Dr Viktor Korolchuk
Reader in Molecular Cell Biology
- Email: email@example.com
- Telephone: +44(0)191 208 1221
- Fax: +44(0)191 208 1101
- Address: Ageing Research Laboratories
(Institute for Cell and Molecular Biosciences)
Campus for Ageing and Vitality
Newcastle upon Tyne
My scientific interests lie in the area of intracellular protein trafficking and degradation pathways. The current focus of research in my laboratory is autophagy (literally self-eating) where portions of cytoplasm are recruited into intracellular vesicles called autophagosomes and transported for degradation by lysosomal hydrolases. Autophagy is now widely accepted to be beneficial for cellular and organismal health by removing damaged and harmful cellular entities while autophagy inhibition due to persistent nutrient signalling could have deleterious effects (see our feature in the Biochemist http://www.biochemist.org/bio/03402/0008/034020008.pdf). The questions being addressed in the lab include: 1) How autophagy is controlled by nutrient-sensing signalling pathways? 2) What are the endogenous autophagy substrates and what are the mechanisms of their recruitment to autophagic vesicles? 3) How autophagy affects other cellular processes and pathways? In addition to basic biological problems we investigate how perturbations of protein trafficking and degradation contribute to neurodegenerative diseases and ageing.
I obtained my PhD from the Institute of Biochemistry in Kiev, Ukraine. During the postdoctoral training at Bristol and Cambridge Universities, UK, I have been studying intracellular trafficking and signalling pathways using a range of biochemical, cell biological and genetic approaches. My contributions to the field include: isolation and characterisation of protein kinases associated with endocytic clathrin-coated vesicles (Traffic, 2002; J Biol Chem, 2005); studies of the topologically unusual protein tetherin, its trafficking and role in the development and maintenance of cell polarity (Traffic, 2003; J Cell Sci, 2007; J Cell Biol, 2009); the role of Drosophila trafficking proteins in the recycling of synaptic vesicles, in neuronal development and in regulation of signalling pathways (J Cell Biol, 2007; J Cell Sci, 2007); characterisation of several mechanisms of autophagy regulation including those by calcium, reactive oxygen species, reactive nitrogen species and in the context of a glycogen storage disease (Autophagy, 2009; Hum Mol Genet, 2010; Mol Cell, 2011); a mechanism of cross-talk between autophagy and the ubiquitin proteasome system (Mol Cell, 2009); a novel mechanism of coordinated control of both mTOR and autophagy by nutrients (Nat Cell Biol, 2011); identification of autophagy regulators that suppress the toxicity caused by mutant huntingtin.
- 1996 High Degree Diploma (MSc in Biology), Kiev State University, Ukraine
- 2000 PhD Biochemistry, Institute of Biochemistry, Kiev, National Academy of Sciences, Ukraine
Membership of societies:
- Biochemical Society
- British Society for Cell Biology
- British Society for Research on Ageing (Executive board member)
- BBSRC, MRC, NIHR
- Intracellular trafficking, autophagy, ubiquitin, protein degradation, nutrient signalling, mTOR, DNA damage, neurodegeneration, ageing.
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RESEARCH FOCUS: AUTOPHAGY IN HEALTH AND DISEASE
Macroautophagy, for simplicity frequently called autophagy, is a mechanism used by cells to survive periods of starvation by degrading cytoplasmic components and releasing much-needed metabolites and energy. Autophagy is a vesicular trafficking pathway in which double-membraned intracellular structures called autophagosomes are formed around portions of cytoplasm containing cellular components destined for degradation. Autophagosomes are transported along microtubules and their lives end when they fuse with lysosomes, where autophagic substrates are degraded. The process of autophagosome formation and maturation is under tight control and is orchestrated by dedicated machinery.
Both basal and induced autophagy is an important determinant of health and longevity. Indeed, perturbations in the autophagy pathway have been recognised as a causative factor in a number of human pathologies including cancer, heart diseases, diabetes and neurodegeneration. Moreover, the ageing itself, being the most important risk factor in the development of many human diseases, has been associated with insufficient clearance of misfolded, toxic and aggregate-prone proteins via the autophagosome- and lysosome-dependent degradation mechanisms. Most excitingly, many of the treatments prolonging lifespan in model organisms are doing so in an autophagy-dependent manner indicating that efficient protein degradation via autophagy may be the major determinant of lifespan extension.
