Simultaneous upregulation of transglutaminase 2 (TG2) and epidermal growth factor receptor (EGFR) have been reported in a number of systems. Moreover. TG2 has been identified as a downstream target gene for EGF/EGFR. However, it is not known whether the relationship between EGFR and TG2 is only one-way or collaborative. Using embryonic fibroblasts derived from TG2 null mice (MEFtg2-/-), co-overexpressing native TG2 and EGFR, here we report that TG2 differentially regulates EGFR protein in the presence and absence of EGF. In the absence of EGF, TG2 facilitates EGFR downregulation whereas in the presence of EGF, TG2 has opposite effect on EGFR and facilitates Akt phosphorylation. TG2 mediated ligand-independent downregulation of EGFR was not observed in MEFtg2-1- cells overexpressing Ser212Ala phospho mutant form of TG2 suggesting a role of TG2 phosphorylation in this process. However, similar to native TG2, Ser212Ala-TG2 mutant was also able to attenuate ligand-dependent down regulation of EGFR in MEtg2-/- cells. Interestingly, overexpression of Ser216Ala-TG2 mutant led to downregulation of EGFR in MEFtg2-/- cells irrespective of the ligand. These results were further confirmed in breast cancer cells expressing high levels of EGFR. Collectively, data presented here suggests that the relationship between EGFR and TG2 is collaborative and phosphorylation of TG2 play a key role in it. Phospho mutant forms of TG2 reported in this study may be utilized as a part of a novel strategy to downregulate EGFR in cancers with enhanced EGFR signaling. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.