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

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.

To identify proteins regulating Rac1 in the context of cell migration, we performed tandem affinity purification (TAP) of native protein complexes containing TAP-tagged Rac1 from MDCKII cells following HGF treatment (a ligand for the growth factor receptor c-Met and a migration stimulus) at a time when Rac1 is active and cells begin to migrate4 (Supplementary Information, Fig. S1a-c). Following TAP, protein complexes were analyzed by mass spectrometry (MS) (Supplementary Information, Fig. S1d and Table I). Amongst known and putative Rac1 interactors, we identified PIAS3 (Protein Inhibitor of Activated STAT3) as a new Rac1-binding protein detected only in HGF-treated samples. PIAS3 interacts with Rac1 in vitro and in vivo (Fig 1a, b; Supplementary Information, Fig. S1e, f) and this interaction was more efficient when Rac1 was in its active form (Fig. 1c; Supplementary Information, Fig. S1g). In addition, activation of HGF signalling using an oncogenic form of the Met receptor (Trp-Met)5, 6 increased this interaction (Fig. 1d).

Figure 1

PIAS3 is a new Rac1-binding protein required for optimal Rac activation and cell migration in response to HGF. (a) GST protein alone or GST-Rac1 were incubated with cell extracts containing FLAG-PIAS3 and protein pulldowns (PD) were analyzed by Western blot (WB). (b) Epitope-tagged proteins were expressed in HEK293T cells and GFP immunoprecipitation (IP) performed. Purified proteins were detected by WB. IN, input. (c) GST-Rac1 beads divided in two samples were incubated with GDP or γGTP for 1h at 30°C and then with equal amounts of cell lysate containing FLAG-PIAS3 for 1 hour at 4°C. PIAS3 interaction was detected by WB. (d) HEK293T cells were transfected with the indicated plasmids and subjected to GFP immunoprecipitation. Samples were analyzed by WB. (e) Individual cells were tracked for at least 24 hours in the presence of HGF. Data shown is average speed of migration of cells from at least three independent experiments (at least 30 cells per experiment). *p<0.001, ** p<0.05, ns indicates no significant difference, two-tailed Student’s t-test. Values are means ± standard deviation. (f) MDCKII cells inducibly expressing pias3 or scr shRNAs were treated with doxycycline (dox), serum was then removed for 18 hours before cells were treated with HGF for 30 minutes and Rac activity measured. Bands were quantified and normalized intensities were calculated relative to control. Values are means ± standard deviation (n=3). (g) MDCKII cells inducibly expressing pias3 or scr shRNAs were treated as above but Rac activity was measured at later time points. (h) COS7 cells transfected with MYC-PIAS3 or empty vector were serum starved for 18 hours before 10 ng/ml of HGF was added for 30 minutes and Rac activity measured. (i) Bar chart shows quantification of the normalized relative amounts of Rac-GTP of (h) determined by scanning densitometry. Error bars indicate standard deviation for 3 independent experiments. ***p<0.001, two-tailed Student’s t-test. Uncropped images of blots are shown in Supplementary Information, Fig. S6.

Next we examined if PIAS3 was necessary for cell migration in response to HGF. We generated stable MDCKII cell lines carrying doxycycline-inducible short hairpin interfering RNAs (shRNAs) to PIAS3 (two independent targets – #1 and #2) or control scrambled shRNA (Scr) (Supplementary Information, Fig. S1h). Downregulation of PIAS3 resulted in cells migrating slower than controls (Fig. 1e) and impaired lamellipodia-membrane ruffle formation (Supplementary Information, Fig. S1i). From the reduced migration and lamellipodia formation, we reasoned that PIAS3 might be influencing Rac-GTP levels. Significantly, while Rac-GTP levels increased in control cells following HGF treatment, no increase was observed in PIAS3 downregulated cells, despite normal activation of the ERK MAPK pathway, indicating a specific requirement of PIAS3 for Rac activation (Fig. 1f). Consistent with this defect in Rac activation, phosphorylation (and hence activation) of p38, a known downstream mediator of Rac signalling7 was impaired in PIAS3 downregulated cells (Supplementary Information, Fig. S1j). HGF stimulates epithelial cell motility initially inducing spreading of cell colonies starting with lamellipodia/membrane ruffle formation, followed by disruption of cell-cell junctions and subsequent cell scattering; Rac1 is involved in both processes8. PIAS3 was required not only for optimal initial Rac activation following HGF-treatment, but also for optimal Rac activation at later time points (Fig. 1g). This continuous involvement of PIAS3 in Rac activation could explain the defect in migration observed throughout the 24 hours of HGF treatment. Rac-GTP levels were elevated in cells overexpressing PIAS3 and also treated with HGF compared to control transfected cells (Fig. 1h, i), confirming a positive role for PIAS3 in augmenting Rac-GTP levels following HGF treatment. In addition to HGF, the modest activation of Rac by EGF or serum was also impaired in PIAS3 downregulated cells (Supplementary Information, Fig. S1k and data not shown). This indicates that the requirement of PIAS3 for optimal Rac activation is not restricted to a particular upstream stimulus. However, downregulation of PIAS3 did not affect Cdc42-GTP levels (Supplementary Information, Fig. S1l). Collectively these data imply that PIAS3 is required for the induction or more likely the maintainenance of Rac activity (since the Rac-PIAS3 interaction is stronger for Rac-GTP over Rac-GDP or Rac1V12 over Rac1 WT) following growth factor treatment and in this way PIAS3 is required for optimal cell migration.

