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Association between Gαi2 and ELMO1/Dock180 connects chemokine signalling with Rac activation and metastasis.

Li H, Yang L, Fu H, Yan J, Wang Y, Guo H, Hao X, Xu X, Jin T, Zhang N - Nat Commun (2013)

Bottom Line: Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration.CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2.In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Medical University Cancer Institute and Hospital and Research Center of Basic Medical Sciences, He Xi District, Tianjin 300060, China.

ABSTRACT
The chemokine CXCL12 and its G-protein-coupled receptor CXCR4 control the migration, invasiveness and metastasis of breast cancer cells. Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration. However, the pathways linking chemokine G-protein-coupled receptor/Gi signalling to actin polymerization and cancer cell migration are not known. Here we show that CXCL12 stimulation promotes interaction between Gαi2 and ELMO1. Gi signalling and ELMO1 are both required for CXCL12-mediated actin polymerization, migration and invasion of breast cancer cells. CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2. In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung. Our findings indicate that a chemokine-controlled pathway, consisting of Gαi2, ELMO1/Dock180, Rac1 and Rac2, regulates the actin cytoskeleton during breast cancer metastasis.

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N-terminal portion of ELMO1 interacts with Gαi2 subunit.(a) Image of SDS–PAGE gel stained by silver stain. Lysates of MDA-MB-231 cells expressing ELMO1-YFP or YFP (as a control) were incubated with beads coupled with anti-GFP antibodies and stained with silver stain. (b) Coimmunoprecipitation assay of ELMO1-YFP, Gαi2 and Dock180. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and were probed with antibodies. Input Gαi2 was used as control. (c) N terminus of ELMO1 was required for the interaction with Gαi2. YFP served as a negative control and ELMO1-YFP was a positive control. (d) CXCL12-induced ELMO1 colocalization with Gαi2 on the plasma membrane by confocal microscopy analysis. After stimulation with 100 ng ml−1 CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. MDA-MB-231 cells were stained with anti-ELMO1, anti-Gαi2 antibodies and probed with an Alexa Fluor 488-conjugated or 546-conjugated secondary antibody. Colocalization efficiency was calculated through Image J software. (e) Knockdown of Gαi2 impaired CXCL12-induced membrane translocation of ELMO1. After stimulation with 100 ng ml−1 CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. Twenty-five images were analysed by ImageJ software. Western blotting analysis of biochemical fractionation showed a clear enrichment of ELMO1 upon CXCL12 stimulation.
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f2: N-terminal portion of ELMO1 interacts with Gαi2 subunit.(a) Image of SDS–PAGE gel stained by silver stain. Lysates of MDA-MB-231 cells expressing ELMO1-YFP or YFP (as a control) were incubated with beads coupled with anti-GFP antibodies and stained with silver stain. (b) Coimmunoprecipitation assay of ELMO1-YFP, Gαi2 and Dock180. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and were probed with antibodies. Input Gαi2 was used as control. (c) N terminus of ELMO1 was required for the interaction with Gαi2. YFP served as a negative control and ELMO1-YFP was a positive control. (d) CXCL12-induced ELMO1 colocalization with Gαi2 on the plasma membrane by confocal microscopy analysis. After stimulation with 100 ng ml−1 CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. MDA-MB-231 cells were stained with anti-ELMO1, anti-Gαi2 antibodies and probed with an Alexa Fluor 488-conjugated or 546-conjugated secondary antibody. Colocalization efficiency was calculated through Image J software. (e) Knockdown of Gαi2 impaired CXCL12-induced membrane translocation of ELMO1. After stimulation with 100 ng ml−1 CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. Twenty-five images were analysed by ImageJ software. Western blotting analysis of biochemical fractionation showed a clear enrichment of ELMO1 upon CXCL12 stimulation.

Mentions: To reveal the potential molecular mechanism of ELMO1 function, we sought to identify proteins that associated with ELMO1. ELMO1-YFP, expressed in CXCL12-stimulated MDA-MB-231 cells, was partially purified from lysates by using anti-green fluorescent protein (GFP) antibodies coupled to beads, and elutes were subjected to SDS gel electrophoresis. The YFP protein expressed in the cells was used as a control (Fig. 2a). Three prominent protein bands, which appeared in the ELMO1-YFP, but not in the control sample, were identified using mass spectrometry (Fig. 2a and Table 1). The identified proteins were ELMO1-YFP, Dock180 and Gαi2 (Table 1). It is well known that Dock180 and ELMO1 form a complex that serves as a GEF for small G-protein Rac to mediate actin polymerization for cell migration17, and Gαi2 is a subunit of heterotrimeric G-proteins that link to chemokine receptors2223. The associations between ELMO1/Dock180 and ELMO1/Gαi2 were confirmed by immunoprecipitation analyses (Fig. 2b). In pulldowns of lysates from cells expressing ELMO1-YFP using an anti-GFP antibody, we found that Dock180 coimmunoprecipitated with ELMO1-YFP but not the YFP control. Similar levels of Dock180 were pulled down from the cells with or without CXCL12 stimulation or PTX treatment, suggesting that ELMO1 and Dock180 formed a stable complex, which was independent of the activation of CXCR4 receptor or heterotrimeric G-proteins. Gαi2 was also coimmunoprecipitated with ELMO1-YFP but not with YFP (control). When the cells were treated with CXCL12 and GTPγS, which activates heterotrimeric G-proteins, the association was increased, whereas the association was clearly decreased upon PTX treatment (Fig. 2b), suggesting that activation of CXCR4 and heterotirmeric G-proteins promoted the association between Gαi2 and the complex of ELMO1 and Dock180.


Association between Gαi2 and ELMO1/Dock180 connects chemokine signalling with Rac activation and metastasis.

