohy- the regulation of paracellular ion permeability (9, 10). The poaldosteronism type 2 (PHA II), characterized by hypertension and paracellular chloride permeability is greater in cells expressing hyperkalemia. Here, we report that long WNK1 inhibited ROMK1 WNK4 mutants than in cells expressing wild-type proteins. Thus, by stimulating its endocytosis. Inhibition of ROMK by long WNK1 hypertension in patients with WNK4 mutations may be caused by was synergistic with, but not dependent on, WNK4. A smaller an increase in NaCl reabsorption through the Na-Cl cotrans- transcript of WNK1 lacking the N-terminal 1–437 amino acids is porter and the paracellular pathway. Wild-type WNK4 inhibits expressed highly in the kidney. Whether expression of the KS- the ROMK1 channel and WNK4 mutants that cause disease WNK1 (kidney-specific, KS) is altered in PHA II is not known. We exhibit increased inhibition of ROMK (11), suggesting that found that KS-WNK1 did not inhibit ROMK1 but reversed the WNK4 mutations cause hyperkalemia by inhibiting ROMK. -
Cul3 Regulates Cytoskeleton Protein Homeostasis and Cell Migration During a Critical
bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902064; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical 2 window of brain development 3 Jasmin Morandell1, Lena A. Schwarz1, Bernadette Basilico1, Saren Tasciyan1, Armel Nicolas1, 4 Christoph Sommer1, Caroline Kreuzinger1, Christoph P. Dotter1, Lisa S. Knaus1, Zoe Dobler1, 5 Emanuele Cacci2, Johann G. Danzl1, Gaia Novarino1@ 6 7 1Institute of Science and Technology (IST) Austria, Klosterneuburg, Austria 8 2 Department of Biology and Biotechnology “Charles Darwin”, Sapienza, University of Rome 9 @Correspondence to: [email protected] 10 11 Abstract 12 De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to 13 autism spectrum disorder (ASD). Here, we used Cul3 mouse models to evaluate the 14 consequences of Cul3 mutations in vivo. Our results show that Cul3 haploinsufficient mice 15 exhibit deficits in motor coordination as well as ASD-relevant social and cognitive impairments. 16 Cul3 mutant brain displays cortical lamination abnormalities due to defective neuronal migration 17 and reduced numbers of excitatory and inhibitory neurons. In line with the observed abnormal 18 columnar organization, Cul3 haploinsufficiency is associated with decreased spontaneous 19 excitatory and inhibitory activity in the cortex. At the molecular level, employing a quantitative 20 proteomic approach, we show that Cul3 regulates cytoskeletal and adhesion protein abundance 21 in mouse embryos. -
WNK4 Kinase Is a Physiological Intracellular Chloride Sensor
WNK4 kinase is a physiological intracellular chloride sensor Jen-Chi Chena,b, Yi-Fen Loa, Ya-Wen Linb, Shih-Hua Lina, Chou-Long Huangc, and Chih-Jen Chenga,1 aDivision of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan; bGraduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan; and cDivision of Nephrology, Department of Medicine, University of Iowa Carver College of Medicine, Iowa City, IA 52242-1081 Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 11, 2019 (received for review October 7, 2018) With-no-lysine (WNK) kinases regulate renal sodium-chloride cotrans- unclear. In transporting epithelia, changes in concentrations of porter (NCC) to maintain body sodium and potassium homeostasis. ions from exit across one membrane (e.g., basolateral) will be Gain-of-function mutations of WNK1 and WNK4 in humans lead to a coupled by parallel entry on the other membrane (e.g., apical). Mendelian hypertensive and hyperkalemic disease pseudohypoal- The tight coupling between apical and basolateral transport to dosteronism type II (PHAII). X-ray crystal structure and in vitro minimize fluctuations of intracellular concentration of solutes − studies reveal chloride ion (Cl ) binds to a hydrophobic pocket and cell volume is a fundamental homeostatic feature of trans- − within the kinase domain of WNKs to inhibit its activity. The mech- porting epithelia. Whether the intracellular concentration of Cl anism is thought to be important for physiological regulation of − ([Cl ]i) in DCT under physiological conditions is within the dy- NCC by extracellular potassium. -
New Strategies to Inhibit KEAP1 and the Cul3-Based E3 Ubiquitin Ligases
