Bcr is a substrate for Transglutaminase 2 cross-linking activity

We here report that activation of TG2 by calcium caused the formation of covalently cross-linked Bcr. Abr, a protein related to Bcr but lacking its N-terminal oligomerization domain, was not cross-linked by TG2 even though it forms a complex with it. A Bcr mutant missing the first 62 amino acid residues remained monomeric in the presence of activated TG2, showing that this specific domain is necessary for the cross-linking reaction. Calcium influx induced by a calcium ionophore in primary human endothelial cells caused cross-linking of endogenous Bcr, which was inhibited by the TG2 inhibitor cystamine. Treatment of cells with cobalt chloride, a hypoxia-mimetic that causes cellular stress, also generated high molecular weight Bcr complexes. Cross-linked Bcr protein appeared in the TritonX-100-insoluble cell fraction and further accumulated in cells treated with a proteasome inhibitor.Bcr thus represents both an interacting partner under non-stressed conditions and a target of transglutaminase activity for TG2 during extreme stress.The breakpoint cluster region (Bcr) protein was originally identified as the amino-terminal part of a fusion protein including the Abl tyrosine kinase, which causes chronic myeloid leukemia and Ph-chromosome-positive acute lymphoblastic leukemia. The fusion of Bcr to Abl deregulates the tyrosine kinase activity of Abl [1]. Although the Bcr protein contributes a varying number of domains to the fusion protein, the N-terminal oligomerization domain of Bcr is considered to be the most critical component that allows the formation of homo-tetramer Bcr/Abl complexes and deregulates the Abl tyrosine kinase [2,3].The normal (non-rearranged) BCR gene encodes a multidomain protein. Apart from the oligomerization domain, it additionally contains serine/threonine protein kinase, tandem DH-PH, C2 and GTPase activating protein (GAP) domains. The latter domain has a relatively well-described function: it down-regulates the activated GTP-bound conformation