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RILP suppresses invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS.

Wang Z, Zhou Y, Hu X, Chen W, Lin X, Sun L, Xu X, Hong W, Wang T - Cell Death Dis (2015)

Bottom Line: We identified RalGDS (Ral guanine nucleotide dissociation stimulator) as a novel interacting partner for RILP, and truncation analysis revealed the N-terminal region of RILP is responsible for interacting with the guanine nucleotide exchange factor (GEF) domain of RalGDS.Further investigations indicated that the overexpression of RILP inhibits the activity of RalA, a downstream target of RalGDS.Our data suggest that RILP suppresses the invasion of breast cancer cells by interacting with RalGDS to inhibit its GEF activity for RalA.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, State Key Laboratory of Cellular Stress Biology, Xiamen University, Xiamen, Fujian, China.

ABSTRACT
RILP (Rab7-interacting lysosomal protein) is a key regulator for late endosomal/lysosomal trafficking, and probably a tumor suppressor in prostate cancer. However, the role of RILP in other cancers and the underlying mechanism for RILP in regulating the invasion of cancer cells remain to be investigated. In this study, we showed that overexpression of RILP in breast cancer cells inhibits the migration and invasion, whereas the depletion of RILP by RNAi-mediated knockdown promotes the migration and invasion. We identified RalGDS (Ral guanine nucleotide dissociation stimulator) as a novel interacting partner for RILP, and truncation analysis revealed the N-terminal region of RILP is responsible for interacting with the guanine nucleotide exchange factor (GEF) domain of RalGDS. Immunofluorescence microscopy revealed that RalGDS can be recruited to the late endosomal compartments by RILP. Further investigations indicated that the overexpression of RILP inhibits the activity of RalA, a downstream target of RalGDS. Our data suggest that RILP suppresses the invasion of breast cancer cells by interacting with RalGDS to inhibit its GEF activity for RalA.

No MeSH data available.


Related in: MedlinePlus

Effect of RILP on the activity of RalA. (a) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-RalA antibody and is indicative of the amount of active RalA. (b) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-Sec5(RBD). The RalA bound by GST-Sec5(RBD) was detected by western blot using anti-RalA antibody and is similarly a reflection of active RalA. (c) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-Exo84(RBD). The bound RalA by GST-Exo84(RBD) was detected by western blot using anti-RalA antibody. (d) MDA-MB-231 cells were transfected with control vector or vector for expressing GFP-RILP, or vectors for co-expressing GFP-RILP and myc-RalGDS. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-RalA antibody. (e) Quantitative analysis of the results from d, demonstrating RalA-GTP was significantly reduced upon overexpression of RILP, but partially restored when RalGDS was co-expressed with RILP. (f) GST-pulldown experiments showing RalA did not bind to GST-RILP. (g) MCF cells were double-transfected with GFP-RILP and HA-RalA WT, or GFP-RILP and RalAQ72L. The resulting lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-HA tag antibody, showing RILP cannot inhibit the activity of RalAQ72L
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fig5: Effect of RILP on the activity of RalA. (a) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-RalA antibody and is indicative of the amount of active RalA. (b) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-Sec5(RBD). The RalA bound by GST-Sec5(RBD) was detected by western blot using anti-RalA antibody and is similarly a reflection of active RalA. (c) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-Exo84(RBD). The bound RalA by GST-Exo84(RBD) was detected by western blot using anti-RalA antibody. (d) MDA-MB-231 cells were transfected with control vector or vector for expressing GFP-RILP, or vectors for co-expressing GFP-RILP and myc-RalGDS. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-RalA antibody. (e) Quantitative analysis of the results from d, demonstrating RalA-GTP was significantly reduced upon overexpression of RILP, but partially restored when RalGDS was co-expressed with RILP. (f) GST-pulldown experiments showing RalA did not bind to GST-RILP. (g) MCF cells were double-transfected with GFP-RILP and HA-RalA WT, or GFP-RILP and RalAQ72L. The resulting lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-HA tag antibody, showing RILP cannot inhibit the activity of RalAQ72L

Mentions: RalGDS is the downstream effector of Ras and stimulates the GDP/GTP exchange of Ral small GTPases.24, 25 The active GTP-bound Ral can specifically interact with a distinct set of downstream effector proteins such as Ral-binding protein 1 (RalBP1), Sec5 and Exo84.26, 27, 28, 29 To investigate the functional implication of the interaction between RILP and RalGDS, the effect of RILP on the RalGDS-mediated activation of RalA was assessed by GST-pulldown assay using GST-RalBP1(RBD), GST-Sec5(RBD) or GST-Exo84(RBD) to measure the amount of the activated RalA-GTP from MCF7 cell lysates. The cell lysates resulted from MCF7 cells expressing GFP or GFP-RILP were subjected for GST-pulldown, Western blot experiments using RalA antibody revealed that GST-RalBP1(RBD), GST-Sec5(RBD) or GST-Exo84(RBD) can efficiently precipitate RalA from the cell lysates containing GFP, however, the amount of RalA bound to GST-RalBP1(RBD), GST-Sec5 (RBD) or GST-Exo84(RBD) was markedly decreased in cell lysates containing GFP-RILP (Figures 5a–c), indicating that overexpression of RILP reduced the amount of GTP-bound RalA.


RILP suppresses invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS.

