U de ada
Accepted 2 March 2015 increased ANXA2 SUMOylation. A survey of protein SUMOylation in hepatic Golgi/endosome (G/E) fractions after insulin injections revealed the presence of a SUMOylation pattern and confirmed the SUMOylation of ANXA2. The construction of an IR/ANXA2/SUMO network (IRASGEN) in the G/E involved in the biogenesis of endosomes without interfering with the SUMOylation -related modifier al domain
SUMOylated. We identified a SUMOylatable site in the N-te domain of ANXA2. We also demonstrate that ANXA2 SUMO is regulated by the ligand insulin in cultured cells. 2. Materials and methods 2.1. Cell culture
LNCaP (LNCaP.FGC, ATCC/CRL-1740) cells were maintained in
RPMI 1640 medium (HyClone) containing 10% FBS, 2.05 mM
Abbreviations: SUMO, small ubiquitin-related modifier; ANXA2, Annexin A2;
IR, insulin receptor
Author contributions: D.C. performed and analysed most of the experiments in this work and also wrote the paper. M.D.B. performed experiments and analysed data.
R.M.T. designed experiments and contributed to the paper. R.L.F. designed experiments, contributed to the paper and led the research program. ⇑ Corresponding author at: Centre de Recherche du CHU de Québec, 2705 Blvd
Laurier, T3-55, Québec, QC G1V 4G2, Canada. Fax: +1 418 654 2753.
E-mail address: email@example.com (R.L. Faure).
FEBS Letters 589 (2015) 985–991 journal homepage: wwfibres within cells. Instead, more dynamic actin structures, in particular those associated with cellular membranes, were noted .
In contrast to ANXA1, ANXA2 interacts with cholesterol and actin . The depletion of ANXA2 with siRNA has shown that it is (IR) [11,12]. In a recent study, we showed that of ANXA1 occurs in the consensus small ubiquitin (SUMO) motif (wKXE/D) located at the C-termin regulated by EGF . We show here thathttp://dx.doi.org/10.1016/j.febslet.2015.03.007 0014-5793/ 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.and is is also rminal ylationThe functions of annexins are linked to their ability to interact with cellular membranes in a regulated manner [1–3]. Annexin
A2 (ANXA2), in association with S100A10, was the first annexin shown to be able to bind and bundle actin filaments in vitro. An
N-terminal sequence (residues 1–30) appears to be involved in the bundling activity . Despite their ability to bundle actin filaments, imaging studies have shown that annexins are not associated with prominent actin bundles and cables such as stress and stabilising F-actin, forcing membrane remodelling and thus endosome biogenesis [5,7,8]. In addition, the activity of ANXA2 at actin assembly points is regulated by phosphorylation at Y24 at the N-terminal domain by vSrc .
Insulin, in addition to initiating metabolic responses, elicits major changes in the F-actin distribution [9,10]. Previous reports have suggested that the tyrosine phosphorylation of ANXA2 at
Y24 is involved in the endocytic traffic of the insulin receptorAvailable online 14 March 2015
Edited by Ivan Sadowski
Endocytosis 1. Introductioncontext reveals the presence of interacting nodes whereby SUMO1 connects ANXA2 to actin and microtubule-mediated changes in membrane topology. Heritable variants associated with type 2 diabetes represent 41% of the IRASGEN thus pointing out the physio-pathological importance of this subnetwork. 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. the invagination of multivesicular bodies [5,6]. ANXA2 plays an important role in early to late endosomal transport by nucleatingArticle history:
Received 11 December 2014
Revised 27 February 2015
Insulin receptor (IR) endocytosis requires a remodelling of the actin cytoskeleton. We show here that ANXA2 is SUMOylated at the K10 located in a non-consensus SUMOylation motif in the N-terminal domain. The Y24F mutation decreased the SUMOylation signal, whereas insulin stimulationAnnexin A2 is SUMOylated on its N-term by insulin
Danielle Caron a, Martial Boutchueng-Djidjou a, Robe aDépartement de Pédiatrie, Laboratoire de biologie cellulaire Centre de recherche du CH b Institut de Biologie Intégrative et des Système (IBIS), Université Laval, Québec, PQ, Can c Laboratory of Cellular and Developmental Genetics, Department of Molecular Biology, d PROTEO, Université Laval, Québec, PQ, Canada a r t i c l e i n f o a b s t r a c tal domain: Regulation
M. Tanguay b,c,d, Robert L. Faure a,⇑
Québec, Université Laval, Québec, PQ, Canada ical Biochemistry and Pathology, Université Laval, Québec, PQ, Canada w.FEBSLetters .org
Laboratories (Laval, Québec). 986 D. Caron et al. / FEBS Letters 589 (2015) 985–991L-glutamine, 1.105/0.1 mg/ml penicillin/streptomycin and were cultured at 37 C in a 5% CO2 constant atmosphere. In some experiments (Fig. 3), cells were grown in the absence of FBS for 2 h prior to insulin stimulation (15 min, 10 ng/ml). 2.2. Cells transfection
Cells were plated 20 h in advance at a 60% confluence. They were washed once with the culture medium and incubated 24 h in medium containing the plasmid:Fugene HD (Roche Diagnostics
Canada, Laval, QC) complex (ratio 2 lg DNA:9 ll Fugene HD) prepared according to the manufacturer’s protocol. The nontransfected (NT) control was realised using empty vector. Cellular lysates were solubilised in Laemmli sample buffer and boiled for 5 min. 2.3. Plasmid constructs
Total cellular RNA from mouse fibroblasts (MF WT) was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Two micrograms of total RNA was used to synthesise the cDNA using Omniscript reverse transcriptase (Qiagen). The pEGFP-SUMO-1GG and pEGFP-SUMO-1D plasmids, expressing enhanced GFP-tagged SUMO-1 coding sequence in its constitutively active form with a diglycine group at the end (SUMO-1GG) or an inactive form (G96D) where the glycine group has been replaced by a stop sequence (SUMO-1D), respectively, were realised using the complete SUMO1 human coding sequence amplified with SUMO-1GG forward primer: 50-GATCTCGAGATGTC