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Bcr is a substrate for Transglutaminase 2 cross-linking activity.

Yi SJ, Groffen J, Heisterkamp N - BMC Biochem. (2011)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular Carcinogenesis, Division of Hematology/Oncology, Ms#54, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles CA 90027, USA.

ABSTRACT

Background: 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. 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.

Results: 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.

Conclusions: Bcr thus represents both an interacting partner under non-stressed conditions and a target of transglutaminase activity for TG2 during extreme stress.

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Ca2+ influx or CoCl2 stimulates Bcr aggregation in cells. A) COS-1 cells transfected with the indicated plasmids were treated with DMSO (Control) or 1 μM ionomycin for 1 h. Antibodies used for Western blots are indicated to the left. B) Localization of Bcr and TG2 in HPAECs using confocal microscopy. Bar, 20 μm. C, D) HPAECs were incubated with the indicated concentrations of A23187 for 1 h (C) or CoCl2 for 24 h (D). Antibodies used for Western blotting are indicated to the left. E) Cellular localization of Bcr in HPAECs after treatment with DMSO (Control), 20 μM A23187 for 1 h or 1000 μM CoCl2 for 24 h using confocal microscopy. DAPI was used to stain nuclei. Arrows point to some of the intracellular Bcr aggregates. Bar, 20 μm.
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Figure 3: Ca2+ influx or CoCl2 stimulates Bcr aggregation in cells. A) COS-1 cells transfected with the indicated plasmids were treated with DMSO (Control) or 1 μM ionomycin for 1 h. Antibodies used for Western blots are indicated to the left. B) Localization of Bcr and TG2 in HPAECs using confocal microscopy. Bar, 20 μm. C, D) HPAECs were incubated with the indicated concentrations of A23187 for 1 h (C) or CoCl2 for 24 h (D). Antibodies used for Western blotting are indicated to the left. E) Cellular localization of Bcr in HPAECs after treatment with DMSO (Control), 20 μM A23187 for 1 h or 1000 μM CoCl2 for 24 h using confocal microscopy. DAPI was used to stain nuclei. Arrows point to some of the intracellular Bcr aggregates. Bar, 20 μm.

Mentions: In the experiments described above, calcium was added to protein lysates. The transglutaminase activity of TG2 can also be activated in the presence of increased levels of intracellular Ca2+. To examine TG2-induced aggregation of Bcr in cells, COS-1 cells were transfected with Bcr alone or together with TG2 and treated with ionomycin, a calcium ionophore, which induces cell death at high concentrations [21]. As shown in Figure 3A, co-expression of TG2 with Bcr caused a significant increase of high molecular weight Bcr complexes in cells. Ionomycin treatment also caused Bcr cross-linking in cells transfected only with Bcr, likely through activation of endogenous TG2 (Figure 3A, lane Bcr/ionomycin).


Bcr is a substrate for Transglutaminase 2 cross-linking activity.

Yi SJ, Groffen J, Heisterkamp N - BMC Biochem. (2011)

Ca2+ influx or CoCl2 stimulates Bcr aggregation in cells. A) COS-1 cells transfected with the indicated plasmids were treated with DMSO (Control) or 1 μM ionomycin for 1 h. Antibodies used for Western blots are indicated to the left. B) Localization of Bcr and TG2 in HPAECs using confocal microscopy. Bar, 20 μm. C, D) HPAECs were incubated with the indicated concentrations of A23187 for 1 h (C) or CoCl2 for 24 h (D). Antibodies used for Western blotting are indicated to the left. E) Cellular localization of Bcr in HPAECs after treatment with DMSO (Control), 20 μM A23187 for 1 h or 1000 μM CoCl2 for 24 h using confocal microscopy. DAPI was used to stain nuclei. Arrows point to some of the intracellular Bcr aggregates. Bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3044668&req=5

Figure 3: Ca2+ influx or CoCl2 stimulates Bcr aggregation in cells. A) COS-1 cells transfected with the indicated plasmids were treated with DMSO (Control) or 1 μM ionomycin for 1 h. Antibodies used for Western blots are indicated to the left. B) Localization of Bcr and TG2 in HPAECs using confocal microscopy. Bar, 20 μm. C, D) HPAECs were incubated with the indicated concentrations of A23187 for 1 h (C) or CoCl2 for 24 h (D). Antibodies used for Western blotting are indicated to the left. E) Cellular localization of Bcr in HPAECs after treatment with DMSO (Control), 20 μM A23187 for 1 h or 1000 μM CoCl2 for 24 h using confocal microscopy. DAPI was used to stain nuclei. Arrows point to some of the intracellular Bcr aggregates. Bar, 20 μm.
Mentions: In the experiments described above, calcium was added to protein lysates. The transglutaminase activity of TG2 can also be activated in the presence of increased levels of intracellular Ca2+. To examine TG2-induced aggregation of Bcr in cells, COS-1 cells were transfected with Bcr alone or together with TG2 and treated with ionomycin, a calcium ionophore, which induces cell death at high concentrations [21]. As shown in Figure 3A, co-expression of TG2 with Bcr caused a significant increase of high molecular weight Bcr complexes in cells. Ionomycin treatment also caused Bcr cross-linking in cells transfected only with Bcr, likely through activation of endogenous TG2 (Figure 3A, lane Bcr/ionomycin).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular Carcinogenesis, Division of Hematology/Oncology, Ms#54, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles CA 90027, USA.

ABSTRACT

Background: 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. 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.

Results: 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.

Conclusions: Bcr thus represents both an interacting partner under non-stressed conditions and a target of transglutaminase activity for TG2 during extreme stress.

Show MeSH