CURRENT RESEARCH PROJECTS IN THE LABORATORY
1) Investigation of mechanisms controlling cellular nutrient responses
The majority of treatments prolonging healthy lifespan of laboratory animals suppress nutrient sensing pathways such as mTOR (mammalian Target Of Rapamycin) and stimulate autophagy. The elaborate network of mTOR signalling events integrates upstream signals such as binding of growth factors to plasma membrane receptors, presence of nutrients such as amino acids, glucose and various metabolites in the extracellular medium as well as the intracellular ATP/ADP ratio signifying cellular energy levels (detected by AMP-dependent kinase, AMPK). We have recently described yet another layer of mTOR control which is mediated by nutrient-imposed changes in intracellular pH. This potentially allows regulation of mTOR activity independently of nutrients and, therefore, offers an opportunity to mimic the effect of dietary restriction on lifespan/ageing through pharmaceutical means. Studying processes regulating cellular nutrient responses and extending the knowledge of their fine molecular detail will increase our chances to fight age-related diseases and ultimately to extend human lifespan.
We are currently addressing the following questions:
What receptors/transporters are involved in implementation of nutrient-dependent changes in intracellular pH?
What is the contribution of this regulatory mechanism to the overall control of mTOR by growth factors, amino acids and energy levels?
What is the potential physiological relevance of this mechanism?
2) Physiologic functions of autophagy
The prominent role of autophagy in cellular and organismal health is commonly explained by the homeostatic function. Indeed, having the capacity to dispose of the large intracellular entities, including protein aggregates and entire organelles such as defective mitochondria, autophagy is a major quality control mechanism clearing damaged and dysfunctional cellular components and thus preventing further cellular damage. However, autophagy is also perfectly positioned to serve regulatory roles. Thus, it can be hypothesised that changes in autophagic flux, induced by upstream signalling events, may lead to changes in the activity of downstream pathways by selective degradation of their regulatory components. This selective recruitment of substrates to autophagosomes can be mediated by adapter molecules such as sequestosome-1/p62, which recruits ubiquitylated proteins for selective autophagosomal degradation. We are interested in characterisation of autophagy-dependent regulatory degradative events as this could result in a conceptually novel model of the control over cellular physiology.
Among the questions that we ask here are the following:
What are the endogenous protein substrates of selective autophagy?
How are endogenous autophagy substrates targeted and recruited to autophagosomes?
How do different substrates define physiological roles of autophagy?
3) Role of autophagy in DNA damage response
One potential mechanism by which autophagy can control the rate of aging is by regulating the DNA damage response (DDR) which contributes to the cellular senescence phenotype. This is supported by the evidence that DDR proteins such as p53 and p38MAPK are shown to affect autophagic activity while autophagy may in turn regulate DDR and cellular senescence. Despite the evidence the molecular details that would explain involvement of autophagy in DDR, senescence and ageing are still largely missing.
In collaboration with Dr. Joao Passos we are using a systematic approach to study the molecular mechanisms underlying the function of autophagy in DDR and senescence. We use systems-based and experimental approaches to identify molecular links between autophagic and DDR machinery. We are testing if DDR proteins interacting with autophagosomal machinery may be targeted to autophagosome-lysosome pathway for degradation. This is achieved by the analysis of the formation and degradation of relevant DDR complexes induced by stressors such as gamma-irradiation in normal or autophagy-impaired cells. We are aiming to evaluate the relative contribution of both autophagy and the proteasome, two major cellular degradative system, to the regulation of DDR complexes. As DDR and autophagy take place in different cellular compartments (nucleus and cytoplasm, respectively), delivery of DDR machinery to cytoplasm, co-localisation with autophagosomes and subsequent degradation is being investigated by biochemical techniques as well as imaging methods such as high-resolution confocal microscopy of fixed and live specimens and by immuno-electron microscopy. Possible feedback mechanisms, whereby DDR proteins are in turn affecting autophagic activity, are also being investigated.
