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.

Transglutaminase activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics

“…Studies have shown that microtubules regulate the expansion of podosome clusters into a belt and that thus disruption of the microtubule network can result in the loss of podosome belt (Destaing et al, ; Destaing, Saltel, Geminard, Jurdic, & Bard, ). Furthermore, previous studies showed that Exoenzyme C3 increased the acetylated α‐tubulin level in osteoclasts (Destaing et al, ), and our group has shown that NC9 regulates detyrosinated Glu‐tubulin in osteoblasts (Al‐Jallad et al, ; Wang, Cui, Hitomi, & Kaartinen, ). Examination of microtubule architecture in osteoclast after NC9 treatment for 24 hr, however, showed no major alterations (Supplementary Figure S4b) and western blotting analysis confirmed that NC9 and Exoenzyme C3 had no effect on the acetylation or detyrosination of α‐tubulin in osteoclasts (Supplementary Figure S4c).…”

Factor XIII-A transglutaminase acts as a switch between preadipocyte proliferation and differentiation

“…bond (isopeptide crosslink/bond) that can induce the formation of multimeric protein networks and change the conformation, structure, solubility, biochemical stability, and cell-adhesion properties of substrate proteins. [23][24][25][26][27][28][29] In addition to fibrin, a major extracellular substrate for FXIII-A is fibronectin (FN). 30 FN is an extracellular glycoprotein capable of regulating various cellular functions, including proliferation and differentiation.…”

Let's cross‐link: diverse functions of the promiscuous cellular transglutaminase factor XIII‐A

“…This matrix forms a scaffold for other proteins to adhere to, including type 1 collagen . Surface‐associated osteoblast FXIII‐A is involved in stabilizing the interaction between microtubules and the plasma membrane, which, in turn, enhances the secretion of collagen . Collagen is the principal component of bone matrix, and it appears that both FXIII‐A activity and fibronectin are essential for normal collagen deposition .…”