Materials and Methods s18

SUPPLEMENTARY LEGENDS

Table I Putative Rac1-binding proteins identified in response to HGF. The total spectral count and protein accession numbers of Rac1-binding proteins from one representative experiment are shown. Only proteins present in HGF-treated samples from three independent experiments are listed. (a) The spectral count for Rac1 was similar in the presence or absence of HGF. (b) Known Rac1 interacting proteins identified in both conditions, but spectral count was greater in the presence of HGF.

Figure S1 Generation of TAP-Rac1 cells, verification of PIAS3 as a Rac1 interacting protein, and effects of PIAS3 downregulation. (a) Schematic illustration of N-terminal TAP tag. (b) Expression levels of TAP-Rac1 and N-TAP in cell lines used for screening. (c) MDCKII cells stably expressing TAP-Rac1 were stimulated with HGF for the indicated times. Levels of activated endogenous (top panel) and exogenous (bottom panel) Rac1 were determined simultaneously from the same cell extracts. (d) Coomassie stained gel after TAP- Rac1 purification used for mass spectrometry. M, Marker. (e) GST or GST-PIAS3 were incubated with cell extracts containing TAP-Rac1. Protein pulldowns (PD) were analyzed by Western blot (WB). CBB, Coomassie Brilliant Blue. (f) Epitope-tagged proteins were expressed in HEK293Ts and FLAG immunoprecipitation performed. Purified proteins were detected by WB. IN, input. * antibody heavy chain. (g) GST-PIAS3 was incubated with lysates from HEK293T cells expressing equivalent levels of either TAP-Rac1 or TAP- Rac1V12 and levels of Rac purified by PD were determined by WB. (h) MDCKII cells inducibly expressing shRNAs against PIAS3 (RNAi #1, RNAi #2) or a scrambled control shRNA (Scr) were treated with doxycycline (dox) where indicated. Cell lysates were probed for PIAS3. Tubulin serves as a loading control. (i) Quantification of lamellipodia-membrane ruffle formation in cells described in (h) after HGF treatment. Error bars indicate standard deviation based on n>300 cells in 3 different experiments. * p<0.05, two-tailed Student’s t- test. (j) MDCKII cells inducibly expressing an shRNA against PIAS3 (RNAi #1) were treated with dox where indicated. Cell lysates were probed for p-p38, total p38 or PIAS3. (k) MDCKII cells, as in (j), were treated with dox where indicated, serum starved and treated with EGF for 30 min and Rac activity measured. Bands were quantified and normalized intensities were calculated relative to control. (l) MDCKII cells, as in (j), were treated as in (k) but with HGF and Cdc42 activity measured.

1 Figure S2 The C-terminus of PIAS3 interacts with Rac. PIAS3 is a SUMO-E3 ligase for Rac and co-localizes with Rac at the leading edge. (a) Schematic illustration of PIAS3 full- length, NPIAS3 and CPIAS3. (b) GST-PIAS3 and GST-N/CPIAS3 were incubated with cell extracts containing Rac1 and protein pulldowns (PDs) were analyzed by Western blot (WB). (c) HEK293T cells were transfected with the indicated plasmids and subjected to GFP immunoprecipitation. Samples were analyzed by WB. IN, input. (d) COS7 cells were transfected with MYC-PIAS3, serum starved and treated with HGF for 30 min. Cells were fixed and stained for MYC and endogenous Rac. Scale bar, 20 µm. (e) Fractionation of MDCKII cells in the presence or absence of HGF for 30 min. C and M refer to cytosol and membrane fractions respectively. -catenin and GAPDH are controls for the purity of membrane and cytosolic fractions respectively. (f) GST-Rac1 protein was incubated at 37oC with E1, S1 and an increasing amount of Ubc9 (E2). Rac1 was detected by WB. (g) GST- Rac1 protein was incubated with E1, E2, and S1 together with increasing amounts of recombinant members of the PIAS family or RanBP2 (another SUMO E3-ligase). Rac1 was detected by WB. (h) GFP-Rac1 was immunoprecipitated from HeLa cells in the presence or absence of pias3 shRNA and in vitro SUMOylation performed as in (f). Rac1 was detected by WB. (i) HeLa cells stably expressing 6His-SUMO-1 were transfected with either GFP or GFP-Rac1. Cells were lysed in the presence of NEM and GFP was immunoprecipitated followed by WB for Rac. GFP-Rac1 immunoprecipitates in the presence of NEM show the presence of three specific Rac1 bands corresponding to SUMOylated Rac. Rac1-nS1 indicates Rac1 modified by SUMO-1.

