Cofilin/Twinstar Phosphorylation Levels Increase in Response to Impaired Coenzyme A Metabolism Katarzyna Siudeja, Nicola A. Grzeschik, Anil Rana, Jannie de Jong, Ody C. M. Sibon* Department of Cell Biology, Radiation and Stress Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Abstract Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture – a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics. Citation: Siudeja K, Grzeschik NA, Rana A, de Jong J, Sibon OCM (2012) Cofilin/Twinstar Phosphorylation Levels Increase in Response to Impaired Coenzyme A Metabolism. PLoS ONE 7(8): e43145. doi:10.1371/journal.pone.0043145 Editor: Florence Janody, Instituto Gulbenkian de Cieˆncia, Portugal Received May 29, 2012; Accepted July 19, 2012; Published August 17, 2012 Copyright: 2012 Siudeja et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Dutch organization for scientific research (NWO) funded the described work in a VICI Grant to Ody Sibon. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interest exist. * E-mail: o.c.m.sibon@umcg.nl Introduction Regulation of actin dynamics is critical for cellular function. Cells respond to various external and internal stimuli by specific remodeling events of the actin cytoskeleton. Actin rearrangements are required for changes in cell shapes and thus actin dynamics are important for a variety of morphogenetic events such as cell divisions, cell migration, adhesion, neuritogenesis (sprouting of neurites) or axon pathfinding. At the same time, the regulation of these processes is tightly linked to the metabolic status of cells and organisms. Numerous data demonstrate the involvement of Rho family GTPases in the control of actin filament nucleation and disassembly [1,2], however relatively little is known about whether and how actin cytoskeleton signaling is influenced by and tuned with the metabolic state of the cell. Coenzyme A (CoA) is an central metabolite present in all living organisms [3]. CoA reacts with carboxyl groups giving rise to thioesters – CoA-activated acyl moieties. About 4% of all known enzymes utilize CoA as a cofactor and CoA thioesters are essential for over 100 different reactions of the intermediary metabolism, such as the tricarboxylic acid cycle (TCA cycle), lipid synthesis and oxidation or the synthesis of some amino acids (reviewed in [3]). Hence, CoA occupies a central position in the regulation of the cellular metabolism. One evolutionary conserved pathway has been described leading to the de novo biosynthesis of CoA. The first step of this pathway is phosphorylation of pantothenic acid (vitamin B5) by pantothenate kinase (PANK). The pathway has gained renewed attention after the discovery that mutations in one of the four human PANK isoforms, PANK2, lead to a severe neurodegenerative disorder, Pantothenate Kinase-Associated Neu- rodegeneration (PKAN) [4]. Recently, we and others have established a Drosophila melanogaster model for PKAN [5,6,7,8] and we have shown that Drosophila dPANK/fbl mutant flies as well as downregulating dPANK/Fbl using an in vitro RNAi approach in Drosophila Schneider’s S2 cells constitute suitable models for studying the consequences of CoA deficiency. The dPANK/fbl gene was initially identified in a screen for male sterility and dPANK/fbl mutants show cell division errors and cytokinesis defects with abnormal F-actin dynamics [5,8]. Additionally, abnormal F- actin accumulation is observed in the ovaries of CoA deficient female flies, which are also sterile [9]. This correlation of CoA metabolism in flies with actin related processes suggests additional, yet not appreciated, influences of CoA levels. Nevertheless the molecular mechanisms of these CoA-related actin abnormalities are not known. Here we use Drosophila S2 cells to study in more detail actin defects caused by CoA deficiency. We demonstrate that phosphorylation of a Drosophila homolog of cofilin, Twinstar (Tsr) [10], is increased in CoA deficient cells. Cofilin is an actin binding protein influencing depolymerization and severing of actin filaments and it plays an essential role in F-actin turnover [11,12] and is has been reported that activity of cofilin is inhibited by phosphorylation [13,14,15]. Our study reveals involvement of Cdi kinase and Slingshot phosphatase in the regulation of actin dynamics in Drosophila, in response to changes in CoA metabolism. Furthermore we show that regulation of cofilin in response to impaired CoA biosynthesis is conserved between Drosophila cells and human neuronal cells. By inhibiting PANK activity during neuronal differentiation in vitro, not only the phosporylation status of human cofilin was affected but also the cellular morphology and PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43145