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SUMOylation of the GTPase Rac1 is required for optimal cell migration.

Castillo-Lluva S, Tatham MH, Jones RC, Jaffray EG, Edmondson RD, Hay RT, Malliri A - Nat. Cell Biol. (2010)

Bottom Line: We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3.Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation.Our data reveal a role for SUMO in the regulation of cell migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, The University of Manchester, Manchester, M20 4BX, UK.

ABSTRACT
The Rho-like GTPase, Rac1, induces cytoskeletal rearrangements required for cell migration. Rac activation is regulated through a number of mechanisms, including control of nucleotide exchange and hydrolysis, regulation of subcellular localization or modulation of protein-expression levels. Here, we identify that the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, interacts with Rac1 and is required for increased Rac activation and optimal cell migration in response to hepatocyte growth factor (HGF) signalling. We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3. Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation. We demonstrate that PIAS3-mediated SUMOylation of Rac1 controls the levels of Rac1-GTP and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion. The finding that a Ras superfamily member can be SUMOylated provides an insight into the regulation of these critical mediators of cell behaviour. Our data reveal a role for SUMO in the regulation of cell migration and invasion.

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Rac1 is SUMOylated in the polybasic region and SUMOylation affects its GTP-levels. (a) GST-Rac1 was incubated at 37°C in vitro in the presence (+) or absence (−) of complete SUMOylation assay components and resolved by SDS-PAGE. (b) The GST-Rac1-SUMO-1 band from (a) was excised and subjected to in gel trypsin digestion. The resultant peptides were analysed by mass spectrometry and MaxQuant data processing to detect modified and unmodified peptides. Column chart indicates the absolute intensity of SUMO-Rac1 branched peptides detected. * indicates that discrimination between lysines is not possible. (c) Schematic illustration of putative Rac1 SUMOylation sites and the mutations created. (d) HeLa cells stably expressing 6His-SUMO-1 were transfected as indicated, lysed in the presence of NEM, GFP immunoprecipitated and analysed by WB. Rac-nS1 indicates multi-mono-SUMOylated Rac1. (e) HeLa cells were transfected as indicated, lysed in the presence of NEM, GFP immunoprecipitated and analysed by WB. GFP-Rac1-nUb indicates Rac1 modified by poly-Ubiquitin. (f-g) COS7 cells transfected with the indicated plasmids were serum starved for 18 hours before 10 ng/ml of HGF was added for 30 minutes and assayed for Rac1 activity. Bar graphs depict quantification of the normalized relative amounts of Rac-GTP determined by scanning densitometry from at least three independent experiments. Error bars indicate ±SEM. * p<0.05, two-tailed Student’s t-test. Uncropped images of blots are shown in Supplementary Information, Fig. S6.
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Figure 4: Rac1 is SUMOylated in the polybasic region and SUMOylation affects its GTP-levels. (a) GST-Rac1 was incubated at 37°C in vitro in the presence (+) or absence (−) of complete SUMOylation assay components and resolved by SDS-PAGE. (b) The GST-Rac1-SUMO-1 band from (a) was excised and subjected to in gel trypsin digestion. The resultant peptides were analysed by mass spectrometry and MaxQuant data processing to detect modified and unmodified peptides. Column chart indicates the absolute intensity of SUMO-Rac1 branched peptides detected. * indicates that discrimination between lysines is not possible. (c) Schematic illustration of putative Rac1 SUMOylation sites and the mutations created. (d) HeLa cells stably expressing 6His-SUMO-1 were transfected as indicated, lysed in the presence of NEM, GFP immunoprecipitated and analysed by WB. Rac-nS1 indicates multi-mono-SUMOylated Rac1. (e) HeLa cells were transfected as indicated, lysed in the presence of NEM, GFP immunoprecipitated and analysed by WB. GFP-Rac1-nUb indicates Rac1 modified by poly-Ubiquitin. (f-g) COS7 cells transfected with the indicated plasmids were serum starved for 18 hours before 10 ng/ml of HGF was added for 30 minutes and assayed for Rac1 activity. Bar graphs depict quantification of the normalized relative amounts of Rac-GTP determined by scanning densitometry from at least three independent experiments. Error bars indicate ±SEM. * p<0.05, two-tailed Student’s t-test. Uncropped images of blots are shown in Supplementary Information, Fig. S6.

Mentions: Frequently, SUMO targets the consensus sequence ΨKXD/E, where Ψ represents a hydrophobic residue and K the acceptor lysine; however, non-consensus sites have been reported22, 23. Rac1 lacks a consensus motif; therefore we adopted a MS approach to identify SUMO-modified site(s). GST-Rac1 was in vitro SUMOylated and the band corresponding to GST-Rac1-SUMO-1 (Fig. 4a) was subjected to in gel trypsin digestion and branched peptides identified by MS. Data was processed using MaxQuant24, and label free intensities for the branched peptides obtained which allowed for approximation of relative abundances. Although 10 of the potential total 17 lysine-SUMO-1 branched peptides were identified, almost 95% of the SUMO-1 modified Rac1 was conjugated at lysines 188, 183 and 184 or 186 (Fig. 4b) present within the C-terminal polybasic region (PBR) of Rac1 (Fig. 4c).


