Importance of Ca2+-Dependent Transamidation Activity in the Protection Afforded by Tissue Transglutaminase against Doxorubicin-Induced Apoptosis

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Abstract
Tissue transglutaminase II (TGase-II), which is capable of both GTP binding and transamidation activities, has been implicated in a variety of biological disorders ranging from cancer to neurodegenerative diseases. Recent studies have suggested that the transamidation activity of TGase-II is necessary for the survival of cancer cells confronted with different stresses and cellular insults. When assayed in vitro, the transamidation activity of TGase-II is Ca2+-dependent. However, at present, little is known with regard to how the regulation by Ca2+ is manifested or if in fact it is important for the cellular functions of TGase-II. Here, we have set out to further examine the Ca2+-mediated regulation of TGase-II's transamidation activity, with our goals being to identify the Ca2+-regulatory sites on the protein and determine whether they are essential for TGase-II to confer survival to human breast cancer cells. On the basis of comparisons between the X-ray crystal structures of TGase-II and TGase-III, we identified three putative Ca2+-regulatory sites on TGase-II. Site-directed mutagenesis was performed to individually alter key residues at each of the sites. These substitutions did not affect the ability of TGase-II to bind guanine nucleotides, nor did they cause any obvious changes in its cellular localization. While substitutions at the different Ca2+-regulatory sites could either slightly enhance or markedly reduce the GTP hydrolytic activity of TGase-II, mutations at each of the three sites inhibited the Ca2+-responsive transamidation activity. We further showed that the same substitutions inhibited the ability of TGase-II to protect human breast cancer cells against the apoptotic activity of doxorubicin. Overall, these findings demonstrate that the Ca2+-mediated regulation of transamidation activity is essential for the ability of TGase-II to confer cell survival.