Polyglutamylation is a posttranslational modification with a broad range of substrates. Juliette van Dijk, Julie Miro, Jean-Marc Strub, Benjamin Lacroix, Alain van Dorsselaer, Bernard Edde, Carsten Janke To cite this version: Juliette van Dijk, Julie Miro, Jean-Marc Strub, Benjamin Lacroix, Alain van Dorsselaer, et al.. Polyg- lutamylation is a posttranslational modification with a broad range of substrates.. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2007, 283 (7), pp.3915-3922. 10.1074/jbc.M705813200. hal-00217037 HAL Id: hal-00217037 https://hal.archives-ouvertes.fr/hal-00217037 Submitted on 25 Jan 2008 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. POLYGLUTAMYLATION IS A POSTTRANSLATIONAL MODIFICATION WITH A BROAD RANGE OF SUBSTRATES* Juliette van Dijk1, Julie Miro1, Jean-Marc Strub2, Benjamin Lacroix1, Alain van Dorsselaer2, Bernard Edde1,3 and Carsten Janke1 From 1CRBM, Universités Montpellier 2 and 1, CNRS, 34293 Montpellier, France; 2LSMBO, CNRS, 67087 Strasbourg, France and 3Université Paris 6, 75252 Paris, France Running head: New substrates of polyglutamylation Address correspondence to: Carsten Janke, CRBM, CNRS, 1919 Route de Mende, 34293 Montpellier, France. Tel: +33 4 67613335; Fax +33 4 67521559; E-mail: [email protected]; Polyglutamylation is a posttranslational reversible monomodifications and are known to modification that generates lateral acidic side function as switches for the activity of many chains on proteins by sequential addition of proteins. Polyglutamylation is a reversible glutamate amino acids. This modification was polymodification generated by sequential covalent first discovered on tubulins and it is important attachment of glutamic acids (up to 20 in some for several microtubule functions. Besides cases) to an internal glutamate residue of the target tubulins, only the nucleosome assembly protein (1). The length of the resulting side chain proteins NAP1 and NAP2 have been shown to is regulated by the balance between the enzymes be polyglutamylated. Here, using a proteomic that catalyze glutamylation, recently identified as approach, we identify a large number of members of the tubulin-tyrosine-ligase-like putative substrates for polyglutamylation in (TTLL) protein family (2-4), and yet unidentified HeLa cells. By analyzing a selection of these deglutamylase enzymes (5). Thus, this putative substrates, we show that several of modification does not only generate “on” and them can serve as in vitro substrates for two of “off” states, but a range of signals that might allow the recently discovered polyglutamylases, for gradual regulation of protein functions. TTLL4 and TTLL5. We further show that The only known targets of polyglutamylation are TTLL4 is the main polyglutamylase enzyme α- and β-tubulins, the structural units of present in HeLa cells and that new substrates of microtubules (MTs (1,6)), and the nucleosome polyglutamylation are indeed modified by assembly proteins, NAP1 and NAP2 (7). Tubulins TTLL4 in a cellular context. No clear consensus are modified in their acidic, glutamate-rich C- polyglutamylation site could be defined from terminus (1), which is the binding site for most the primary sequence of the here-identified new MT-associated proteins (MAPs; reviewed in (8)). substrates of polyglutamylation. However, we Tubulin polyglutamylation was therefore proposed demonstrate that glutamate-rich stretches are to generate functionally divergent MTs by important for a protein to become regulating the affinity between MAPs and MTs polyglutamylated. Most of the newly identified (4,9-11). It was also shown to be important for substrates of polyglutamylation are nucleo- centriole stability (12), axonemal motility cytoplasmic shuttling proteins, including many (2,13,14) and neurite outgrowth (15). The role of chromatin binding proteins. Our work reveals NAP polyglutamylation has not yet been that polyglutamylation is a much more addressed, but as for tubulins, it may regulate the widespread posttranslational modification than affinity of NAPs for their binding partners. initially thought and thus that it might be a Additional polyglutamylated proteins probably regulator of many cellular processes. exist as the polyglutamylation-specific antibody GT335 (16) recognizes protein bands besides One fundamental aspect of proteomic complexity those of tubulins and NAPs on western blots of comes from the various processing events that HeLa cell extracts (4,7). Here, using a proteomic many proteins undergo