Molecular Cell, Volume 33

Supplemental Data

Src-Induced Disassembly of Adherens Junctions Requires Localized Phosphorylation and Degradation of the Rac Activator Tiam1

Simon A. Woodcock, Claire Rooney, Michalis Liontos, Yvonne Connolly, Vassilis Zoumpourlis, Anthony D. Whetton, Vassilis G. Gorgoulis, and Angeliki Malliri

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Constructs

Full-length, and N-terminally truncated (C1199), mouse Tiam1 carrying a C-terminal HA tag in

pcDNA3 were provided by J. Collard. The full-length construct was used as a template to mutate

Y384 to phenylalanine using the QuikChange site-directed mutagenesis kit (Stratagene)

according to manufacturer’s directions, and these constructs were used for transient expression.

Wild-type and Y384F Tiam1 carrying C-terminal HA tags were cloned from pcDNA3 into the

retroviral LZRS-IRES-Zeo expression vector for stable expression. Mammalian expression

constructs encoding wild-type (WT) and active (YF) cSrc, and active (YF) cYes were provided

by A. Welman. Constructs for the expression of GST fused to the wild-type or mutant (R86K)

SH2 domain of Grb2 in pGex-2T, and the wild-type Grb2-Myc in pcDNA3, were provided by E.

de Vet. Dominant-negative Grb2, encoding the SH2 domain only fused to GFP in pEGFP-N1,

was provided by M. Martin. Wild-type Sos1-Myc in pcDNA3 was provided by E. Nishida.

Vinculin-GFP in the retroviral LZRS expression vector was provided by I. van den Bout.

Cell Culture and Cell Lines

H293T, MDCK II and ts-vSrc MDCK cells (Behrens et al., 1993) (a gift from J. Behrens) were

maintained in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) in the presence of 10%

fetal bovine serum (FBS; GIBCO). Wild-type and Tiam1 knock-out mouse embryonic fibroblasts

(Malliri et al., 2002) (MEFs), and wild-type and Capn4 knock-out MEFs (Dourdin et al., 2001)

(a gift from P.A. Greer) were maintained in DMEM in the presence of 10% FBS and 0.1mM 2-

Mercaptoethanol (GIBCO).

Antibodies

Immunoblotting was performed using the following primary antibodies: anti-HA (Clone 12CA5,

CRUK, 1:5000), anti-pERK (CST, 1:1000), anti-total-ERK (Sigma, 1:12,000), anti-Myc (Clone

9E10, CRUK, 1:5000), anti-Sos1 (BD, 1:500), anti-Tiam1 (C16 Santa Cruz, 1:1000), anti-

pY384-Tiam1 (ordered against peptide ‘CDAARQGV(p)YEN’ from Biogenes, 1:1000), anti-

pY416-Src (CST, 1:1000), anti-β-Actin (Clone AC-15, Sigma, 1:5000), anti-Tubulin (Clone

TAT-1, CRUK, 1:10,000), anti-P-Tyr (Clone PY20, BD, 1:5000), anti-Grb2 (BD, 1:2000), anti-

Src (Clone 3-27, CRUK, 1:1000), anti-Calpain 4 (1:1000, a gift from P.A. Greer), and

subsequently horseradish peroxidise-conjugated anti-mouse or anti-rabbit secondary antibodies

(GE Healthcare, 1:5000) and visualised by enhanced chemiluminescence (Perkin Elmer). Band

intensities were quantified using the Genetools software (SynGene). For immunohistochemical

analysis the following antibodies were used: anti-Tiam1 (C16 Santa Cruz, 1:100), anti-pY384-

Tiam1 (Biogenes, 1:100), anti-pY416-Src (CST, 1:100), and anti-Src (Clone 3-27, CRUK,

1:100). For immunofluorescence analysis fixed cells were stained using the following primary

antibodies: anti-HA-Alexa Fluor 594 conjugate (Clone 16B12, Molecular Probes, 1:500), anti-

Vinculin-Fluorescein conjugate (Clone hVIN-1, Sigma, 1:100), anti-pY384-Tiam1 (Biogenes,

1:200), anti-E-cadherin-Fluorescein conjugate (Clone 36, BD, 1:150), anti-phospho-ERK (CST,

1:400), and subsequently with highly cross-adsorbed Alexa Fluor 488 or 568-conjugated anti-

rabbit secondary antibodies or Fab fragments (Molecular Probes, 1:500) where appropriate. F-

actin was stained using Phalloidin-Alexa Fluor 568 conjugate (Molecular Probes).

