Supplementary Information
Supplementary Materials and Methods
Constructs
N-terminal TAP tag (N-TAP) cloned in pcDNA4/TO (Invitrogen) was a gift from
Prof. Clevers. Mouse full-length Rac1V12 was cloned in frame with the N-TAP (to
yield TAP-Rac1V12). The full-length construct was used as a template to mutate V12
to G12 using the QuickChange site-directed mutagenesis kit (Stratagene) according to
the manufacturer’s instructions. These constructs were used for stable expression in
mammalian cells. The inducible mammalian expression constructs encoding wildtype HA-HACE1 or HA-HACE1C876S were obtained by subcloning HA-HACE1
from pMSCVhyg-HA-HACE1 (1) into pCDNA4/TO vector. The HACE1∆HECT and
HACE1∆ANK were provided by Dr Sorensen. The Tpr-Met plasmid was provided by
Dr Trusolino. For the HACE1 protein purification from insect cells, full-length
HACE1 was cloned in pFastBac-CHis-TOPO (Invitrogen). GST-Rac1WT and GSTRac1Q61L constructs were provided by Dr Sánchez-Madrid. GST-Rac1Q61LK147R
was derived from GST-Rac1Q61L using QuickChange site direct mutagenesis kit
(Stratagene) according to the manufacturer’s instructions. GFP-HACE1, GFPRac1WT, GFP-Rac1V12 and GFP-Rac1K147R were obtained by cloning HACE1,
Rac1WT, Rac1V12 or Rac1K147R respectively into pEGFP-C1 (Clontech) and
subsequently subcloned into pLZRS-IRES-Neo vector. The mutant MYCRac1K147R was obtained using the QuickChange site-directed mutagenesis kit
(Stratagene) according to the manufacturer’s instructions. The full-length MYC-Rac1
construct was used as a template to mutate K147 to R147. pMT1076xHis-ub
expression vector has been described elsewhere (2). The Halo-Rac1 construct was
1
obtained by cloning Rac1 into pFN21A HaloTag® CMV Flexi® Vector (Promega,
Cat. # G282A). The MDCKII inducible HACE1 system was generated by cotransfecting pCDNA4/TO-HA-HACE1 or HACE1C867S mutant with pCDNA6/TR.
HACE1 specific double stranded short hairpin DNA oligos N1 and N2 have been
described elsewhere (3) and were obtained from Sigma-Aldrich. The Scr, N1 and N2
shRNA constructs were cloned in the pLKO.1-puro vector. The siRNA
oligonucleotides targeting Rac1 were manufactured by Dharmacon and transfected
into cells using DharmaFECT1 transfection reagent (Dharmacon) according to the
manufacturer’s instructions. The target sequence of oligos were as follows: Nontarget control: Scr siRNA (D-001210-02), On-target plus siRNA Rac1 #1 (J-00356014), #2 (J-003560-15).
Cell Culture and Cell Lines
HEK293T, MDCKII, and COS7 cells were maintained in Dulbecco's Modified Eagle
Medium (DMEM; Invitrogen) in the presence of 10% fetal bovine serum (FBS;
GIBCO). HACE1-null MEFs (KOHACE1) or control cells (WT) and MEFs WT or
KO for Rac1 (a kind gift from Dr Vidali), were maintained in the same medium
containing 70μM β-mercaptoethanol. Deletion of Rac1 was induced by incubating
cells with Ad5CMCre-eGFP (Gene Transfer Vector Core; University of Iowa). Cell
lines expressing the inducible system were maintained in DMEM supplemented with
10% tetracycline-free FBS (Autogen Bioclear). HEK293Ts were transiently
transfected using LT1 transfection reagent (Mirus). For growth factor treatment,
serum was removed for 16 hours before cells were stimulated with 10ng/ml HGF (R
& D Systems) for the times indicated. For the MDCKII HACE1 inducible system,
cells were treated with 1 μg/ml doxycycline (dox) at the same time that serum was
2
removed for 16 hours. For the downregulation of HACE1, HEK293T cells were
transiently transfected for 72 hours before samples were harvested. Human
fibronectin plates (354402, BD Biocoat) were used for the fibronectin seeding
experiments. For cycloheximide (CHX) experiments 100 µg/ml final concentration of
CHX (SIGMA C4859) was used.
TAP purification
TAP purification assays were performed as previously described (4).
GST (glutathione S-transferase) pull-down assays
GST-pull-down assays were performed as previously described (5).
Immunoprecipitation under denaturing conditions
Immunoprecipitation under denaturing conditions were performed as previously
described (6).