Biochemistry: Subcellular fractionation, purification of proteins from complex biological samples; chromatographic and electrophoresis techniques; immunoblotting.
Molecular biology: Cloning, site-directed mutagenesis, qPCR, production and purification of recombinant proteins using variety of expression systems.
Cell biology: Fluorescent and confocal microscopy, immunocytochemistry; flow cytometry; electron microscopy.
In vivo approaches: Drosophila genetics, imaging techniques.
THE RESEARCH TEAM
Dr. Bernadette Carroll, postdoctoral Research Associate
Dr. Yoana Rabanal, postdoctoral Research Associate
Ms. Lucia Sedlackova, PhD student
Dr. Graeme Hewitt
Dr. Diego Manni
Dr. Gisela Otten
Dr. Alvaro Martinez Guimera
Dr. Alison Howard
A PhD position is currently available to start in October 2018 in the lab to study molecular mechanisms of autophagy/mitophagy in skin senescence. The project is in collaboration with Durham University and Procter & Gamble. For informal enquiries please use contact details below.
No postdoctoral vacancies are currently available but we always welcome an interest from enthusiastic students and postdocs who are interested in applying for funding. Applicants should have a strong background in molecular cell biology or genetics and a record of successful research in the areas of molecular biology, cell biology, biochemistry, or genetics.
To discuss available options forward your curriculum vitae, bibliography, and the names and addresses of referees to: Dr. Viktor Korolchuk, Institute for Cell and Molecular Biosciences, Newcastle University, Campus for Ageing and Vitality, Newcastle upon Tyne, NE4 5PL or by e-mail addressed to firstname.lastname@example.org.
Biology of Ageing, undergraduate course for biomedical students (Deputy Module Leader)
MBBS Life Cycle Unit 3: Ageing, undergraduate course for medical students (Module Leader)
- Rabanal-Ruiz Y, Korolchuk VI. mTORC1 and nutrient homeostasis: The central role of the lysosome. International Journal of Molecular Sciences 2018, 19(3), 818.
- Carroll B, Korolchuk VI. Nutrient sensing, growth and senescence. FEBS journal 2018, ePub ahead of print.
- Carroll B, Otten E, Manni D, Stefanatos R, Menzies F, Smith G, Jurk D, Kenneth N, Wilkinson S, Passos J, Attems J, Veal E, Teyssou E, Seilhean D, Millecamps S, Eskelinen E-L, Bronowska A, Rubinsztein DC, Sanz A, Korolchuk V. Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis. Nature Communications 2018, 9, 256.
- Hallam D, Collin j, Bojic S, Chichagova V, Buskin A, Xu Y, Lafage L, Otten EG, Anyfantis G, Mellough C, Przyboski S, Alhart S, Korolchuk V, Lotery A, Saretzki G, McKibbin M, Armstrong L, Steel D, Kavanagh D, Lako M. An iPSC patient specific model of CFH (Y402H) polymorphism displays characteristic features of AMD and indicates a beneficial role for UV light exposure. Stem Cells 2017, Accepted article.
- Carroll B, Korolchuk VI. Dysregulation of mTORC1/autophagy axis in senescence. Aging 2017, 9(8), 1851-1852.
- Korolchuk VI, Miwa S, Carroll B, von Zglinicki T. Mitochondria in cell senescence: Is mitophagy the weakest link?. EBioMedicine 2017, 21, 7-13.
- Rabanal-Ruiz Y, Otten EG, Korolchuk VI. mTORC1 as the main gateway to autophagy. Essays in Biochemistry 2017, 61(6), 565-584.
- Carroll B, Nelson G, Rabanal-Ruiz Y, Kucheryavenko O, Dunhill-Turner NA, Chesterman CC, Zahari Q, Zhang T, Conduit SE, Mitchell CA, Maddocks ODK, Lovat P, von Zglinicki T, Korolchuk VI. Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing. Journal of Cell Biology 2017, ePub ahead of print.
- Lane JD, Korolchuk VI, Murray JT. Signalling mechanisms in autophagy: An introduction to the issue. Essays in Biochemistry 2017, 61(6), 561-563.
