Increased expression and activity of the ubiquitous enzyme, tissue transglutaminase (TG2), is consistently seen in a variety of models of apoptosis. The p53 oncoprotein is also involved in apoptosis. Here we investigated the interaction of TG2 with p53 and show that the p53 is a substrate for the recently identified serine/threonine kinase activity of TG2. Phosphospecific antibodies indicated that TG2 phosphorylated p53 at Set 15 and Ser 20, residues that are critically important in the interaction of p53 with Mdm2. The TG2-induced phosphorylation was abrogated by high Ca2+ concentrations and inhibited by cystamine, a known inhibitor of TG2 cross-linking activity. Furthermore, we demonstrate that TG2-induced phosphorylation of p53 reduces the ability of p53 to interact with Mdm2. Although TG2 cross-linking activity has been clearly implicated in apoptosis, our observations reported here suggest TG2 modification of p53 could be an additional mechanism whereby TG2 could facilitate apoptosis. (c) 2005 Elsevier Inc. All rights reserved.