title: Detyrosinated Glu-tubulin is a substrate for cellular Factor XIIIA transglutaminase in differentiating osteoblasts.
abstract: Microtubule components α- and β-tubulin undergo a number of posttranslational modifications that modulate their dynamics and cellular functions. These modifications include polyamination and covalent crosslinking by transglutaminase enzymes. We have demonstrated previously that the less dynamic and more stable tubulin form-detyrosinated Glu-tubulin-is found in high molecular weight, oligomeric complexes in bone-forming osteoblasts during differentiation and along with deposition of collagenous extracellular matrix. In this study, we report that oligomeric Glu-tubulin has high nocodazole tolerance, indicating further increased stability. We show that α-tubulin, which gives rise to Glu-tubulin, is a transglutaminase substrate in in vitro assays and that it is crosslinked into oligomers (dimers, trimers and tetramers) by transglutaminase 2 and Factor XIIIA; β-tubulin was not crosslinked by transglutaminase activity. The oligomeric Glu-tubulin was specifically localized to the plasma membrane of osteoblasts as analyzed by subcellular fractionation, cell surface biotinylation experiments and total internal reflection fluorescence (TIRF) microscopy. Glu- and α-tubulin co-localized with cellular Factor XIIIA as analyzed by conventional and TIRF microscopy. The Factor XIIIA-specific substrate peptide bF11 co-localized with α-tubulin and acted as a competitive inhibitor to oligomerization of Glu-tubulin, attenuating its formation in cells. This was associated with significantly decreased type I collagen deposition and decreased secretory activity as measured by synaptotagmin VII levels on the osteoblast plasma membrane. Our results suggest that Glu-tubulin may exist as covalently stabilized form which may be linked to the secretion and elaboration of collagenous extracellular matrix.
authors.affiliation: Division of Biomedical Sciences, Faculty of Dentistry, McGill University, 3640 University Street, Montreal, QC, H3A 2B2, Canada.
authors.forename: Shuai
authors.initials: S
authors.lastname: Wang
authors.forename: Cui
authors.initials: C
authors.lastname: Cui
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Hitomi
authors.forename: Mari T
authors.initials: MT
authors.lastname: Kaartinen
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D026122:Factor XIIIa / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000378:metabolism*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D011503:Transglutaminases / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
chemical_list: D024042:Collagen Type I; D014404:Tubulin; D026122:Factor XIIIa; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins