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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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CXCL12 induces membrane translocation and activation of Rac.(a) Coimmunoprecipitation assay of ELMO1-YFP, Rac1 and Rac2. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and probed with antibodies. (b) Knockdown of ELMO1 impaired CXCL12-induced membrane translocation of Rac1/2. 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 Image J software. Western blotting of biochemical fractionation showed that a clear enrichment of Rac1/2 upon CXCL12 stimulation and knockdown ELMO1 impaired CXCL12-induced Rac1/2 translocation. (c) Overexpression of ELMO1-YFP pulled down activated Rac1 and Rac2. Rac activation assay was performed by Rac Activation Assay Biochem Kit (Cytoskeleton, Inc.). Cleared lysates were obtained by centrifugation and incubated with PAK–PBD beads with rotation at 4 °C for 1 h. (d) siELMO1 had no effect in membrane translocation of AKT and PDK1. Western blotting showed a clear enrichment of AKT and PDK1 upon CXCL12 stimulation, and knockdown ELMO1 did not impair CXCL12-induced AKT and PDK1 translocation. (e) Western blotting analysis of phosphorylation of AKT308 or 473, ERK1/2 and PDK1. Control/MDA-MB-231 cells were stimulated by 10 ng ml−1 EGF or 100 ng ml−1 CXCL12 for 1 h. siELMO1/MDA-MB-231 cells were stimulated by 100 ng ml−1 CXCL12 for 1 h. Total AKT, ERK1/2 and PDK1 served as control.
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f3: CXCL12 induces membrane translocation and activation of Rac.(a) Coimmunoprecipitation assay of ELMO1-YFP, Rac1 and Rac2. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and probed with antibodies. (b) Knockdown of ELMO1 impaired CXCL12-induced membrane translocation of Rac1/2. 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 Image J software. Western blotting of biochemical fractionation showed that a clear enrichment of Rac1/2 upon CXCL12 stimulation and knockdown ELMO1 impaired CXCL12-induced Rac1/2 translocation. (c) Overexpression of ELMO1-YFP pulled down activated Rac1 and Rac2. Rac activation assay was performed by Rac Activation Assay Biochem Kit (Cytoskeleton, Inc.). Cleared lysates were obtained by centrifugation and incubated with PAK–PBD beads with rotation at 4 °C for 1 h. (d) siELMO1 had no effect in membrane translocation of AKT and PDK1. Western blotting showed a clear enrichment of AKT and PDK1 upon CXCL12 stimulation, and knockdown ELMO1 did not impair CXCL12-induced AKT and PDK1 translocation. (e) Western blotting analysis of phosphorylation of AKT308 or 473, ERK1/2 and PDK1. Control/MDA-MB-231 cells were stimulated by 10 ng ml−1 EGF or 100 ng ml−1 CXCL12 for 1 h. siELMO1/MDA-MB-231 cells were stimulated by 100 ng ml−1 CXCL12 for 1 h. Total AKT, ERK1/2 and PDK1 served as control.

Mentions: Chemokine GPCR has been shown to promote actin polymerization by activating the small G-proteins Rac1 and Rac2 (ref. 11). We therefore examined whether ELMO1 regulated the functions of Rac1 and Rac2 in breast cancer cells. Using an immunoprecipitation assay (Fig. 3a), we found that Rac1 and Rac2 associated with ELMO1-YFP, but not with the YFP control. In addition, CXCL12 and GTPγS stimulation, which activates Gαi signalling, promoted the association of both Rac1 and Rac2 with ELMO1-YFP, whereas PTX treatment, which blocks Gαi signalling, inhibited these associations (Fig. 3a). Using a biochemical fractionation analysis (Fig. 3b), we found that CXCL12 simulation resulted enrichment of Rac1 and Rac2 in the membrane fraction and that this membrane enrichment did not occur in ELMO1 knockdown (siELMO1) cells. Using confocal fluorescence microscopy, we confirmed that CXCL12-induced membrane translocation of Rac1 and Rac2, and that these translocations were suppressed in siELMO1 cells (Fig. 3b and Supplementary Fig. S4). We then examined the role of ELMO1 in CXCL12-induced activation of Rac1 and Rac2 using a Rac activation assay (Fig. 3c). We found that CXCL12 stimulation induced activation of both Rac1 and Rac2, and that these activations were impaired when Gαi signalling was blocked by PTX or when ELMO1 was knocked down (siELMO1; Fig. 3c). Together, these results suggest that CXCL12 activates a pathway in which activated Gαi2 and its association with the ELMO1/Dock180 complex promotes the membrane translocation and activation of Rac1 and Rac2 in breast cancer cells.


