Importance of Ca(2+)-dependent transamidation activity in the protection afforded by tissue transglutaminase against doxorubicin-induced apoptosis.

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 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.

• Authors: Sunando Datta, Marc A Antonyak, Richard A Cerione
• Journal: Biochemistry (Biochemistry) Vol. 45 Issue 44 Pg. 13163-74 (Nov 07 2006) ISSN: 0006-2960 [Print] United States
• PMID: 17073438 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Amides
• Antineoplastic Agents
• Doxorubicin
• Protein Glutamine gamma Glutamyltransferase 2
• Transglutaminases
• GTP-Binding Proteins
• Calcium

Topics

• Amides (metabolism)
• Antineoplastic Agents (pharmacology)
• Apoptosis (drug effects)
• Calcium (metabolism)
• Cell Line, Tumor
• Doxorubicin (pharmacology)
• Fluorescent Antibody Technique
• GTP-Binding Proteins
• Humans
• Models, Molecular
• Mutagenesis, Site-Directed
• Protein Glutamine gamma Glutamyltransferase 2
• Transglutaminases (chemistry, genetics, metabolism)