Insect cell protein purification
Wild-type HACE1 was expressed as a poly-His-fusion protein in Sf9 insect cells
following the protocol supplied from Bac-to-Bac® TOPO® expression system
(Invitrogen). Insect cells were pelleted and lysed in Buffer A containing 50mM TrisHCl pH8.0, 5mM MgCl2, 300mM NaCl, 1mM ß-mercaptoethanol, 1% Triton-X,
5mM Imidazole, 10% Glycerol and protease inhibitor cocktail (Roche). Lysates were
incubated on ice for 30 minutes prior to sonication at 5 amplitude micron for 6x 5s
and clarification at 25,000g for 20 minutes at 4°C. HACE1 recombinant protein was
then purified using Ni2+-NTA agarose beads (Qiagen) following the protocol provided
3
by the supplier. Protein was eluted with Buffer A containing 100mM Imidazole and
subjected to desalting and buffer exchange using PD-10 column (GE Healthcare) to a
final storage buffer containing 50mM Tris-HCl, pH7.5, 150mM NaCl, 5mM MgCl2,
1mM ß-mercaptoethanol and 10% Glycerol.
Histidine pull down using nickel affinity purification
6His-Ubiquitin binding proteins were purified from cell lysates by nickel affinity
chromatography using Dynabeads His-Tag (Invitrogen) beads. Cells were pelleted
and lysed in 1 ml 8M Urea buffer containing 0.3% SDS in PBS. Lysates were
sonicated 2-3 minutes using a bioruptor at the highest level (20 amplitude micron) and
clarified at 13K rpm for 20 minutes at 20oC. Lysates were incubated with 100µl of
beads for 2 hours at room temperature. Protein was eluted with SDS loading buffer
containing 20mM EDTA.
Fluorescent labelling of Halo-tagged Rac1 protein
MDCKII cells were transfected with pFN21A-Halo-Rac1WT or –Halo-Rac1V12
expression vectors and 24 hours later cells were labelled with 50 nM of fluorescent
HaloTag® TMR Ligand (Promega, Cat. # G8252) in Leibovitz media (Invitrogen) for
10 minutes. Cells were then washed 3 times with fresh Leibovitz media to remove
unbound ligands before being visualized by live-cell imaging on a Deltavision
microscope.
Membrane staining
MEFs WT or KOHACE1 were stained with CellMask Deep Red plasma membrane
stain (Invitrogen, cat. # C10046) for 5 min in Leibovitz media at 2.5 µg/ml final
4
concentration and washed 3 times in Leibovitz media before being visualized by livecell imaging on a Deltavision microscope.
Quantification of GFP-Rac1 signal at the cell leading edge
Live-cell imaging of WT, KOHACE1 or KORac1 MEFs expressing GFP-Rac1WT or
GFP-Rac1K147R was performed on a Deltavision microscope using 100x oil lens. Zstacks of 0.2µm per section were taken for a total height of 7 µm for each cell, which
were then converted into maximal projections using Imaris software. ImageJ was used
to quantify GFP-Rac1 signal at the cell edge. Cell border was first selected by using
Wand Tool on ImageJ with setting set to Legacy and Tolerance value set to 20.0. A
band of 30 pixels-wide within the cell was then made from this selected cell border.
Average intensity of this region was calculated by measuring the Mean Gray Value.
This value was then subtracted with background value which was obtained by
measuring the Mean Gray Value of the band of 30 pixels-wide outside the cell. At
least 30 cells were used for the quantification from 3 independent experiments (10
cells each) and the average values were plotted on graph and independent t-test was
performed using SPSS.
Quantification of Halo-tagged Rac1V12 signal at membrane ruffles.
Live-cell imaging of MDCKII cells expressing Halo-Rac1V12 and GFP-HACE1 or
Halo-Rac1V12 and GFP alone was performed on a Deltavision microscope as above.
Z-stacks of 0.2µm per section for a total height of 10µm, were taken for each cell for
every minute for 4 minutes total. Z-stacks were then converted into maximal
projection on Imaris software. ImageJ was used to quantify Halo-Rac1V12 signal at
the membrane ruffles by manually selecting the ruffle regions and then analyzed using
5
‘Intensity vs Time Monitor’ function. Average Intensity values of Halo-Rac1V12
signals for different time points were normalized to 0 min time point which was set as
1. At least 30 cells were quantified from 3 independent experiments (10 cells per
experiment).
