Methylated α-tubulin antibodies recognize a new microtubule modification on mitotic microtubules.
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 α-tubulin, which is trimethylated (α-TubK40me3) by SETD2. In the course of these studies, we generated polyclonal (α-TubK40me3 pAb) and monoclonal (α-TubK40me3 mAb) antibodies to a methylated α-tubulin peptide (GQMPSD-Kme3-TIGGGDC). Here, we characterize these antibodies, and the specific mono-, di- or tri-methylated 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 α-tubulin migrated faster than unmethylated α-tubulin, suggesting methylation may be a signal for additional processing of α-tubulin and/or microtubules. As the first reagents that specifically recognize methylated α-tubulin, these antibodies are a valuable tool for studying this new modification of the cytoskeleton, and the function of methylated microtubules.
a Center for Precision Environmental Health, Departments of Cellular and Molecular Biology and Medicine, Baylor College of Medicine , Houston , TX , USA.
In Young
IY
Park
a Center for Precision Environmental Health, Departments of Cellular and Molecular Biology and Medicine, Baylor College of Medicine , Houston , TX , USA.
Pratim
P
Chowdhury
a Center for Precision Environmental Health, Departments of Cellular and Molecular Biology and Medicine, Baylor College of Medicine , Houston , TX , USA.
Durga Nand
DN
Tripathi
a Center for Precision Environmental Health, Departments of Cellular and Molecular Biology and Medicine, Baylor College of Medicine , Houston , TX , USA.
Reid T
RT
Powell
a Center for Precision Environmental Health, Departments of Cellular and Molecular Biology and Medicine, Baylor College of Medicine , Houston , TX , USA.
Ruhee
R
Dere
b Division of Hematology/Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville , TN , USA.
Esteban A
EA
Terzo
b Division of Hematology/Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville , TN , USA.
W Kimryn
WK
Rathmell
a Center for Precision Environmental Health, Departments of Cellular and Molecular Biology and Medicine, Baylor College of Medicine , Houston , TX , USA.
Cheryl Lyn
CL
Walker
D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D008745:Methylation; D008870:Microtubules / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008938:Mitosis / Q000502:physiology; D011499:Protein Processing, Post-Translational; D014404:Tubulin / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D000911:Antibodies, Monoclonal; D014404:Tubulin; D008239:Lysine
PTM; SETD2; methylation; microtubule; α-TubK40me3; α-tubulin