Molecular determinants for α-tubulin methylation by SETD2

• DOI: 10.1016/j.jbc.2021.100898
• Publication Date: 2021-06-19T14:44:25Z
• Authors: Sarah Kearns; Frank M. Mason; W. Kimryn Rathmell; In Young Park; Cheryl Walker; Kristen J. Verhey; Michael A. Cianfr...
• Keywords: 0301 basic medicine; Histone-Lysine N-Methyltransferase; Methylation; Histones; 03 medical and health sciences; Tubulin; Catalytic Domain; COS Cells; Chlorocebus aethiops; Mutation; Animals; Humans; Protein Processing, Post-Translational; Research Article; Protein Binding

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in defects. However, catalytic mechanism methyl addition is unclear. We used a truncated version human wild type (tSETD2) containing SET and C-terminal Set2–Rpb1–interacting (SRI) domains investigate biochemical methylation. found recombinant tSETD2 had higher activity toward dimers than polymerized microtubules. Using single-isotype tubulin, we demonstrated restricted lysine 40 α-tubulin. then introduced pathogenic mutations into probe recognition substrates. A mutation domain (R1625C) allowed bind but not methylate it, whereas SRI (R2510H) caused loss both binding Further investigation role substrate within this region differential effects ability versus partner RNA polymerase II methylating histones vivo, suggesting distinct mechanisms SETD2. Finally, also requires negatively charged tail Together, study provides framework understanding how serves as dual Microtubules dynamic cytoskeletal polymers maintain shape, serve tracks intracellular trafficking, provide structural form elements cilia. How achieve their varied cellular comes, part, from code multiple isoforms α- β-tubulin post-translational (PTMs) (1Verhey K.J. Gaertig J. The code.Cell Cycle. 2007; 6: 2152-2160Crossref PubMed Scopus (389) Google Scholar, 2Gadadhar S. Bodakuntla Natarajan K. Janke C. at glance.J. Cell Sci. 2017; 130: 1347-1353Crossref (148) 3Roll-Mecak A. cells exploit diversity build functional microtubule mosaics.Curr. Opin. 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Structure/function analysis recurrent protein critical conserved residue maintaining stability Lys-36 trimethylation.J. Chem. 291: 21283-21295Abstract (38) 19Park I.Y. Powell R.T. Tripathi D.N. Dere Ho Blasius T.L. Y.C. Hacker Verhey Bedford M.T. Jonasch Walker C.L. Dual remodeling SETD2.Cell. 166: 950-962Abstract (130) Scholar), pathogenicity due methylation.Our previous work can α-tubulin (αTubK40me3) (19Park dividing cells, αTubK40me3 localizes minus ends KO resulted genomic instability defects such multipolar spindles, lagging chromosomes anaphase, chromosome bridging cytokinesis, micronuclei 20Chiang Y.-C. Park Ohi haploinsufficiency early driver carcinoma.Cancer Res. 78: 3135-3146Crossref (30) These phenotypes correlated drastic reduction Reintroduction WT rescued well (18Hacker contrast, R2510H unable rescue or increase tubulin-dependent manner.Many aspects still unexplained, recognizes differentiates between Structural information has been difficult obtain, given large size presence unstructured regions; date, only two structurally characterized, crystallography (PDB ID: 4H12) NMR 2A7O) (21Zheng Ibáñez Blum Dong Hajian T. Allali-Hassani Amaya M.F. Siarheyeva Yu Brown P.J. Schapira Vedadi al.Sinefungin derivatives inhibitors structure probes SETD2.J. Am. Soc. 2012; 134: 18004-18014Crossref 22Li Phatnani H.P. Guan Sage Greenleaf Zhou Solution Set2-Rpb1 interacting Set2 interaction hyperphosphorylated Rpb1.Proc. 2005; 102: 17636-17641Crossref (96) Here, took reconstitution approach define minimal components vitro. purified (23Yu N. Signorile Basu Ottema Lebbink J.H.G. 