Antibodies
Immunoblotting and immunofluorescence were performed as described previously
(5). Immunoblotting was preformed using the following primary antibodies: anti-Myc
(Clone 9E10, CRUK, 1:5000), anti-HA (ab9110, abcam, 1:5000), anti-Rac1 (clone
102, BD Biosciences, 1:1000), anti-Rho GDI (Clone A-20, Santa Cruz, 1:5000), antiß-catenin (610154, BD Biosciences), anti-HACE1 (sab2101010, SIGMA 1:1000),
anti-PAP (p 1291, SIGMA, 1:5000), anti-c-Met (Clone C-12, Santa Cruz, 1:5000),
anti-caveolin-1 (610493, BD Biosciences), anti-Tubulin (Clone TAT-1, CRUK,
1:10,000), anti-GFP (ab290 Abcam, 1:5000), anti-6His (631212, Clontech, 1:10000),
anti-ubiquitin (clone P4D1 Enzo Life Sciences), anti-ERK and anti-p-ERK (Cell
Signaling, 1:2000) and subsequently with horseradish peroxidise-conjugated antimouse, anti-rabbit or anti-sheep secondary antibodies (GE Healthcare, 1:5000) and
visualised by enhanced chemiluminescence (Perkin Elmer).
Subcellular fractionation
Fractionation assays were performed as previously described (4).
In vitro Ubiquitylation assay
In vitro ubiquitylation assay was performed as previously described (7) with the
following modifications: the in vitro reaction was reconstituted using 1.75µg E1
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activating enzyme UBE1 (Ubiquigent), 2µg E2 conjugating enzyme UbcH7/Ube2L3
(Ubiquigent), 0.8µg HACE1 or HACE1C876S, 0.5µg wild-type Rac1 or its mutants
(Q61L and Q61L K147), 20µg bovine ubiquitin (Sigma) in a reaction buffer of final
volume 100µl containing 50mM Tris-HCl, pH7.5, 5mM MgCl2 and 5mM DTT and
3mM ATP. The reaction was incubated at 37 °C for 2 hours and then subjected to
GST-pull down using Gluthathione Sepharose beads (GE Healthcare) at 4°C for 1
hour in a buffer containing 50mM Tris-HCl, pH7.5, 150mM NaCl, 25mM NaF, 2mM
NaH2PO4, 2mM EDTA, 1% Triton-X and 10mM N-Ethyl Maleimide. Proteins were
eluted with sample buffer and heated at 70°C for 10 min prior to SDS PAGE and
Western blotting.
Rac1 activity
Rac1 activity assays were performed as previously described (4).
Cell Migration Assay
Cells were seeded sparsely and images were acquired every 30 minutes for 24 hours
at 37oC using a time-lapse system consisting of a Zeiss Axiovert 200M microscope
under the control of Metamorph software (Molecular Devices, PA, USA). Single cells
were manually tracked using ImageJ software (NIH).
Systematic identification of proteins from Tandem Affinity Purified complexes
by LC-MS/MS analysis and database searches.
Gel lanes from 1D gels (Nupage 4−12%) were manually cut into 40 × 1.5 mm bands
using a razor blade. Each band was placed in a well of a 96-well plate (PRO10003,
Genomic Solutions), and gel bands were processed as previously described (4).
7
Supplementary References
1 Anglesio MS, Evdokimova V, Melnyk N, Zhang L, Fernandez CV, Grundy PE et
al. Differential expression of a novel ankyrin containing E3 ubiquitin-protein ligase,
Hace1, in sporadic Wilms' tumor versus normal kidney. Hum Mol Genet 2004; 13:
2061-2074.
2 Mendez J, Stillman B. Chromatin association of human origin recognition complex,
cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of
prereplication complexes in late mitosis. Mol Cell Biol 2000; 20: 8602-8612.
3 Zhang L, Anglesio MS, O'Sullivan M, Zhang F, Yang G, Sarao R et al. The E3
ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple
cancers. Nat Med 2007; 13: 1060-1069.
4 Castillo-Lluva S, Tatham MH, Jones RC, Jaffray EG, Edmondson RD, Hay RT et
al. SUMOylation of the GTPase Rac1 is required for optimal cell migration. Nat Cell
Biol 2010; 12: 1078-1085.
5 Woodcock SA, Rooney C, Liontos M, Connolly Y, Zoumpourlis V, Whetton AD et
al. SRC-induced disassembly of adherens junctions requires localized
phosphorylation and degradation of the rac activator tiam1. Mol Cell 2009; 33: 639653.
6 Jaffray EG, Hay RT. Detection of modification by ubiquitin-like proteins. Methods
2006; 38: 35-38.
7 Wang HR, Zhang Y, Ozdamar B, Ogunjimi AA, Alexandrova E, Thomsen GH et al.
Regulation of cell polarity and protrusion formation by targeting RhoA for
degradation. Science 2003; 302: 1775-1779.
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Supplementary Figure Legends
Table I
List of putative Rac1-binding proteins identified by mass spectrometry potentially
enriched in HGF-treated samples.