Li H, Yang L, Fu H, Yan J, Wang Y, Guo H, Hao X, Xu X, Jin T, Zhang N - Nat Commun (2013)

N-terminal portion of ELMO1 interacts with Gαi2 subunit.(a) Image of SDS–PAGE gel stained by silver stain. Lysates of MDA-MB-231 cells expressing ELMO1-YFP or YFP (as a control) were incubated with beads coupled with anti-GFP antibodies and stained with silver stain. (b) Coimmunoprecipitation assay of ELMO1-YFP, Gαi2 and Dock180. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and were probed with antibodies. Input Gαi2 was used as control. (c) N terminus of ELMO1 was required for the interaction with Gαi2. YFP served as a negative control and ELMO1-YFP was a positive control. (d) CXCL12-induced ELMO1 colocalization with Gαi2 on the plasma membrane by confocal microscopy analysis. After stimulation with 100 ng ml−1 CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. MDA-MB-231 cells were stained with anti-ELMO1, anti-Gαi2 antibodies and probed with an Alexa Fluor 488-conjugated or 546-conjugated secondary antibody. Colocalization efficiency was calculated through Image J software. (e) Knockdown of Gαi2 impaired CXCL12-induced membrane translocation of ELMO1. After stimulation with 100 ng ml−1 CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. Twenty-five images were analysed by ImageJ software. Western blotting analysis of biochemical fractionation showed a clear enrichment of ELMO1 upon CXCL12 stimulation.
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f2: N-terminal portion of ELMO1 interacts with Gαi2 subunit.(a) Image of SDS–PAGE gel stained by silver stain. Lysates of MDA-MB-231 cells expressing ELMO1-YFP or YFP (as a control) were incubated with beads coupled with anti-GFP antibodies and stained with silver stain. (b) Coimmunoprecipitation assay of ELMO1-YFP, Gαi2 and Dock180. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and were probed with antibodies. Input Gαi2 was used as control. (c) N terminus of ELMO1 was required for the interaction with Gαi2. YFP served as a negative control and ELMO1-YFP was a positive control. (d) CXCL12-induced ELMO1 colocalization with Gαi2 on the plasma membrane by confocal microscopy analysis. After stimulation with 100 ng ml−1 CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. MDA-MB-231 cells were stained with anti-ELMO1, anti-Gαi2 antibodies and probed with an Alexa Fluor 488-conjugated or 546-conjugated secondary antibody. Colocalization efficiency was calculated through Image J software. (e) Knockdown of Gαi2 impaired CXCL12-induced membrane translocation of ELMO1. After stimulation with 100 ng ml−1 CXCL12 for 1 h at 37 °C, cells were fixed, permeabilized and blocked in 3% BSA. Twenty-five images were analysed by ImageJ software. Western blotting analysis of biochemical fractionation showed a clear enrichment of ELMO1 upon CXCL12 stimulation.
Mentions: To reveal the potential molecular mechanism of ELMO1 function, we sought to identify proteins that associated with ELMO1. ELMO1-YFP, expressed in CXCL12-stimulated MDA-MB-231 cells, was partially purified from lysates by using anti-green fluorescent protein (GFP) antibodies coupled to beads, and elutes were subjected to SDS gel electrophoresis. The YFP protein expressed in the cells was used as a control (Fig. 2a). Three prominent protein bands, which appeared in the ELMO1-YFP, but not in the control sample, were identified using mass spectrometry (Fig. 2a and Table 1). The identified proteins were ELMO1-YFP, Dock180 and Gαi2 (Table 1). It is well known that Dock180 and ELMO1 form a complex that serves as a GEF for small G-protein Rac to mediate actin polymerization for cell migration17, and Gαi2 is a subunit of heterotrimeric G-proteins that link to chemokine receptors2223. The associations between ELMO1/Dock180 and ELMO1/Gαi2 were confirmed by immunoprecipitation analyses (Fig. 2b). In pulldowns of lysates from cells expressing ELMO1-YFP using an anti-GFP antibody, we found that Dock180 coimmunoprecipitated with ELMO1-YFP but not the YFP control. Similar levels of Dock180 were pulled down from the cells with or without CXCL12 stimulation or PTX treatment, suggesting that ELMO1 and Dock180 formed a stable complex, which was independent of the activation of CXCR4 receptor or heterotrimeric G-proteins. Gαi2 was also coimmunoprecipitated with ELMO1-YFP but not with YFP (control). When the cells were treated with CXCL12 and GTPγS, which activates heterotrimeric G-proteins, the association was increased, whereas the association was clearly decreased upon PTX treatment (Fig. 2b), suggesting that activation of CXCR4 and heterotirmeric G-proteins promoted the association between Gαi2 and the complex of ELMO1 and Dock180.

Bottom Line: Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration.CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2.In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung.

View Article: PubMed Central - PubMed

Affiliation: Tianjin Medical University Cancer Institute and Hospital and Research Center of Basic Medical Sciences, He Xi District, Tianjin 300060, China.

ABSTRACT
The chemokine CXCL12 and its G-protein-coupled receptor CXCR4 control the migration, invasiveness and metastasis of breast cancer cells. Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration. However, the pathways linking chemokine G-protein-coupled receptor/Gi signalling to actin polymerization and cancer cell migration are not known. Here we show that CXCL12 stimulation promotes interaction between Gαi2 and ELMO1. Gi signalling and ELMO1 are both required for CXCL12-mediated actin polymerization, migration and invasion of breast cancer cells. CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2. In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung. Our findings indicate that a chemokine-controlled pathway, consisting of Gαi2, ELMO1/Dock180, Rac1 and Rac2, regulates the actin cytoskeleton during breast cancer metastasis.

Show MeSH
Related in: MedlinePlus