Wang Z, Zhou Y, Hu X, Chen W, Lin X, Sun L, Xu X, Hong W, Wang T - Cell Death Dis (2015)

Effect of RILP on the activity of RalA. (a) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-RalA antibody and is indicative of the amount of active RalA. (b) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-Sec5(RBD). The RalA bound by GST-Sec5(RBD) was detected by western blot using anti-RalA antibody and is similarly a reflection of active RalA. (c) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-Exo84(RBD). The bound RalA by GST-Exo84(RBD) was detected by western blot using anti-RalA antibody. (d) MDA-MB-231 cells were transfected with control vector or vector for expressing GFP-RILP, or vectors for co-expressing GFP-RILP and myc-RalGDS. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-RalA antibody. (e) Quantitative analysis of the results from d, demonstrating RalA-GTP was significantly reduced upon overexpression of RILP, but partially restored when RalGDS was co-expressed with RILP. (f) GST-pulldown experiments showing RalA did not bind to GST-RILP. (g) MCF cells were double-transfected with GFP-RILP and HA-RalA WT, or GFP-RILP and RalAQ72L. The resulting lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-HA tag antibody, showing RILP cannot inhibit the activity of RalAQ72L
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fig5: Effect of RILP on the activity of RalA. (a) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-RalA antibody and is indicative of the amount of active RalA. (b) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-Sec5(RBD). The RalA bound by GST-Sec5(RBD) was detected by western blot using anti-RalA antibody and is similarly a reflection of active RalA. (c) MCF cells were transfected with control vector or vector for expressing GFP-RILP. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-Exo84(RBD). The bound RalA by GST-Exo84(RBD) was detected by western blot using anti-RalA antibody. (d) MDA-MB-231 cells were transfected with control vector or vector for expressing GFP-RILP, or vectors for co-expressing GFP-RILP and myc-RalGDS. Cells were lysed and the lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-RalA antibody. (e) Quantitative analysis of the results from d, demonstrating RalA-GTP was significantly reduced upon overexpression of RILP, but partially restored when RalGDS was co-expressed with RILP. (f) GST-pulldown experiments showing RalA did not bind to GST-RILP. (g) MCF cells were double-transfected with GFP-RILP and HA-RalA WT, or GFP-RILP and RalAQ72L. The resulting lysates were subjected for GST-pulldown assay using GST-RalBP1(RBD). The RalA bound by GST-RalBP1(RBD) was detected by western blot using anti-HA tag antibody, showing RILP cannot inhibit the activity of RalAQ72L
Mentions: RalGDS is the downstream effector of Ras and stimulates the GDP/GTP exchange of Ral small GTPases.24, 25 The active GTP-bound Ral can specifically interact with a distinct set of downstream effector proteins such as Ral-binding protein 1 (RalBP1), Sec5 and Exo84.26, 27, 28, 29 To investigate the functional implication of the interaction between RILP and RalGDS, the effect of RILP on the RalGDS-mediated activation of RalA was assessed by GST-pulldown assay using GST-RalBP1(RBD), GST-Sec5(RBD) or GST-Exo84(RBD) to measure the amount of the activated RalA-GTP from MCF7 cell lysates. The cell lysates resulted from MCF7 cells expressing GFP or GFP-RILP were subjected for GST-pulldown, Western blot experiments using RalA antibody revealed that GST-RalBP1(RBD), GST-Sec5(RBD) or GST-Exo84(RBD) can efficiently precipitate RalA from the cell lysates containing GFP, however, the amount of RalA bound to GST-RalBP1(RBD), GST-Sec5 (RBD) or GST-Exo84(RBD) was markedly decreased in cell lysates containing GFP-RILP (Figures 5a–c), indicating that overexpression of RILP reduced the amount of GTP-bound RalA.

Bottom Line: We identified RalGDS (Ral guanine nucleotide dissociation stimulator) as a novel interacting partner for RILP, and truncation analysis revealed the N-terminal region of RILP is responsible for interacting with the guanine nucleotide exchange factor (GEF) domain of RalGDS.Further investigations indicated that the overexpression of RILP inhibits the activity of RalA, a downstream target of RalGDS.Our data suggest that RILP suppresses the invasion of breast cancer cells by interacting with RalGDS to inhibit its GEF activity for RalA.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, State Key Laboratory of Cellular Stress Biology, Xiamen University, Xiamen, Fujian, China.

ABSTRACT
RILP (Rab7-interacting lysosomal protein) is a key regulator for late endosomal/lysosomal trafficking, and probably a tumor suppressor in prostate cancer. However, the role of RILP in other cancers and the underlying mechanism for RILP in regulating the invasion of cancer cells remain to be investigated. In this study, we showed that overexpression of RILP in breast cancer cells inhibits the migration and invasion, whereas the depletion of RILP by RNAi-mediated knockdown promotes the migration and invasion. We identified RalGDS (Ral guanine nucleotide dissociation stimulator) as a novel interacting partner for RILP, and truncation analysis revealed the N-terminal region of RILP is responsible for interacting with the guanine nucleotide exchange factor (GEF) domain of RalGDS. Immunofluorescence microscopy revealed that RalGDS can be recruited to the late endosomal compartments by RILP. Further investigations indicated that the overexpression of RILP inhibits the activity of RalA, a downstream target of RalGDS. Our data suggest that RILP suppresses the invasion of breast cancer cells by interacting with RalGDS to inhibit its GEF activity for RalA.

No MeSH data available.


Related in: MedlinePlus