Figure S1
(A, B) HEK293T cells were transfected with the indicated plasmids and subjected to
GFP or HA inmunoprecipitation. Samples were analyzed by WB. (C) HEK293T cells
were
transfected
with
the
indicated
plasmids
and
subjected
to
GFP
inmunoprecipitation. Samples were analyzed by WB. HA-HACE1 bands were
quantified
and
normalized
intensities
were
calculated
relative
to
the
immunoprecipitated GFP-Rac1 bands (values indicated beneath lanes). (D) HEK293T
cells were transfected with the indicated plasmids and GFP was immunoprecipitated
from lysates made in denaturing conditions to disrupt any non-covalent protein
complexes. GFP-Rac1-nUb was detected by WB and indicates Rac1 modified by
ubiquitin. GFP-Rac1, GFP, HA-HACE1 and His-ubiquitin were detected by WB.
Figure S2
(A, B) MDCKII cells were serum starved and either treated with dox in parallel (+
dox) for 16 hours to induce overexpression of HACE1 or the HACE1C876S mutant
or left uninduced (- dox). Cells were subsequently treated with cycloheximide and
where indicated HGF (10 ng/ml) for 30 minutes. Proteins were detected by WB. Bar
charts show quantification of the normalized relative amounts of total Rac1
determined by scanning densitometry. Error bars indicate standard deviation for 4
9
independent experiments. *P<0.05, two-tailed Student’s t-test. ns indicates no
significant difference. (C) HEK293T cells were transfected with Scr or HACE1 (N2)
shRNA for 3 days. Proteins were detected by WB. Rac1 bands were quantified and
normalized intensities were calculated relative to Tubulin (values indicated beneath
lanes).
Figure S3
(A, B) WB showing expression levels of Rac1, Cdc42 and RhoA in HACE1-null
(KOHACE1) or control (WT) MEFs. * indicates non-specific band. (C) Individual
KOHACE1, WT or KOHACE1 MEFs reconstituted with HA-HACE1 were tracked
for at least 24 hours. Data shown is average speed of migration of n> 300 cells from
at least three independent experiments. * P<0.05, two-tailed Student’s t-test. Values
are means ± standard deviation. (D) Representative images of KOHACE1 MEFs or
those reconstituted with HA-HACE1 seeded on plastic for 16 hours. * indicates
accumulation of actin and Rac1 at the leading edge of migrating cells. Scale bar, 20
µm. (E) Lysates of WT and KOHACE1 MEFs transfected with Scr or Rac1 siRNA
were analyzed for protein levels of Rac1 by WB. (F) Individual KOHACE1 MEFs
transfected with Scr or two different siRNA oligos for Rac1 (#1, #2) were tracked for
24 hours. Data shown is average speed of migration of cells from at least three
independent experiments. * P<0.05, two-tailed Student’s t-test. Values are means ±
standard deviation. (G) WT or KOHACE1 MEFs expressing GFP-Rac1 were stained
with a plasma membrane dye (see methods above) and live-cell imaging was
performed. Arrows indicate localisation of GFP-Rac1, whereas arrowheads indicate
membrane staining. (H) WT or KOHACE1 MEFs were serum starved for 16 hours
before cells were treated with HGF for 5 minutes and Rac1 activity measured. Rac1-
10
GTP bands were quantified and normalized intensities were calculated relative to lane
1. Error bars represent standard error mean from 3 independent experiments. (I)
Migration tracks from 10 random cells from experiment in Figure 3F. (J) HEK293T
cells were transfected with the indicated plasmids and subjected to GFP
immunoprecipitation. Samples were analyzed by WB. * Background band.
Figure S4
(A) In vitro ubiquitylation assay. GST-Rac1Q61L or the mutant GSTRac1Q61LK147R proteins were incubated at 37ºC for 2 hours in the presence of
complete ubiquitylation reaction components (E1, E2, ubiquitin, HACE1 or
HACE1C876S mutant, ATP) and then subjected to GST-pull down. Rac1ubiquitylated proteins were resolved by SDS-PAGE. Ubiquitin or GST were detected
by WB. (B) HEK293T cells were transfected with the indicated plasmids and TAPRac1 activity measured. (C) Individual cells with the indicated genotypes were
tracked for 24 hours. Data shown is the average speed of migration of cells based on
n>300 cells from 3 different experiments. Error bars indicate standard deviation. *
P<0.05, two-tailed Student’s t-test. (D) MDCKII cells expressing Halo-Rac1V12 and
GFP or Halo-Rac1V12 and GFP-HACE1 were labelled with fluorescent TMR ligand
and visualized live for the indicated times. Only Halo-Rac1V12 signals at membrane
ruffles, but not those at cell-cell adhesion, were analyzed. Shown are the maximal
projections of representative cells. Scale bar, 15 µm. Boxed areas are shown beneath
whole cell images and depict regions where prominent membrane ruffling was
observed. (E) Halo-Rac1V12 signals at the membrane ruffles were quantified (see
methods above) and plotted against time.
11