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Life 72: 4237-4255Crossref (150) methylated tSETD2. tSETD2–R2510H neither pull down nor tubulin. Because makes protein–protein interactions (RNA Pol II) (22Li investigated residues could distinguish II. positively more where aromatic binding. (CTT) α-tubulin, likely through electrostatic interactions. establishes molecular basis changes derived SRI-domain regulation.ResultsSETD2 methylates vitroTo reconstitute vitro, (tSETD2, aa1418–2564, Fig. 1, B) domains. smaller made it amenable purification, indicated When expressed FLAG-tagged (tSETD2-FLAG) localized nucleus interphase dispersed throughout (Figs. S1 S2), similar localization full-length endogenous (30Li Diao Qiu Jiang Han Hou Ge Ou Histone meiotic maturation mouse oocyte.J. Physiol. 234: 661-668Crossref (10) 31Li Gu adversely affects development embryos.J. Biochem. 121: 797-803Crossref Recombinant tSETD2-FLAG HEK293 yielding single species size-exclusion chromatography (Fig. 1C) band detected SDS-PAGE 1D). confirmed SETD2-FLAG via Western blot against FLAG epitope 1D).To measure enzymatic tSETD2-FLAG, adopted fluorescence-based assay monitors S-adenosyl homocysteine production, product transfer. assay, fluorescence directly corresponds 1E). Thus, incubated porcine brain donor SAM. As positive control, peptide (residues 21–44) (32Eram M.S. Kuznetsova Lima-fernandes Kennedy Chau Arrowsmith C.H. Kinetic methyltransferases, ASH1L SETD2.Biochim. Biophys. Acta. 1850: 1842-1848Crossref (31) negative measured SAM any substrate. case, indicative automethylation subtracted out substrate-containing reactions. Both substrates produced time 1E), indicating reconstituted substrate.tSETD2 displays polymerOur showed glutathione-S-transferase-tagged (1392–2564) capable soluble dimeric states Scholar); however, relative these tested. To do this, maintained taxol compared unpolymerized podophyllotoxin (33Jordan M.A. Thrower Wilson Effects vinblastine, nocodazole spindles. Implications dynamics mitosis.J. 1992; 401-416Crossref 34Ojima Chakravarty Inoue Lin Horwitz S.B. Kuduk S.D. Danishefsky S.J. common pharmacophore cytotoxic natural products stabilize microtubules.Proc. 1999; 96: 4256-4261Crossref (281) 2, A–C). 5 μM (5.08 ± 0.06 nmol/min) concentration (3.19 0.05 D E). This finding consistent K40 luminal microtubules, being accessible dimer context. Microtubule may occur incorporation microtubules.Figure 2tSETD2-FLAG polymer. A, schematic showing (enzymatic red purple) either B C, verification polymer state. Porcine stabilized treated (podo) prevent polymerization. B, micrographs (left) taxol-stabilized (right) Tris-based buffer polymerization (see Experimental procedures). scale bar represents 100 nm. were pelleted centrifugation, pellet supernatant fractions separated stained Coomassie. E, assay. (with podophyllotoxin, gray) taxol, black) substrates, monitored time. D, representative traces tSETD2-Flag activity. dot indicates experiment, error bars show average SD across total three experiments. SRI, Set2–Rpb1–interacting; tSETD2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SETD2 sites other K40We previously determined (αTubK40) using followed MS contains numerous premodified proteins, thus, whether additional β-tubulin.To if utilized Recent advances purification purify αTub1B/βTub3 (hereafter referred αβ-tubulin) baculovirus system 3, B). mutate known purifying αβ-tubulin alanine [αβ-tubulin(αK40A)]. αβ-tubulin, K40A, observing polymerize GMPCPP-tubulin seeds S3).Figure 3αTubK40 primary αTubA1B-βTub3B (αβ-tubulin) (black) αK40A (yellow) mutant, Hi Five analyzed (A) (B) Coomassie-stained gel. αβ-tubulin(αK40A) tubulins. Each value n = 4 table lists sequence number unmodified monomethylated peptides identified experiment. raw data deposited at: https://osf.io/m62x7/. K40; K40, 40; tSETD2, SETD2; (PPT)The decreased when provided αβ-tubulin(αK40 A) (1.28 0.19 (3.73 0.04 3C). suggests although αTubK40 β-tubulin. identify potential site(s), carried reactions K40A experiments lacking parallel. tub