- Ward C, Martinez-Lopez N, Otten EG, Carroll B, Maetzel D, Singh R, Sarkar S, Korolchuk VI. Autophagy, lipophagy and lysosomal lipid storage disorders. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 2016, 1861(4), 269-284.
- Carroll B, Maetzel D, Maddocks ODK, Otten G, Ratcliff M, Smith GR, Dunlop EA, Passos JF, Davies OR, Jaenisch R, Tee AR, Sarkar S, Korolchuk VI. Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity. eLIFE 2016, 5, e11058.
- Klinonsky DJ, et, al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd Ed.). Autophagy 2016, 12(1), 1-222.
- Correia-Melo C, Marques FDM, Anderson R, Hewitt G, Hewitt R, Cole J, Carroll BM, Miwa S, Birch J, Merz A, Rushton MD, Charles M, Jurk D, Tait SWG, Czapiewski R, Greaves L, Nelson G, Bohlooly-Y M, Rodriguez-Cuenca S, Vidal-Puig A, Mann D, Saretzki G, Quarato G, Green DR, Adams PD, von Zglinicki T, Korolchuk VI, Passos JF. Mitochondria are required for pro-ageing features of the senescent phenotype. EMBO Journal 2016, 35(7), 724-742.
- Otten EG, Manni D, Korolchuk VI. Mitochondrial degradation, autophagy and neurodegenerative disease. In: Reeve A; Simcox E; Duchen M; Turnbull D, ed. Mitochondrial Dysfunction in Neurodegenerative Disorders: Second Edition. Cham: Springer International Publishing, 2016, pp.255-278.
- Santin Y, Sicard P, Vigneron F, Guilbeau-Frugier C, Dutaur M, Lairez O, Couderc B, Manni D, Korolchuk VI, Lezoualc'h F, Parini A, Mialet-Perez J. Oxidative Stress by Monoamine Oxidase-A Impairs Transcription Factor EB Activation and Autophagosome Clearance, Leading to Cardiomyocyte Necrosis and Heart Failure. Antioxidants & Redox Signaling 2016, 25(1), 10-27.
- Brown A, Patel S, Ward C, Lorenz A, Ortiz M, DuRoss A, Wieghardt F, Esch A, Otten EG, Heiser LM, Korolchuk VI, Sun C, Sarkar S, Sahay G. PEG-lipid micelles enable cholesterol efflux in Niemann-Pick Type C1 disease-based lysosomal storage disorder. Scientific Reports 2016, 6, 31750.
- Hewitt G, Korolchuk VI. Repair, Reuse, Recycle: The Expanding Role of Autophagy in Genome Maintenance. Trends in Cell Biology 2016, 27(5), 340-351.
- Hewitt G, Carroll B, Sarallah R, Correia-Melo C, Ogrodnik M, Nelson G, Otten EG, Manni D, Antrobus R, Morgan BA, von Zglinicki T, Jurk D, Seluanov A, Gorbunova V, Johansen T, Passos JF, Korolchuk VI. SQSTM1/p62 mediates crosstalk between autophagy and the UPS in DNA repair. Autophagy 2016, 12(10), 1917-1930.
- Carroll B, Korolchuk VI, Sarkar S. Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis. Amino Acids 2015, 47(10), 2065-2088.
- Hewitt G, Carroll B, Korolchuk VI. Mechanisms of cross-talk between intracellular protein degradation pathways. In: Hayat, M, ed. Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging : Regulation of Autophagy and Selective Autophagy. San Diego: Academic Press, 2015, pp.103-119.
- Vicinanza M, Korolchuk VI, Ashkenazi A, Puri C, Menzies FM, Clarke JH, Rubinsztein DC. PI(5)P regulates autophagosome biogenesis. Molecular Cell 2015, 57(2), 219-234.
- Lu W, Zhang Y, McDonald DO, Jing HE, Carroll B, Robertson N, Zhang Q, Griffin H, Sanderson S, Lakey JH, Morgan NV, Reynard LN, Zheng L, Murdock HM, Turvey SE, Hackett SJ, Prestidge T, Hall JM, Cant AJ, Matthews HF, Koref MFS, Simon AK, Korolchuk VI, Lenardo MJ, Hambleton S, Su HC. Dual Proteolytic Pathways Govern Glycolysis and Immune Competence. Cell 2014, 159(7), 1578-1590.
