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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 vitro and in vivo. (a) GST-Rac1 protein was incubated at 37°C in vitro in the presence of complete SUMOylation assay components (E1, E2, SUMO-1 (S1) or SUMO-2 (S2)) and resolved by SDS-PAGE. Unmodified sample (Um) contains all the assay components but without incubation at 37°C. Rac1-S1/2 indicates Rac1 modified by SUMO-1 or -2. (b) GFP-Rac1 was immunoprecipitated from HeLa cells in the presence or absence of PIAS3 and in vitro SUMOylation assay was performed as in (a). Rac1 was detected by Western blot (WB). (c) Immunoprecipitated GFP-Rac1 or Rac1V12 was subjected to in vitro SUMOylation as in (a) and Rac1 was detected by WB. (d) HeLa cells transfected as indicated were lysed in the presence of NEM, and GFP was immunoprecipitated. The presence in immunoprecipitates of exogenous Rac1 or GFP was detected by WB analysis. IN, input. (e) GFP-SUMO-1 was immunoprecipitated from HeLa cells transfected as indicated, divided into two and incubated in the presence or absence of SENP1 for 2 hours at 25°C. (f) HeLa cells stably expressing 6His-SUMO-1 were transfected with Trp-Met and 6His-SUMO-1-modified proteins were purified from lysates. Endogenous Rac1-SUMO-1 was detected by WB. (g) MDCKII cells stably expressing scr shRNA or pias3 shRNA were treated as in Fig. 1f in the presence of the crosslinker DSS and HGF and Rac-GTP immunoprecipitation (IP) was performed. GTP-Rac-SUMO-1 bands were detected by WB. (h) MDCKII cells were grown to confluency and incubated for 18 hours in low calcium medium. Rac activity was measured after calcium re-addition for the indicated times. Pulldows (PD) of active Rac were analyzed by WB for Rac and SUMO-1. Blots at the bottom of (h) represent part of the blot above showing the unmodified Rac-GTP band at lower exposure, as well as levels of total Rac. Quantification of the Rac-GTP bands, both SUMO-modified and unmodified, is depicted in the histogram normalized to total Rac. Rac-nS1 in (b-h) is multi-mono-SUMOylated Rac1. Uncropped images of blots are shown in Supplementary Information, Fig. S6.
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Figure 3: Rac1 is SUMOylated in vitro and in vivo. (a) GST-Rac1 protein was incubated at 37°C in vitro in the presence of complete SUMOylation assay components (E1, E2, SUMO-1 (S1) or SUMO-2 (S2)) and resolved by SDS-PAGE. Unmodified sample (Um) contains all the assay components but without incubation at 37°C. Rac1-S1/2 indicates Rac1 modified by SUMO-1 or -2. (b) GFP-Rac1 was immunoprecipitated from HeLa cells in the presence or absence of PIAS3 and in vitro SUMOylation assay was performed as in (a). Rac1 was detected by Western blot (WB). (c) Immunoprecipitated GFP-Rac1 or Rac1V12 was subjected to in vitro SUMOylation as in (a) and Rac1 was detected by WB. (d) HeLa cells transfected as indicated were lysed in the presence of NEM, and GFP was immunoprecipitated. The presence in immunoprecipitates of exogenous Rac1 or GFP was detected by WB analysis. IN, input. (e) GFP-SUMO-1 was immunoprecipitated from HeLa cells transfected as indicated, divided into two and incubated in the presence or absence of SENP1 for 2 hours at 25°C. (f) HeLa cells stably expressing 6His-SUMO-1 were transfected with Trp-Met and 6His-SUMO-1-modified proteins were purified from lysates. Endogenous Rac1-SUMO-1 was detected by WB. (g) MDCKII cells stably expressing scr shRNA or pias3 shRNA were treated as in Fig. 1f in the presence of the crosslinker DSS and HGF and Rac-GTP immunoprecipitation (IP) was performed. GTP-Rac-SUMO-1 bands were detected by WB. (h) MDCKII cells were grown to confluency and incubated for 18 hours in low calcium medium. Rac activity was measured after calcium re-addition for the indicated times. Pulldows (PD) of active Rac were analyzed by WB for Rac and SUMO-1. Blots at the bottom of (h) represent part of the blot above showing the unmodified Rac-GTP band at lower exposure, as well as levels of total Rac. Quantification of the Rac-GTP bands, both SUMO-modified and unmodified, is depicted in the histogram normalized to total Rac. Rac-nS1 in (b-h) is multi-mono-SUMOylated Rac1. Uncropped images of blots are shown in Supplementary Information, Fig. S6.

