Methylated α-tubulin antibodies recognize a new microtubule modification on mitotic microtubules

Abstract

Posttranslational modifications (PTMs) on microtubules differentiate these cytoskeletal elements for a variety of cellular functions. We recently identified SETD2 as a dual-function histone and microtubule methyltransferase, and methylation as a new microtubule PTM that occurs on lysine 40 of a-tubulin, which is trimethylated (a-TubK40me3) by SETD2. In the course of these studies, we generated polyclonal (a-TubK40me3 pAb) and monoclonal (a-TubK40me3 mAb) antibodies to a methylated a-tubulin peptide (GQMPSD-Kme3-TIGGGDC). Here, we characterize these antibodies, and the specific mono-, di-or trimethylated lysine residues they recognize. While both the pAb and mAb antibodies recognized lysines methylated by SETD2 on microtubules and histones, the clone 18 mAb was more specific for methylated microtubules, with little cross-reactivity for methylated histones. The clone 18 mAb recognized specific subsets of microtubules during mitosis and cytokinesis, and lacked the chromatin staining seen by immunocytochemistry with the pAb. Western blot analysis using these antibodies revealed that methylated a-tubulin migrated faster than unmethylated a-tubulin, suggesting methylation may be a signal for additional processing of a-tubulin and/or microtubules. As the first reagents that specifically recognize methylated a-tubulin, these antibodies are a valuable tool for studying this new modification of the cytoskeleton, and the function of methylated microtubules.

SETD2 Haploinsufficiency for Microtubule Methylation Is an Early Driver of Genomic Instability in Renal Cell Carcinoma

“…Cells were lysed by gentle pipetting several times and incubated on ice for 10 mins in a cell fractionation buffer (320 mM sucrose, 10mM Tris-HCl (pH8.0), 2mM magnesium acetate, 3 mM calcium chloride, 0.1 mM EDTA, 0.5% NP-40, fresh 1mM dithiothreitol, protease inhibitor cocktail, 1 mM PMSF, and 1 mM sodium orthovanadate). Cytoplasmic fractions were collected after centrifugation at 1,000 g for 5 min and were used for western blots using antibodies against α-TubK40me3 (18, 23) or α-tubulin (MA1-19162, Thermo Fisher Scientific, Waltham, MA). The nuclear proteins were extracted in cell lysis buffer (20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1mM EDTA, 1 mM EGTA, 1% Triton X-100, and protease inhibitor cocktail) by sonication for 3 cycles of 30 secs on/off using Bioruptor 300 (Diagenode, Denville, NJ) and used for western blots using antibodies against SETD2 (HPA042451, Sigma-Aldrich, St. Louis, MO), H3K36me3 (61101, Active Motif, Carlsbad, CA) or histone H3 (ab1791, Abcam, Cambridge, MA).…”

SETD2: a complex role in blood malignancy

“…The recent guidelines of the National Heart, Lung, and Blood Institute to treat the complications of SCD did not adequately address this issue. 5,6 In addition, a recent Cochrane review of fluid replacement therapy for acute episodes of pain in people with SCD found no randomized controlled trials that have assessed the safety and efficacy of different routes, types, or quantities of fluid.…”

A Survey on Tubulin and Arginine Methyltransferase Families Sheds Light on P. lividus Embryo as Model System for Antiproliferative Drug Development

“…Recently, methylation has been found on tubulins occurring in lysine and arginine amino acid residues [22,75,76,77,78,79,80] and it has not been characterized in detail.…”

“…All these features, including post-transcriptional and post-translation mechanisms, represent a hallmark of the tubulin multigene family and the polymer structural diversity [74,75,76].…”