- Dalle Pezze P, Nelson G, Otten EG, Korolchuk VI, Kirkwood TBL, von Zglinicki T, Shanley DP. Dynamic Modelling of Pathways to Cellular Senescence Reveals Strategies for Targeted Interventions. PLoS Computational Biology 2014, 10(8), e1003728.
- Sarkar S, Maetzel D, Korolchuk VI, Jaenisch R. Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder, Niemann-Pick type C1 disease. Autophagy 2014, 10(6), 1137-1140.
- Carroll B, Hewitt G, Korolchuk VI. Autophagy and ageing: implications for age-related neurodegenerative diseases. Essays in Biochemistry: Autophagy: Molecules and Mechanisms 2013, 55, 119-131.
- Sarkar S, Carroll B, Buganim Y, Maetzel D, Ng AH, Cassady JP, Cohen MA, Chakraborty S, Wang H, Spooner E, Ploegh H, Gsponer J, Korolchuk VI, Jaenisch R. Impaired Autophagy in the Lipid-Storage Disorder Niemann-Pick Type C1 Disease. Cell Reports 2013, 5(5), 1302-1315.
- Ivanov A, Pawlikowski J, Manoharan I, vanTuyn J, Nelson DM, Rai TS, Shah PP, Hewitt G, Korolchuk VI, Passos JF, Wu H, Berger SL, Adams PD. Lysosome-mediated processing of chromatin in senescence. Journal of Cell Biology 2013, 202(1), 129-143.
- Jurk D, Wang C, Miwa S, Maddick M, Korolchuk V, Tsolou A, Gonos ES, Thrasivoulou C, Saffrey JM, Cameron K, von Zglinicki T. Postmitotic neurons develop a p21-dependent senescence-like phenotype driven by a DNA damage response. Aging Cell 2012, 11(6), 996-1004.
- Rocha JJE, Korolchuk VI, Robinson IM, O'Kane CJ. A Phagocytic Route for Uptake of Double-Stranded RNA in RNAi. PLoS ONE 2011, 6(4), e19087.
- Sarkar S, Korolchuk VI, Renna M, Imarisio S, Fleming A, Williams A, Garcia-Arencibia M, Rose C, Luo S, Underwood BR, Kroemer G, O'Kane CJ, Rubinsztein DC. Complex inhibitory effects of nitric oxide on autophagy. Molecular Cell 2011, 43(1), 19-32.
- Korolchuk VI, Saiki S, Lichtenberg M, Siddiqi FH, Roberts EA, Imarisio S, Jahreiss L, Sarkar S, Futter M, Menzies FM, O'Kane CJ, Deretic V, Rubinsztein DC. Lysosomal positioning coordinates cellular nutrient responses. Nature Cell Biology 2011, 13(4), 453-460.
- Underwood BR, Imarisio S, Fleming A, Rose C, Krishna G, Heard P, Quick M, Korolchuk VI, Renna M, Sarkar S, García-Arencibia M, O'Kane CJ, Murphy MP, Rubinsztein DC. Antioxidants can inhibit basal autophagy and enhance neurodegeneration in models of polyglutamine disease. Human Molecular Genetics 2010, 19(17), 3413-3429.
- Knecht E, Aguado C, Sarkar S, Korolchuk VI, Criado-García O, Vernia S, Boya P, Sanz P, de Córdoba SR, Rubinsztein DC. Impaired autophagy in Lafora disease. Autophagy 2010, 6(7), 991-993.
- Aguado C, Sarkar S, Korolchuk VI, Criado O, Vernia S, Boya P, Sanz P, de Córdoba SR, Knecht E, Rubinsztein DC. Laforin, the most common protein mutated in Lafora disease, regulates autophagy. Human Molecular Genetics 2010, 19(14), 2867-2876.
- Korolchuk VI, Menzies FM, Rubinsztein DC. Mechanisms of cross-talk between the ubiquitin-proteasome and autophagy-lysosome systems. FEBS Letters 2010, 584(7), 1393-1398.
- Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, Jimenez-Sanchez M, Korolchuk VI, Lichtenberg M, Luo SQ, Massey DCO, Menzies FM, Moreau K, Narayanan U, Renna M, Siddiqi FH, Underwood BR, Winslow AR, Rubinsztein DC. Regulation of Mammalian Autophagy in Physiology and Pathophysiology. Physiological Reviews 2010, 90(4), 1383-1435.
- Rollason R, Korolchuk V, Hamilton C, Jepson M, Banting G. A CD317/tetherin–RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells. Journal of Cell Biology 2009, 184(5), 721-736.
- Korolchuk VI, Menzies FM, Rubinsztein DC. A novel link between autophagy and the ubiquitin-proteasome system. Autophagy 2009, 5(6), 862-863.
- Korolchuk VI, Mansilla A, Menzies FM, Rubinsztein DC. Autophagy Inhibition Compromises Degradation of Ubiquitin-Proteasome Pathway Substrates. Molecular Cell 2009, 33(4), 517-527.
- Rubinsztein CD, Cuervo AM, Ravikumar B, Sarkar S, Korolchuk VI, Kaushik S, Klionsky DJ. In search of an “autophagomometer”. Autophagy 2009, 5(5), 585-589.
- Ravikumar B, Futter M, Jahreiss L, Korolchuk VI, Lichtenberg M, Luo S, Massey DCO, Menzies FM, Narayanan U, Renna M, Jimenez-Sanchez M, Sarkar S, Underwood B, Winslow A, Rubinsztein DC. Mammalian macroautophagy at a glance. Journal of Cell Science 2009, 122(11), 1707-1711.
- Sarkar S, Korolchuk V, Renna M, Winslow A, Rubinsztein DC. Methodological considerations for assessing autophagy modulators: A study with calcium phosphate precipitates. Autophagy 2009, 5(3), 307-313.
- Imarisio S, Carmichael J, Korolchuk V, Chen CW, Saiki S, Rose C, Krishna G, Davies JE, Ttofi E, Underwood BR, Rubinsztein DC. Huntington's disease: from pathology and genetics to potential therapies. Biochemical Journal 2008, 412, 191-209.
- Korolchuk VI, Schütz MM, Gómez-Llorente C, Rocha J, Lansu NR, Collins SM, Wairkar YP, Robinson IM, O'Kane CJ. Drosophila Vps35 function is necessary for normal endocytic trafficking and actin cytoskeleton organisation. Journal of Cell Science 2007, 120(24), 4367-4376.
- Rollason R, Korolchuk V, Hamilton C, Schu P, Banting G. Clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif. Journal of Cell Science 2007, 120(21), 3850-3858.
- Koh TW, Korolchuk VI, Wairkar YP, Jiao W, Evergren E, Pan HL, Zhou Y, Venken KJT, Shupliakov O, Robinson IM, O'Kane CJ, Bellen HJ. Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development. Journal of Cell Biology 2007, 178(2), 309-322.
- Palmer CL, Lim W, Hastie PGR, Toward M, Korolchuk VI, Burbidge SA, Banting G, Collingridge GL, Isaac JTR, Henley JM. Hippocalcin functions as a calcium sensor in hippocampal LTD. Neuron 2005, 47(4), 487-494.
- Korolchuk VI, Cozier G, Banting G. Regulation of CK2 activity by phosphatidylinositol phosphates. Journal of Biological Chemistry 2005, 280(49), 40796-40801.
- Kupzig S, Korolchuk V, Rollason R, Sugden A, Wilde A, Banting G. BST-2/HM1.24 is a raft-associated apical membrane protein with an unusual topology. Traffic 2003, 4(10), 694-709.
- Korolchuk V, Banting G. Kinases in clathrin-mediated endocytosis. In: Molecular Mechanisms of Exocytosis and Endocytosis. 2003, University of Edinburgh: Biochemical Society Transactions: Portland Press Ltd.
- Korolchuk VI, Banting G. CK2 and GAK/auxilin2 are major protein kinases in clathrin-coated vesicles. Traffic 2002, 3(6), 428-439.