Most PIAS3 substrates reported so far are nuclear proteins, frequently transcription factors9, 10, although PIAS3 can also be detected in the cytoplasm. In contrast, Rac is mainly cytoplasmic, although nuclear Rac has been reported11, 12. To identify whether PIAS3 regulates Rac activity in the cytoplasm, we used a truncated version of PIAS3, CPIAS3, that is exclusively nuclear (Fig. 2a, b)13. In contrast to overexpression of full-length PIAS3, no increase in Rac-GTP levels occurred following CPIAS3 overexpression in response to HGF (Fig. 2c, d), even though Rac1 interacts with CPIAS3 in vitro and in vivo (Supplementary Information, Fig. S2a-c). Furthermore, PIAS3 co-localizes with Rac1 in the leading edge of cells and translocates from the cytosol to the membrane in response to HGF (Supplementary Information, Fig. S2d, e). Together, these results suggest that PIAS3 regulates Rac activity in the cytoplasm, and most likely at sites of membrane protrusion.

Figure 2

PIAS3 regulates Rac1 activity in the cytoplasm. (a) Schematic illustration of PIAS3 full-length and CPIAS3. (b) Representative images of PIAS3 and CPIAS3 localization in COS7 cells. Scale bar, 20 μm. (c) COS7 cells transfected with empty vector, MYC-PIAS3 or CPIAS3 were serum starved for 18 hours before 10 ng/ml of HGF was added for 30 minutes and Rac activity measured. (d) Bar chart shows quantification of the normalized relative amounts of Rac-GTP determined by scanning densitometry of (c). Error bars indicate standard deviation for 3 independent experiments. * p<0.05, ns indicates no significant difference, two-tailed Student’s t-test. Uncropped images of blots are shown in Supplementary Information, Fig. S6.

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).

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.

We next evaluated whether Rac1 could be SUMOylated in vivo. SUMOylation is reversed by the SUMO-specific proteases SENP1-3 and SENP5-716; therefore we added N-ethylmaleimide (NEM) to the lysis buffer to inhibit these SUMO-deconjugating enzymes17. This allowed detection of higher molecular weight Rac1 bands potentially representing SUMO-modified Rac1 from cells co-expressing 6-His-SUMO-1 and GFP-Rac1 (Supplementary Information, Fig. S2i). To further demonstrate in vivo SUMOylation of Rac1, TAP-Rac1 was expressed with GFP-SUMO-1 or a GFP-SUMO-1 mutant lacking the diglycine C-terminal motif required for its conjugation to substrates (SUMO-1ΔGG). Probing GFP immunoprecipitates for Rac revealed up to three bands in lysates from GFP-SUMO-1, but not GFP-SUMO-1ΔGG expressing cells (Fig. 3d). Further, incubating the above GFP immunoprecipitates with the active domain of SENP118 resulted in loss of the higher molecular weight bands (Fig. 3e), confirming that these represented SUMO conjugates. Since SUMO-1 is not thought to form polymeric chains in vivo19 the presence of multiple bands suggested that Rac1 could be SUMOylated at multiple sites.