SUMOylation of the GTPase Rac1 is required for optimal cell migration.

Castillo-Lluva S, Tatham MH, Jones RC, Jaffray EG, Edmondson RD, Hay RT, Malliri A - Nat. Cell Biol. (2010)

Rac1 is SUMOylated in the polybasic region and SUMOylation affects its GTP-levels. (a) GST-Rac1 was incubated at 37°C in vitro in the presence (+) or absence (−) of complete SUMOylation assay components and resolved by SDS-PAGE. (b) The GST-Rac1-SUMO-1 band from (a) was excised and subjected to in gel trypsin digestion. The resultant peptides were analysed by mass spectrometry and MaxQuant data processing to detect modified and unmodified peptides. Column chart indicates the absolute intensity of SUMO-Rac1 branched peptides detected. * indicates that discrimination between lysines is not possible. (c) Schematic illustration of putative Rac1 SUMOylation sites and the mutations created. (d) HeLa cells stably expressing 6His-SUMO-1 were transfected as indicated, lysed in the presence of NEM, GFP immunoprecipitated and analysed by WB. Rac-nS1 indicates multi-mono-SUMOylated Rac1. (e) HeLa cells were transfected as indicated, lysed in the presence of NEM, GFP immunoprecipitated and analysed by WB. GFP-Rac1-nUb indicates Rac1 modified by poly-Ubiquitin. (f-g) COS7 cells transfected with the indicated plasmids were serum starved for 18 hours before 10 ng/ml of HGF was added for 30 minutes and assayed for Rac1 activity. Bar graphs depict quantification of the normalized relative amounts of Rac-GTP determined by scanning densitometry from at least three independent experiments. Error bars indicate ±SEM. * p<0.05, two-tailed Student’s t-test. Uncropped images of blots are shown in Supplementary Information, Fig. S6.
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Figure 4: Rac1 is SUMOylated in the polybasic region and SUMOylation affects its GTP-levels. (a) GST-Rac1 was incubated at 37°C in vitro in the presence (+) or absence (−) of complete SUMOylation assay components and resolved by SDS-PAGE. (b) The GST-Rac1-SUMO-1 band from (a) was excised and subjected to in gel trypsin digestion. The resultant peptides were analysed by mass spectrometry and MaxQuant data processing to detect modified and unmodified peptides. Column chart indicates the absolute intensity of SUMO-Rac1 branched peptides detected. * indicates that discrimination between lysines is not possible. (c) Schematic illustration of putative Rac1 SUMOylation sites and the mutations created. (d) HeLa cells stably expressing 6His-SUMO-1 were transfected as indicated, lysed in the presence of NEM, GFP immunoprecipitated and analysed by WB. Rac-nS1 indicates multi-mono-SUMOylated Rac1. (e) HeLa cells were transfected as indicated, lysed in the presence of NEM, GFP immunoprecipitated and analysed by WB. GFP-Rac1-nUb indicates Rac1 modified by poly-Ubiquitin. (f-g) COS7 cells transfected with the indicated plasmids were serum starved for 18 hours before 10 ng/ml of HGF was added for 30 minutes and assayed for Rac1 activity. Bar graphs depict quantification of the normalized relative amounts of Rac-GTP determined by scanning densitometry from at least three independent experiments. Error bars indicate ±SEM. * p<0.05, two-tailed Student’s t-test. Uncropped images of blots are shown in Supplementary Information, Fig. S6.
Mentions: Frequently, SUMO targets the consensus sequence ΨKXD/E, where Ψ represents a hydrophobic residue and K the acceptor lysine; however, non-consensus sites have been reported22, 23. Rac1 lacks a consensus motif; therefore we adopted a MS approach to identify SUMO-modified site(s). GST-Rac1 was in vitro SUMOylated and the band corresponding to GST-Rac1-SUMO-1 (Fig. 4a) was subjected to in gel trypsin digestion and branched peptides identified by MS. Data was processed using MaxQuant24, and label free intensities for the branched peptides obtained which allowed for approximation of relative abundances. Although 10 of the potential total 17 lysine-SUMO-1 branched peptides were identified, almost 95% of the SUMO-1 modified Rac1 was conjugated at lysines 188, 183 and 184 or 186 (Fig. 4b) present within the C-terminal polybasic region (PBR) of Rac1 (Fig. 4c).

Bottom Line: We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3.Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation.Our data reveal a role for SUMO in the regulation of cell migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, The University of Manchester, Manchester, M20 4BX, UK.

ABSTRACT
The Rho-like GTPase, Rac1, induces cytoskeletal rearrangements required for cell migration. Rac activation is regulated through a number of mechanisms, including control of nucleotide exchange and hydrolysis, regulation of subcellular localization or modulation of protein-expression levels. Here, we identify that the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, interacts with Rac1 and is required for increased Rac activation and optimal cell migration in response to hepatocyte growth factor (HGF) signalling. We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3. Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation. We demonstrate that PIAS3-mediated SUMOylation of Rac1 controls the levels of Rac1-GTP and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion. The finding that a Ras superfamily member can be SUMOylated provides an insight into the regulation of these critical mediators of cell behaviour. Our data reveal a role for SUMO in the regulation of cell migration and invasion.

Show MeSH
Related in: MedlinePlus