In Vitro Kinase Assay

For the in vitro kinase assay, immunoprecipitations (IPs) were carried out as described in

Experimental Procedures, beads were further washed with Kinase buffer (50mM Tris-HCl pH

7.5, 10mM MgCl2, 0.01% (v/v) Triton-X-100, and 10% (v/v) glycerol) containing 2mM sodium

vanadate. Aliquots of beads were incubated in Kinase buffer containing 2mM DTT, 2mM

sodium vanadate and where indicated 200μM ATP and/or 200ng recombinant Src (SrcN1,

Human, Calbiochem) at 30°C for 30 minutes. Samples were then eluted and processed as with

IPs as described in Experimental Procedures.

In Vitro Calcium-Dependent Protease Assay

For the in vitro calcium-dependent protease assay, cells were lysed in GST-FISH buffer (50mM

Tris-HCl pH 7.5, 100mM NaCl, 2mM MgCl2, 1% (v/v) Nonidet P-40, and 10% (v/v) glycerol).

Aliquots of the cleared lysate were incubated for 15 minutes on ice with or without calpain

inhibitors (ALLN (50 μM), MDL-28170 (50 μM)) before 5 mM CaCl2 was added (except to

control lysate). Reactions were incubated for 1 hr at 37°C to allow activation of endogenous

calcium-dependent proteases before stopping the reaction by the addition of 1x SDS-PAGE

sample buffer and heating. Samples were then resolved using SDS-PAGE as described in

Experimental Procedures.

In Vitro Calpain Protease Assay

For the in vitro Calpain protease assay, IPs were carried out as described in Experimental

Procedures but beads were washed using Proteolysis buffer (50mM Tris-HCl pH 7.5, 150mM

NaCl, and 0.1% (v/v) Triton-X-100). Aliquots of the beads were then incubated in Proteolysis

buffer containing 6mM CaCl2 or 3mM EDTA, with various concentrations of recombinant

Calpain 2 (Calpain-2, Rat, Calbiochem) as indicated, at 37°C for 1 hr. Samples were then eluted

and processed as with IPs as described in Experimental Procedures.

Mass Spectrometry

H293T cells were transfected with wild-type Tiam1-HA only or co-transfected with wild-type

Tiam1-HA and active cSrc (cSrcYF). Tiam1 was immunoprecipitated and processed as in

Experimental Procedures, and after SDS-PAGE the gel was stained using Coomasie (SimplyBlue

SafeStain, Invitrogen). The putative bands containing Tiam1 were excised, destained and

tryptically digested. LC-MS/MS to identify phosphotyrosyl peptides was performed using a

MIDASTM workflow (Unwin et al., 2005) on a 4000 Q-Trap tandem mass spectrometer (Applied

Biosystems).

Biochemical Fractionation

Cells treated as indicated were permeabilised on ice using digitonin buffer (50mM Tris-HCl pH

7.5, 150mM NaCl, 1mM EDTA and 0.01% digitonin) for 5 minutes. After a brief centrifugation

the cytoplasmic fraction was removed. The insoluble pellet was resuspended in IP lysis buffer

(containing 1% Triton, see Experimental Procedures) and incubated on ice for a further 15

minutes. After centrifugation the membrane fraction was removed. Both buffers contained a

protease inhibitor cocktail (Sigma), phosphatase inhibitor cocktails 1 and 2 (Sigma) and 5mM

sodium vanadate (Sigma).

Quantification of Vinculin-Stained Focal Adhesion Area

Immunofluorescence images of stained vinculin were background subtracted, an overlay image

was created and a threshold was set to limit analysis to focal adhesions and their areas were

calculated using ImageJ software.

FRAP Analysis

A time lapse system consisting of a Zeiss Axiovert 200M microscope under the control of

Metamorph software was used for FRAP experiments. Cells expressing GFP-tagged vinculin

were plated on glass-bottom dishes and were imaged at 35°C in Leibovitz's L-15 medium

(Invitrogen) supplemented with 10% FBS. Initial fluorescence intensity was measured with a

300W Xenon light source at 100ms exposures followed by photobleaching of focal adhesions

with a Melles Griot air cooled tuneable 200mW Argon ion laser at 100% power for 3 seconds.

The fluorescence recovery was then followed with fluorescence imaging at 100ms until the

fluorescence intensities recovered to a plateau. Images were transferred into ImageJ and

background subtracted. Corrected recovery fluorescence intensities were normalized to prebleach

intensity. The mobile fraction was calculated for individual adhesions according to MF = (Finf –

F0)/(Fi – F0) where Finf is the fluorescence intensity in the bleached region after full recovery, Fi

is the fluorescence intensity before bleaching and F0 fluorescence intensity immediately post

bleaching, and was presented as a percentage.