Since PIAS3 was identified as a Rac interactor in response to HGF, we analyzed if activation of the Met-HGF pathway induced the appearance of endogenous Rac-SUMO-1 conjugates. Endogenous Rac-SUMO conjugates were visualized in HeLa cells stably expressing 6His-SUMO-120 only following transfection with Trp-Met (Fig. 3f). Furthermore, we detected co-localisation of endogenous SUMO-1 and endogenous Rac1 in membrane ruffles of HGF-treated MDCKII cells (Supplementary Information, Fig. S3a). To visualize SUMOylation of endogenous Rac1 by addition of HGF alone and to determine the role of PIAS3 in this process, MDCKII cells were treated with HGF in the presence of a chemical cross-linker (DSS) to preserve SUMO-conjugated Rac during immunoprecipitation with a Rac-GTP specific antibody or CRIB-pull-down of GTP-Rac. Endogenous GTP-Rac1-SUMO-1 was detected in control cells following HGF treatment, but not in PIAS3 downregulated cells (Fig. 3g; Supplementary Information, Fig. S3b). We next determined whether SUMOylation of endogenous Rac occurs under other conditions that induce Rac activation. Calcium-switch stimulates cell-cell adhesion in confluent epithelial cells and Rac1 activation21. Following calcium re-addition and endogenous Rac-GTP pull-down we detected endogenous Rac-SUMO conjugates (Fig. 3h). Quantitation of SUMO-conjugated and unmodified Rac-GTP indicated that the SUMO-modified fraction is a significant proportion of Rac-GTP (histogram, Fig. 3h).

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).

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.

Replacement of the putative SUMOylation lysines within the PBR with arginines (same charge) abolished most of the Rac1 SUMOylation in vitro (Supplementary Information, Fig. S3c-e). Moreover, lysines 96, 123, 130, 132, and 133 were not confirmed as SUMO-sites (Supplementary Information, Fig. S3f and data not shown). Interestingly, the in vitro SUMOylation assay showed one SUMO-Rac1 band, however, MS analysis identified more than one lysine modified by SUMO-1. This suggested that SUMOylation can occur on any of the lysines within the PBR. The monomeric SUMOylation of Rac1 in vitro is in contrast to the multiple SUMO-conjugate bands detected in vivo, presumably resulting from multi-monoSUMOylation. This difference implies the existence of additional factors in vivo that support multi-monoSUMOylation.

To confirm that the lysines present in the PBR were SUMOylated in vivo we generated a GFP-Rac1 mutant in which the four lysines of the PBR were replaced with arginines (GFP-Rac1ΔSUMO) (Fig. 4c). The Rac1ΔSUMO mutation was sufficient to decrease Rac1 SUMOylation (Fig. 4d) but not ubiquitylation (Fig. 4e; Supplementary Information, Fig. S3g). The PBR of Rac1 is implicated in binding certain effectors25-27, as well as plasma membrane and nuclear localization11, 28, 29. Rac1ΔSUMO mutation did not disrupt membrane or nuclear localization of Rac (Supplementary Information, Fig. S4a-c), nor did it affect Rac interaction with PIAS3, the effector IQGAP, or the Guanine nucleotide Exchange Factors (GEFs) β-Pix and Tiam1 (Supplementary Information, Fig. S4d-g).

We next investigated the connection between Rac1-GTP levels and SUMO modification. Interestingly, GFP-Rac1ΔSUMO was less GTP-bound than wild-type (Fig. 4f), suggesting that Rac SUMOylation was required to increase or maintain part of its activity. Consistent with this, a Rac1 chimaera mimicking constitutive SUMOylation (GFP-SUMO-1-Rac1; Supplementary Information, Fig. S4h) was more GTP-bound than wild-type Rac1 (Fig. 4g). Moreover, GFP-SUMO-1-Rac1 was localized similarly to wild-type GFP-Rac1 (Supplementary Information, Fig. S4i, j). These results suggest that Rac1 SUMOylation was not necessary for its localization but was required for its optimal GTP loading.

Subsequently, we addressed whether the defect in activation of non-SUMOylated mutant Rac affected its ability to promote cell migration. Rac1-null mouse embryonic fibroblasts (MEFs) derived from Rac1 conditional knock-out mice are unable to form lamellipodia-membrane ruffles and are defective in cell migration (Supplementary Information, Fig. S5a and30). We detected a higher molecular weight Rac1 species in these cells potentially corresponding to Rac1-SUMO-1 (Supplementary Information, Fig. S5b). Rac1-depleted MEFs were transfected with GFP, GFP-Rac1 WT or GFP-Rac1ΔSUMO (Fig. 5a) and GFP positive cells analyzed for the presence of lamellipodia-membrane ruffles (Fig. 5b). Whereas Rac1 WT rescued the lamellipodia-ruffle formation to around 85% of wild-type levels, non-SUMOylated Rac1 restored it to only 45% (Fig. 5c). Moreover, unlike Rac1 WT, the ΔSUMO mutant was unable to rescue significantly the migration defect of Rac1-depleted cells (Fig. 5d; Supplementary Information, Fig. S5c), or their invasion in response to HGF (Fig. 5e; see also Supplementary Information, Fig. S5d where the induction of invasion by HGF is demonstrated). However, the ΔSUMO mutant was equally competent as Rac1 WT to rescue the clonogenic defect of Rac1-depleted MEFs (Supplementary Information, Fig. S5e, f).