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)

CXCL12 induces membrane translocation and activation of Rac.(a) Coimmunoprecipitation assay of ELMO1-YFP, Rac1 and Rac2. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and probed with antibodies. (b) Knockdown of ELMO1 impaired CXCL12-induced membrane translocation of Rac1/2. 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 Image J software. Western blotting of biochemical fractionation showed that a clear enrichment of Rac1/2 upon CXCL12 stimulation and knockdown ELMO1 impaired CXCL12-induced Rac1/2 translocation. (c) Overexpression of ELMO1-YFP pulled down activated Rac1 and Rac2. Rac activation assay was performed by Rac Activation Assay Biochem Kit (Cytoskeleton, Inc.). Cleared lysates were obtained by centrifugation and incubated with PAK–PBD beads with rotation at 4 °C for 1 h. (d) siELMO1 had no effect in membrane translocation of AKT and PDK1. Western blotting showed a clear enrichment of AKT and PDK1 upon CXCL12 stimulation, and knockdown ELMO1 did not impair CXCL12-induced AKT and PDK1 translocation. (e) Western blotting analysis of phosphorylation of AKT308 or 473, ERK1/2 and PDK1. Control/MDA-MB-231 cells were stimulated by 10 ng ml−1 EGF or 100 ng ml−1 CXCL12 for 1 h. siELMO1/MDA-MB-231 cells were stimulated by 100 ng ml−1 CXCL12 for 1 h. Total AKT, ERK1/2 and PDK1 served as control.
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f3: CXCL12 induces membrane translocation and activation of Rac.(a) Coimmunoprecipitation assay of ELMO1-YFP, Rac1 and Rac2. The immunoprecipitation was performed by using the μMACS GFP isolation kit. The eluted proteins were separated by SDS–PAGE and probed with antibodies. (b) Knockdown of ELMO1 impaired CXCL12-induced membrane translocation of Rac1/2. 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 Image J software. Western blotting of biochemical fractionation showed that a clear enrichment of Rac1/2 upon CXCL12 stimulation and knockdown ELMO1 impaired CXCL12-induced Rac1/2 translocation. (c) Overexpression of ELMO1-YFP pulled down activated Rac1 and Rac2. Rac activation assay was performed by Rac Activation Assay Biochem Kit (Cytoskeleton, Inc.). Cleared lysates were obtained by centrifugation and incubated with PAK–PBD beads with rotation at 4 °C for 1 h. (d) siELMO1 had no effect in membrane translocation of AKT and PDK1. Western blotting showed a clear enrichment of AKT and PDK1 upon CXCL12 stimulation, and knockdown ELMO1 did not impair CXCL12-induced AKT and PDK1 translocation. (e) Western blotting analysis of phosphorylation of AKT308 or 473, ERK1/2 and PDK1. Control/MDA-MB-231 cells were stimulated by 10 ng ml−1 EGF or 100 ng ml−1 CXCL12 for 1 h. siELMO1/MDA-MB-231 cells were stimulated by 100 ng ml−1 CXCL12 for 1 h. Total AKT, ERK1/2 and PDK1 served as control.
Mentions: Chemokine GPCR has been shown to promote actin polymerization by activating the small G-proteins Rac1 and Rac2 (ref. 11). We therefore examined whether ELMO1 regulated the functions of Rac1 and Rac2 in breast cancer cells. Using an immunoprecipitation assay (Fig. 3a), we found that Rac1 and Rac2 associated with ELMO1-YFP, but not with the YFP control. In addition, CXCL12 and GTPγS stimulation, which activates Gαi signalling, promoted the association of both Rac1 and Rac2 with ELMO1-YFP, whereas PTX treatment, which blocks Gαi signalling, inhibited these associations (Fig. 3a). Using a biochemical fractionation analysis (Fig. 3b), we found that CXCL12 simulation resulted enrichment of Rac1 and Rac2 in the membrane fraction and that this membrane enrichment did not occur in ELMO1 knockdown (siELMO1) cells. Using confocal fluorescence microscopy, we confirmed that CXCL12-induced membrane translocation of Rac1 and Rac2, and that these translocations were suppressed in siELMO1 cells (Fig. 3b and Supplementary Fig. S4). We then examined the role of ELMO1 in CXCL12-induced activation of Rac1 and Rac2 using a Rac activation assay (Fig. 3c). We found that CXCL12 stimulation induced activation of both Rac1 and Rac2, and that these activations were impaired when Gαi signalling was blocked by PTX or when ELMO1 was knocked down (siELMO1; Fig. 3c). Together, these results suggest that CXCL12 activates a pathway in which activated Gαi2 and its association with the ELMO1/Dock180 complex promotes the membrane translocation and activation of Rac1 and Rac2 in breast cancer cells.

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