Importance of Ca(2+)-dependent transamidation activity in the protection afforded by tissue transglutaminase against doxorubicin-induced apoptosis.
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 Ca(2+)-dependent. However, at present, little is known with regard to how the regulation by Ca(2+) 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 Ca(2+)-mediated regulation of TGase-II's transamidation activity, with our goals being to identify the Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-mediated regulation of transamidation activity is essential for the ability of TGase-II to confer cell survival.
Datta
Sunando
S
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Antonyak
Marc A
MA
Cerione
Richard A
RA
D000577:Amides / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000577:Amides; D000970:Antineoplastic Agents; D004317:Doxorubicin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D002118:Calcium