Mentions: In higher eukaryotes PIAS proteins act as SUMO E3-ligases10. SUMOylation is a covalent modification leading to the attachment of SUMO to specific lysine residues of target proteins14. In vitro SUMOylation followed by Western blotting revealed a band consistent with the attachment of SUMO-1 (S1) or SUMO-2/3 (S2/3) to Rac1, which was absent when E1, E2, or SUMO protein was not present (Fig. 3a). Furthermore, SUMOylation was enhanced by increasing the E2-conjugating enzyme Ubc9 (Supplementary Information, Fig. S2f). In most cases, SUMOylation of a substrate is enhanced by a SUMO E3-ligase10, 15. Rac1 SUMOylation was enhanced by increasing amounts of PIAS3 (Supplementary Information, Fig. S2g). Other PIAS family members also enhanced in vitro Rac1 SUMOylation, although PIAS3 was the most efficient (Supplementary Information, Fig. S2g). Additionally, in vitro SUMOylation of GFP-Rac1 immunoprecipitated from HeLa cells was enhanced upon co-transfection of FLAG-PIAS3 (Fig. 3b). Conversely, downregulation of PIAS3 decreased in vitro SUMOylation of GFP-Rac1 (Supplementary Information, Fig. S2h). These results indicate that PIAS3 works as a SUMO E3-ligase for Rac1. Furthermore, consistent with the finding that PIAS3 binds more efficiently to GTP-bound Rac1 (Fig. 1c; Supplementary Information, Fig. S1g), Rac1V12 was a better substrate for SUMOylation (Fig. 3c).


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 vitro and in vivo. (a) GST-Rac1 protein was incubated at 37°C in vitro in the presence of complete SUMOylation assay components (E1, E2, SUMO-1 (S1) or SUMO-2 (S2)) and resolved by SDS-PAGE. Unmodified sample (Um) contains all the assay components but without incubation at 37°C. Rac1-S1/2 indicates Rac1 modified by SUMO-1 or -2. (b) GFP-Rac1 was immunoprecipitated from HeLa cells in the presence or absence of PIAS3 and in vitro SUMOylation assay was performed as in (a). Rac1 was detected by Western blot (WB). (c) Immunoprecipitated GFP-Rac1 or Rac1V12 was subjected to in vitro SUMOylation as in (a) and Rac1 was detected by WB. (d) HeLa cells transfected as indicated were lysed in the presence of NEM, and GFP was immunoprecipitated. The presence in immunoprecipitates of exogenous Rac1 or GFP was detected by WB analysis. IN, input. (e) GFP-SUMO-1 was immunoprecipitated from HeLa cells transfected as indicated, divided into two and incubated in the presence or absence of SENP1 for 2 hours at 25°C. (f) HeLa cells stably expressing 6His-SUMO-1 were transfected with Trp-Met and 6His-SUMO-1-modified proteins were purified from lysates. Endogenous Rac1-SUMO-1 was detected by WB. (g) MDCKII cells stably expressing scr shRNA or pias3 shRNA were treated as in Fig. 1f in the presence of the crosslinker DSS and HGF and Rac-GTP immunoprecipitation (IP) was performed. GTP-Rac-SUMO-1 bands were detected by WB. (h) MDCKII cells were grown to confluency and incubated for 18 hours in low calcium medium. Rac activity was measured after calcium re-addition for the indicated times. Pulldows (PD) of active Rac were analyzed by WB for Rac and SUMO-1. Blots at the bottom of (h) represent part of the blot above showing the unmodified Rac-GTP band at lower exposure, as well as levels of total Rac. Quantification of the Rac-GTP bands, both SUMO-modified and unmodified, is depicted in the histogram normalized to total Rac. Rac-nS1 in (b-h) is multi-mono-SUMOylated Rac1. Uncropped images of blots are shown in Supplementary Information, Fig. S6.