Figure S3 SUMO-1 and Rac co-localise at the leading edge. Rac is SUMOylated in its polybasic region (PBR). (a) Representative images of MDCKII cells in the presence or absence of HGF stained for endogenous Rac and SUMO-1 proteins. Scale bars, 15 µm. (b) MDCKII cells stably expressing control (scr) shRNA or pias3 shRNA were treated with HGF in the presence of DSS for 30 min and subjected to pulldown (PD) of the active form of Rac (GTP-Rac). Samples were analyzed by Western blot (WB). (c) Putative Rac1 SUMOylation sites and the mutations created. (d) GST-Rac1 and GST-Rac1 with mutations in the PBR were subjected to in vitro SUMOylation assay. Modified proteins were detected with a Rac specific antibody. (e) Quantification of intensities of Rac1-SUMO-1 bands from (d). (f) GST-Rac1 and GST-Rac1 with mutations in some other lysines identified by mass

2 spectrometry were subjected to in vitro SUMOylation assay. Modified proteins were detected with a Rac specific antibody. (g) HeLa cells were transfected with the indicated plasmids and 6His-Ubiquitin proteins were purified from lysates. GFP-Rac1-nUb was detected by WB and indicates Rac1 modified by Ubiquitin.

Figure S4 Mutation of the Rac1 SUMOylation sites does not affect known functions of the polybasic region of Rac. (a) Representative images of COS7 cells transfected with the indicated plasmids. Note the presence of more membrane ruffles in the Rac1 WT compared with the SUMO mutant (white arrowheads). Scale bar, 10 µm. (b) Fractionation of COS7 cells transfected with TAP-Rac1 WT or TAP-Rac1ΔSUMO. C and M refer to cytosolic and membrane fractions respectively. -catenin and RhoGDI serve as a control for the membrane and cytosolic fractions respectively. (c) Percentage of COS7 cells expressing the indicated plasmids with nuclear or cytoplasmic localization. Error bars indicate standard deviation based on n>300 cells from 3 different experiments. (d-g) HeLa cells were transfected with the indicated plasmids. GFP immunprecipitations and inputs (IN) were probed with the indicated antibodies. (h) Schematic illustration of GFP-Rac1 and the chimaeric GFP-SUMO- 1-Rac1. (i) Representative images of COS7 cells transfected with the indicated plasmids. Scale bar, 20 µm. (j) Fraction of COS7 cells expressing the indicated plasmids with cytoplasmic localization. Error bars indicate standard deviation based on n>200 cells from 3 different experiments. ns indicates no significant difference.

Figure S5 Rac depletion impairs membrane ruffle formation, cell migration and invasion. SUMOylation of Rac1 is required for optimal motility, but not cell proliferation. (a) Representative images of conditional mouse embryonic fibroblasts (MEFs) before (c/c) and after Rac1 depletion (k/k). Cells were stained for actin. Scale bars, 20 µm. (b) MEFs as in (a) were lysed in normal lysis buffer or in 8M Urea (denaturing conditions). Rac1 was detected by Western blot (WB) using a specific Rac1 antibody. (c) Migration tracks from 10 random cells from Fig. 5d of the main text. (d) c/c and k/k MEFs (k/k) were analyzed for invasion at 40 μm in the presence or absence of HGF. Error bars indicate standard deviation based on n=5 wells. A representative from 3 independent experiments is shown. * p<0.05, two-tailed Student’s t-test. (e) Colony formation of cells as in Fig. 5a of the main text at day 10 after infection and selection. Images are representative of at least three independent experiments. (f) Quantitation of (e) by crystal violet absorbance at 590nm. Error bars

3 indicate standard deviation for 3 independent experiments. *p<0.001, two-tailed Student’s t- test. (g) Average speed of migration of c/c MEFs (at least 100 cells) expressing either scr or pias3 shRNA tracked for 24 hours is shown. Error bars indicate ±SEM. *p<0.001, two-tailed Student’s t-test. (h) Quantification of lamellipodia-membrane ruffle formation of k/k cells stably expressing control shRNA (scr) or pias3 shRNA following rescue with the GFP-Rac1 WT construct for 24 hours. Cells were subsequently treated with HGF. Error bars indicate ±SEM based on n>200 cells from 2 different experiments. (i) Model. HGF induces Rac1 activation through the action of GEFs; GAPs return Rac1 to its inactive state. With PIAS3 present and WT Rac1, GTP-loading stimulates binding between Rac and PIAS3 and SUMOylation of Rac. Consequently, Rac1 remains longer in its GTP-bound form (perhaps because it is localized by SUMO-1 to an environment free from GAPs or because SUMO-1 antagonizes GAP binding). Thus, SUMOylation results in stabilization of a pool of GTP- bound Rac1 which promotes membrane protrusion and cell migration. SENPs deSUMOylate Rac1, so that it becomes a substrate once more for GAPs.

Figure S6 Uncropped image blots.