following their synthesis. approach, we identify new substrates for Posttranslational modifications, such as polyglutamylation and show that they are modified phosphorylation, acetylation and methylation, are by two polyglutamylases from the TTLL protein 1 family. No clear “glutamylation motif” could be 75 µm inner diameter, 15 cm length, LC Packings) defined in their primary sequences, but all the and analyzed by MS/MS on a Q-Tof II mass here-identified new substrates contain glutamate- spectrometer (Micromass Ltd., Manchester, UK). rich stretches that are most likely the acceptor sites Data analysis was performed with Mascot (Matrix for the modification. Our study opens the door to Science Ltd., London, UK) against the NCBI (The further investigations of the role of National Center for Biotechnology Information) polyglutamylation as a general regulatory event of Database. Double and triple charged peptides were a broad range of cellular functions. used for the research in database and the peptide tolerance was set to 0.2 Da for MS and to 0.3 Da for MS/MS. One missed cleavage by trypsin was Experimental Procedures accepted and carbamidomethylated cysteine and oxidized methionine were set as variable Purification of GT335-reactive proteins from modifications. All entries were selected for the HeLa cells by immuno-affinity chromatography- taxonomy. The Mascot score cut-off value for a 10 mg of GT335 antibody was coupled to an N- positive protein hit was set to 50 and peptides with hydroxysuccinimide-activated Hi-Trap column a score below 40 were manually interpreted to (1 ml, GE Healthcare) according to manufacturer’s validate or discard the identification. Identified instructions. HeLa cells were extracted in PBS proteins were submitted to a second research containing 0.1% Triton X-100 and protease including the polyglutamylation as variable inhibitors (aprotinin, leupeptin, and 4-(2- modification. Additions of 1 to 8 glutamic acids aminoethyl)-benzenesulfonyl fluoride, each at were considered and a new research has been done 10 µg/ml). The high speed supernatant (15 mg of each time. protein) was loaded onto the column and after In vitro polyglutamylation of the GT335-affinity- extensive washing with PBS, bound proteins were purified fraction- The GT335-affinity-purified eluted with PBS containing 0.7 M NaCl and fraction (final protein concentration of 0.3 mg/ml) concentrated to 1 mg/ml on an Amicon was incubated for 2 hours at 30°C in the presence ultrafiltration device (4 ml, cut-off 10 kDa, of 50 mM Tris-HCl, pH 9.0, 0.5 mM ATP, Millipore). 2.4 mM MgCl2, 0.5 mM dithiothreitol, and 60 µM Western blotting- Proteins fused to EYFP were L-[3H]-glutamate (45–55 Ci/mmol, GE immunodetected with rabbit anti-GFP antibody Healthcare). Following SDS-PAGE, gels were (1:5,000, Torrey Pines Biolabs) and HRP- exposed to MP films (GE Healthcare) for 40 days conjugated anti-rabbit IgG (GE Healthcare) at -80°C after enhancement with Amplify (GE followed by detection with enhanced chemo- Healthcare). luminescence (GE Healthcare). GT335 antibody Immuno-precipitation experiments- For the was biotinylated with NHS-LC-Biotin (Pierce) depletion of TTLLs, the GT335-affinity-purified according to manufacturer’s instructions and fraction (10 µg of total protein) was incubated two incubated simultaneously with HRP-labeled hours at 4°C on a rotary agitator with protein G streptavidin (GE Healthcare). For producing magnetic beads (Dynabeads) that were coupled to polyclonal anti-TTLL4 and anti-TTLL5 anti- anti-TTLL4 or anti-TTLL5 antibodies (2 µg). bodies, bacterially expressed and purified full- After washing with PBS, the TTLL-depleted length mouse TTLL4 or fragment 692-1152 of fraction was polyglutamylated in vitro as mouse TTLL5 were injected into rabbits. Anti- described above. For immuno-precipitation of the TTLL antibodies were then purified from antisera EYFP fusions of NAPs and their chimeras, the against their respective targets. high-speed supernatant of HeLa cell extracts was Mass spectrometric analysis- To identify the incubated under similar conditions with beads protein content of the GT335-affinity-purified linked to anti-GFP antibody. Following extensive fraction, the Coomassie stained protein bands were washing, the beads were directly resuspended in excised from the gel and submitted to in-gel Laemmli buffer. trypsin digestion (17). Peptides were then In vitro polyglutamylation assay with recombinant concentrated on a C18 pre-column, separated on a proteins-