Abstract
BackgroundBreakpoint cluster region (Bcr) is a multi-domain protein that contains a C-terminal GTPase activating protein (GAP) domain for Rac. Transglutaminase 2 (TG2) regulates Bcr by direct binding to its GAP domain. Since TG2 has transglutaminase activity that has been implicated in the response to extreme stress, we investigated if Bcr can also act as a substrate for TG2.ResultsWe 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.ConclusionsBcr thus represents both an interacting partner under non-stressed conditions and a target of transglutaminase activity for TG2 during extreme stress.
Highlights
Breakpoint cluster region (Bcr) is a multi-domain protein that contains a C-terminal GTPase activating protein (GAP) domain for Rac
We previously showed that Transglutaminase 2 (TG2) interacts with Bcr when TG2 is in the open, non-G-nucleotide bound conformation, which is the conformation adopted in the presence of calcium [7]
These results suggest that under normal physiological conditions, TG2 interacts with Bcr even though the calcium concentration is not sufficient for activation of the transglutaminase activity
Summary
Breakpoint cluster region (Bcr) is a multi-domain protein that contains a C-terminal GTPase activating protein (GAP) domain for Rac. Transglutaminase 2 (TG2) regulates Bcr by direct binding to its GAP domain. To understand how the GAP activity of Bcr is regulated, we performed a yeast two-hybrid screen with fulllength Bcr and isolated transglutaminase 2 (TG2), an interesting multi-functional and multi-domain member of the transglutaminase family [7]. Unlike other transglutaminases, it is expressed in a variety of tissues and cells and undergoes a GTP-binding/GTPase cycle. We found that this conformation has decreased affinity for binding with Bcr
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