Dissociation Assay

To assess cell–cell adhesion strength in ts-vSrc MDCK pools a dissociation assay was performed

essentially as described previously (Takeda et al., 1995), however instead of confluent

monolayers, cells were seeded for 2 days with vSrc off in order to form small colonies of 10-20

cells before shifting to 35°C for 0 or 2 hrs. The extent of cell dissociation was presented as the

ratio Np/Nc, where Np and Nc are the total number of particles and cells respectively.

Migration Assays

For wounding assays cells were grown in a six-well dish to confluency in the presence of 10%

fetal bovine serum. Subsequently, a wound was made using a plastic pipette tip, and fresh serum-

containing medium was added. Closure of the wound was monitored by microscopy, with

images taken at 0 and 16 hours after the wound was made. Migration was quantified by

measuring the distance the wound margin advanced over the time-course. For single cell tracking

experiments, cells were seeded sparsely and phase contrast images were acquired every 30

minutes for 24 hours at 35°C using a time lapse system consisting of a Zeiss Axiovert 200M

microscope under the control of Metamorph software. Movies were made using Imaris, and

imported into ImageJ. Individual single cells were manually tracked for at least 5 hours using

ImageJ in order to calculate their average speeds.

Detection of Calpain Activity in Cells

Cells were incubated for 20 minutes with the cell-permeable fluorogenic calpain substrate BOC-

LM-CMAC (25μM, Molecular Probes). Cleavage of this substrate by calpain results in an

increase in fluorescence that was detected using a Deltavision system.

SUPPLEMENTAL REFERENCES

Behrens, J., Vakaet, L., Friis, R., Winterhager, E., Van Roy, F., Mareel, M. M., and Birchmeier,

W. (1993). Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine

phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-

sensitive v-SRC gene. J Cell Biol 120, 757-766.

Dourdin, N., Bhatt, A. K., Dutt, P., Greer, P. A., Arthur, J. S., Elce, J. S., and Huttenlocher, A.

(2001). Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient

embryonic fibroblasts. J Biol Chem 276, 48382-48388.

Malliri, A., van der Kammen, R. A., Clark, K., van der Valk, M., Michiels, F., and Collard, J. G.

(2002). Mice deficient in the Rac activator Tiam1 are resistant to Ras-induced skin tumours.

Nature 417, 867-871.

Takeda, H., Nagafuchi, A., Yonemura, S., Tsukita, S., Behrens, J., Birchmeier, W., and Tsukita,

S. (1995). V-src kinase shifts the cadherin-based cell adhesion from the strong to the weak state

and beta catenin is not required for the shift. J Cell Biol 131, 1839-1847.

Unwin, R. D., Griffiths, J. R., Leverentz, M. K., Grallert, A., Hagan, I. M., and Whetton, A. D.

(2005). Multiple reaction monitoring to identify sites of protein phosphorylation with high

sensitivity. Mol Cell Proteomics 4, 1134-1144.

Figure S1. Identification and Characterization of Tiam1 Phosphorylation on Y384

(A) H293T cells were transfected with either vector only or Tiam1-HA with or without active

cSrcYF. Exogenous Tiam1 was immunoprecipitated from lysates with anti-HA antibody and

detected with anti-P-Tyr or anti-Tiam1. (B) Product ion spectrum of a peptide precursor ion of

546.7. Sequence confirmation is derived from a number of fragment ion masses labelled. The site

of tyrosine phosphorylation is confirmed as Y384 by the mass difference between y4 and y5

equivalent to intact phosphotyrosine at 243 Da. (C) Alignment of Tiam1 sequences from various

vertebrates shows Y384 and its surrounding amino acids are well conserved through evolution.

(D) Cell lysates were prepared from parental H293T cells at sequential time points following

pervanadate treatment (100μM). Levels of total and Y384-phosphorylated endogenous Tiam1 in

the lysate are shown. Tubulin levels are shown as a loading control. (E) H293T cells were

transfected with wild-type Tiam1-HA with or without active cYesYF. Tiam1 was detected in

lysates by immunoblotting with anti-pY384-Tiam1 or anti-HA. Levels of active (anti-pSFK) and

total cYes are shown. Actin levels are shown as a loading control.

Figure S2. Localization and Effects of Y384 Phosphorylation on Tiam1 Distribution

(A) Parental ts-vSrc cells were cultured at the restrictive temperature (41°C) for 2 days and either

fixed (vSrc – off), or shifted to the permissive temperature (35°C) for 2 hrs and fixed (vSrc – on).