Signalling 2013: from Structure to Function 103 New strategies to inhibit KEAP1 and the Cul3-based E3 ubiquitin ligases Peter Canning*1,2 and Alex N. Bullock*1 *Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K. Abstract E3 ubiquitin ligases that direct substrate proteins to the ubiquitin–proteasome system are promising, though largely unexplored drug targets both because of their function and their remarkable specificity. CRLs [Cullin– RING (really interesting new gene) ligases] are the largest group of E3 ligases and function as modular multisubunit complexes constructed around a Cullin-family scaffold protein. The Cul3-based CRLs uniquely assemble with BTB (broad complex/tramtrack/bric-a-brac)` proteins that also homodimerize and perform the role of both the Cullin adapter and the substrate-recognition component of the E3. The most prominent member is the BTB–BACK (BTB and C-terminal Kelch)–Kelch protein KEAP1 (Kelch-like ECH-associated protein 1), a master regulator of the oxidative stress response and a potential drug target for common conditions such as diabetes, Alzheimer’s disease and Parkinson’s disease. Structural characterization of BTB–Cul3 complexes has revealed a number of critical assembly mechanisms, including the binding of an N-terminal Cullin extension to a bihelical ‘3-box’ at the C-terminus of the BTB domain. Improved understanding of the structure of these complexes should contribute significantly to the effort to develop novel therapeutics targeted to CRL3-regulated pathways. Cullin–RING ligases The multisubunit CRLs (Cullin–RING ligases) represent Specific patterns of mono- or poly-ubiquitylation are used the largest class of E3 ligase. -
Mutations Affecting the Conserved Acidic Wnk1 Motif Cause Inherited Hyperkalemic Hyperchloremic Acidosis Supplemental Informatio
MUTATIONS AFFECTING THE CONSERVED ACIDIC WNK1 MOTIF CAUSE INHERITED HYPERKALEMIC HYPERCHLOREMIC ACIDOSIS SUPPLEMENTAL INFORMATIONS OF METHODS IN VITRO FUNCTIONAL CHARACTERIZATION Vectors The pGH19-L- and KS-WNK1-∆11 plasmids carrying the A634T, D635E, Q636E, Q636R or D635N mutations were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and checked by direct sequencing. The primers used are referenced in Supplemental Table 7B. We subcloned the smaller fragment of the SnaB1-BamH1 digestion from pGH19-L-WNK1- ∆11 and pGH19-KS-WNK1-∆11 plasmids carrying the mutation into pCDNA3-L and KS-WNK1- ∆11 plasmids linearised by SnaB1-BamH1. The LigaFast Rapid DNA Ligation System (Promega) was used according to the manufacturer's instructions. A pCMV6-mKLHL3 (Origene) plasmid encoded Myc- and Flag-tagged mouse KLHL3. We deleted the Flag tag by subcloning the HindIII- EcoRV digestion fragment of pCMV6-mKLHL3 into the same plasmid digested with HindIII-PmeI. pcDNA-hCUL3 encodes HA-tagged human CUL3 is a gift from C. Rochette-Egly (Department of Functional Genomics and Cancer, University of Strasbourg, Illkirch, France). Flag-hKLHL3 cDNA was purchased from the MRC-PPU facility of the University of Dundee and was subcloned into pCDNA5/FRT/(His)6-Protein C vector (derived from pCDNA5/FRT/V5-His (Invitrogen) as described by Derivery and Gautreau (1). All tags are fused at the N-terminus of KLHL3 cDNA. The Ubiquitin-HA expressing vector was a gift from Gervaise Loirand (INSERM UMR1087, Nantes, France) Mutant proteins in HEK293T cells Cell culture conditions The stable and inducible cell lines derived were grown in DMEM medium (Gibco) supplemented with 10% (v/v) Fetal Calf Serum (Biological Industries), Penicillin/Streptomycin (0.1 mg/ml each) (Gibco), Hygromycin B 200 µg/ml (Invivogen), Blasticidin 7,5 µg/ml (Invivogen). -
LINGO-1 and AMIGO3, Potential Therapeutic Targets for Neurological and Dysmyelinating Disorders?
[Downloaded free from http://www.nrronline.org on Wednesday, February 28, 2018, IP: 147.188.108.81] NEURAL REGENERATION RESEARCH August 2017, Volume 12, Issue 8 www.nrronline.org INVITED REVIEW LINGO-1 and AMIGO3, potential therapeutic targets for neurological and dysmyelinating disorders? Simon Foale, Martin Berry, Ann Logan, Daniel Fulton, Zubair Ahmed* Neuroscience and Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK How to cite this article: Foale S, Berry M, Logan A, Fulton D, Ahmed Z (2017) LINGO-1 and AMIGO3, potential therapeutic targets for neu- rological and dysmyelinating disorders?. Neural Regen Res 12(8):1247-1251. Funding: This work was supported by a grant from The University of Birmingham. Abstract *Correspondence to: Zubair Ahmed, Ph.D., Leucine rich repeat proteins have gained considerable interest as therapeutic targets due to their expres- [email protected]. sion and biological activity within the central nervous system. LINGO-1 has received particular attention since it inhibits axonal regeneration after spinal cord injury in a RhoA dependent manner while inhibiting orcid: leucine rich repeat and immunoglobulin-like domain-containing protein 1 (LINGO-1) disinhibits neuron 0000-0001-6267-6492 outgrowth. Furthermore, LINGO-1 suppresses oligodendrocyte precursor cell maturation and myelin (Zubair Ahmed) production. Inhibiting the action of LINGO-1 encourages remyelination both in vitro and in vivo. Accord- ingly, LINGO-1 antagonists show promise as therapies for demyelinating diseases. An analogous protein doi: 10.4103/1673-5374.213538 to LINGO-1, amphoterin-induced gene and open reading frame-3 (AMIGO3), exerts the same inhibitory effect on the axonal outgrowth of central nervous system neurons, as well as interacting with the same re- Accepted: 2017-07-17 ceptors as LINGO-1.