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Figure 3: Rac1 is SUMOylated in vitro and in vivo. (a) GST-Rac1 protein was incubated at 37°C in vitro in the presence of complete SUMOylation assay components (E1, E2, SUMO-1 (S1) or SUMO-2 (S2)) and resolved by SDS-PAGE. Unmodified sample (Um) contains all the assay components but without incubation at 37°C. Rac1-S1/2 indicates Rac1 modified by SUMO-1 or -2. (b) GFP-Rac1 was immunoprecipitated from HeLa cells in the presence or absence of PIAS3 and in vitro SUMOylation assay was performed as in (a). Rac1 was detected by Western blot (WB). (c) Immunoprecipitated GFP-Rac1 or Rac1V12 was subjected to in vitro SUMOylation as in (a) and Rac1 was detected by WB. (d) HeLa cells transfected as indicated were lysed in the presence of NEM, and GFP was immunoprecipitated. The presence in immunoprecipitates of exogenous Rac1 or GFP was detected by WB analysis. IN, input. (e) GFP-SUMO-1 was immunoprecipitated from HeLa cells transfected as indicated, divided into two and incubated in the presence or absence of SENP1 for 2 hours at 25°C. (f) HeLa cells stably expressing 6His-SUMO-1 were transfected with Trp-Met and 6His-SUMO-1-modified proteins were purified from lysates. Endogenous Rac1-SUMO-1 was detected by WB. (g) MDCKII cells stably expressing scr shRNA or pias3 shRNA were treated as in Fig. 1f in the presence of the crosslinker DSS and HGF and Rac-GTP immunoprecipitation (IP) was performed. GTP-Rac-SUMO-1 bands were detected by WB. (h) MDCKII cells were grown to confluency and incubated for 18 hours in low calcium medium. Rac activity was measured after calcium re-addition for the indicated times. Pulldows (PD) of active Rac were analyzed by WB for Rac and SUMO-1. Blots at the bottom of (h) represent part of the blot above showing the unmodified Rac-GTP band at lower exposure, as well as levels of total Rac. Quantification of the Rac-GTP bands, both SUMO-modified and unmodified, is depicted in the histogram normalized to total Rac. Rac-nS1 in (b-h) is multi-mono-SUMOylated Rac1. Uncropped images of blots are shown in Supplementary Information, Fig. S6.
Mentions: In higher eukaryotes PIAS proteins act as SUMO E3-ligases10. SUMOylation is a covalent modification leading to the attachment of SUMO to specific lysine residues of target proteins14. In vitro SUMOylation followed by Western blotting revealed a band consistent with the attachment of SUMO-1 (S1) or SUMO-2/3 (S2/3) to Rac1, which was absent when E1, E2, or SUMO protein was not present (Fig. 3a). Furthermore, SUMOylation was enhanced by increasing the E2-conjugating enzyme Ubc9 (Supplementary Information, Fig. S2f). In most cases, SUMOylation of a substrate is enhanced by a SUMO E3-ligase10, 15. Rac1 SUMOylation was enhanced by increasing amounts of PIAS3 (Supplementary Information, Fig. S2g). Other PIAS family members also enhanced in vitro Rac1 SUMOylation, although PIAS3 was the most efficient (Supplementary Information, Fig. S2g). Additionally, in vitro SUMOylation of GFP-Rac1 immunoprecipitated from HeLa cells was enhanced upon co-transfection of FLAG-PIAS3 (Fig. 3b). Conversely, downregulation of PIAS3 decreased in vitro SUMOylation of GFP-Rac1 (Supplementary Information, Fig. S2h). These results indicate that PIAS3 works as a SUMO E3-ligase for Rac1. Furthermore, consistent with the finding that PIAS3 binds more efficiently to GTP-bound Rac1 (Fig. 1c; Supplementary Information, Fig. S1g), Rac1V12 was a better substrate for SUMOylation (Fig. 3c).

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