Figure 5

SUMOylation of the polybasic region is required for optimal cell migration and invasion. (a) Representative Western blot (WB) showing expression levels of GFP-Rac1 WT or GFP-Rac1ΔSUMO in Rac1-depleted cells (k/k). c/c represents Rac1-WT MEFs. (b) Representative images of Rac1-depleted MEFs (k/k) reconstituted with GFP-Rac1 WT or GFP-Rac1ΔSUMO. Images were taken 24 hours later. Scale bar, 20 μm. (c) Fraction of k/k cells expressing the indicated constructs with lamellipodia-membrane ruffles 24 hours after transfection. Error bars indicate standard deviation based on n = 110-150 cells from 3 independent experiments. (d) Cells as in (a) grown on glass bottom dishes were tracked for at least 24 hours. Box-whisker plots of cell velocities of n = 90-100 cells from three independent experiments are shown. (e) Cells as in (a) were analyzed for invasion at 40 μm in the presence of HGF. Error bars indicate standard deviation based on n=5 wells. A representative from 3 independent experiments is shown. (f) Representative WB showing expression levels of GFP-Rac1 WT in Rac1-depleted cells (k/k) also stably expressing scr shRNA or pias3 shRNA. Please note lower levels of expression of exogenously expressed GFP-Rac1 WT compared to endogenous levels of Rac1 of control c/c MEFs. (g) Individual cells as in (f) were tracked for 24 hours. Data shown is average speed of migration of at least 50 cells per experiment. A representative experiment is shown here. (c-e, g) *p<0.001, ** P<0.05, ns indicates no significant difference, two-tailed Student’s t-test. Uncropped images of blots are shown in Supplementary Information, Fig. S6.

Similarly to MDCKII, PIAS3 downregulation in MEFs resulted in reduced migration and membrane ruffle formation (Supplementary Information, Fig. S5g and data not shown). To determine whether PIAS3 was required for the ability of Rac1 WT to rescue cell migration in Rac1-depleted cells, PIAS3 was downregulated and cells then infected with GFP-Rac1 WT (Fig. 5f) before being analyzed for migration and membrane ruffle formation. Significantly, while Rac1 WT partly restored the migration and membrane ruffling of Rac1-depleted cells additionally expressing control shRNA, it was unable to do so in cells expressing shRNA targeting PIAS3 (Fig. 5g; Supplementary Information, Fig. S5h). These results suggest that PIAS3, likely through promoting Rac SUMOylation, is to a great extent responsible for the ability of Rac1 WT to rescue the phenotype of the Rac1-depleted cells. However, it can not be excluded that PIAS3 exerts some Rac-independent effects on cell motility.

Our study demonstrates that Rac1, and particularly GTP-bound Rac, can be SUMOylated. While SUMOylated-Rac represents a small fraction of total Rac, it can be a significant fraction of GTP-bound Rac (Fig. 3g, h). However, since not all Rac-GTP is SUMOylated (Fig. 3g, h), since Rac1ΔSUMO retained some GTP-binding (Fig. 4f), because PIAS3 interacts more with Rac-GTP than Rac-GDP (Fig. 1c), and also because active Rac1 is a better SUMOylation substrate than Rac1 WT (Fig. 3c), our data imply that SUMOylation is not essential for activation, but rather influences the maintenance of the active state. Consistent with this, levels of active Rac1 following HGF treatment were reduced by downregulation of PIAS3 (Fig. 1f and 3g), and augmented by PIAS3 overexpression (Fig. 1h, i and 2c, d), mirroring the effects of PIAS3 on levels of Rac SUMOylation (Figs 3b, g; Supplementary Information, Fig. S2h, S3b). Further, a SUMO-mimicking Rac1 chimaera was more GTP-bound than Rac1 WT (Fig. 4g). Potentially, Rac SUMOylation could either increase its binding to a GEF or conversely decrease binding to a GTPase activating protein (although such effects need to be demonstrated; for model see Supplementary Information, Fig. S5i). Significantly, we show that Rac SUMOylation is important for optimal lamellipodia-ruffle formation, cell migration and invasion (Fig. 5b-e), but not for clonogenicity (Supplementary Information, Fig. S5e, f). This conclusion is strengthened by the observation that PIAS3 is required for Rac1 WT to restore membrane ruffling and migration in Rac1-depleted MEFs (Fig. 5g). SUMOylation may possibly increase active Rac levels above a threshold required for lamellipodia formation, cell migration and invasion and/or target a pool of Rac towards particular biological functions through influencing binding to effector molecules, consistent with findings that SUMOylation of other proteins influences their interactions31. Overall, our study advances our understanding of how GTPases and in turn cell migration are regulated and could provide new targets for future therapeutic intervention.