Fixed cells were stained using anti-pY384-Tiam1 to detect endogenous Tiam1. The plane shown

is 0.6 µm above basal. (B) Pools of ts-vSrc MDCK cells stably expressing Tiam1WT-HA or

Tiam1Y384F-HA were cultured at the restrictive temperature for 2 days and either fixed (vSrc –

off), or shifted to the permissive temperature for 2 hours and fixed (vSrc – on). Fixed cells were

stained with anti-HA. Images shown are maximal projections of the HA-Tiam1 stainings. (C)

Cells cultured as in B were fractionated into cytoplasmic (C) and membrane (M) fractions (see

Supplemental Experimental Procedures). Levels of exogenous Tiam1 in each fraction were

detected by immunoblotting with anti-HA, band intensities were quantified and are presented as

percentages of total Tiam1. The fractionation of GAPDH (cytoplasmic) and β-Catenin

(membrane) into the appropriate fractions is shown. Scale Bars, 15 µm.

Figure S3. Phosphorylation of Tiam1 on Y384 Affects the Turnover of Focal Adhesion

Components

(A) Pools of ts-vSrc MDCK cells stably expressing Tiam1WT-HA (WT) or Tiam1Y384F-HA

(Y384F) were cultured at the restrictive temperature for 2 days before activating vSrc for 4 hours

at the permissive temperature (vSrc – on). Cells were fixed and co-stained with anti-HA and anti-

Vinculin. Images of colonies are shown at the basal surface. (B) Quantification of vinculin

stained focal adhesion area from at least 30 cells (n=3) located at the edge of colonies as seen in

A. Data are represented as mean ±SEM (*p<0.05). (C) Pools of ts-vSrc MDCK cells stably

expressing Vinculin-GFP together with either Tiam1WT-HA or Tiam1Y384F-HA were cultured at

the restrictive temperature for 2 days before activating vSrc for 4 hours at the permissive

temperature. To assess the turnover of vinculin-GFP at focal adhesions in cells at the edge of

colonies, the focal adhesions were bleached and the recovery of fluorescence was measured.

Normalised recovery of vinculin-GFP in multiple focal adhesions (from at least 15 cells (n=4))

was quantified, with lines indicating the single exponential fit of the data. (D) The mobile

fraction was calculated for individual adhesions from FRAP analysis performed as described in

C, and is presented as a percentage of pre-bleach intensity. Data are represented as mean ±SEM

(*p<0.05; n=4). (E) Pools of ts-vSrc MDCK cells stably expressing Tiam1WT-HA or Tiam1Y384F-

HA were seeded sparsely and cultured at the restrictive temperature for 2 days before activating

vSrc for 4 hours at the permissive temperature (vSrc – on). Cells were fixed and co-stained with

anti-HA and anti-Vinculin. Images of single cells are shown at the basal surface. (F)

Quantification of vinculin stained focal adhesion area from at least 30 isolated cells is shown

(n=3) as seen in E. Data are represented as mean ±SEM, and show no significant difference.

Scale bars, 15 µm.

Figure S4. Effects of Tiam1 Y384 Phosphorylation on Cell Migration

(A) Confluent pools of ts-vSrc MDCK cells stably expressing Tiam1WT (WT) or Tiam1Y384F

(Y384F) were wounded and allowed to migrate into the wound at the permissive temperature

(vSrc - on). Phase contrast images are shown at 0 and 16 hours after wounding. (B) Confluent

pools of ts-vSrc MDCK cells stably expressing Tiam1WT (WT) or Tiam1Y384F (Y384F) were

wounded and allowed to migrate into the wound for 16 hours at the restrictive temperature (vSrc

- off). Quantification of the relative migration compared to WT Tiam1 is shown. Data are

represented as mean ±SEM, and show no significant difference. (C) Pools of ts-vSrc MDCK

cells stably expressing Tiam1WT-HA (WT) or Tiam1Y384F-HA (Y384F) were seeded sparsely.

Phase contrast images were acquired every 30 minutes with vSrc on, individual single cells were

tracked for at least 5 hours to calculate their average speed. Quantification of the individual

single cell speeds of at least 30 cells (n=3) is shown. Data are represented as mean ±SEM, and

show no significant difference.

Figure S5. Y384-Phosphorylated Tiam1 Binds the Grb2/Sos Complex

(A) Lysates from H293T cells transfected with WT Tiam1-HA, with or without cSrcYF, were

incubated with either immobilised GST-Grb2-SH2WT or GST-Grb2-SH2* domains. Bound

exogenous Tiam1 was detected by IB, with relative levels in the lysate also shown. (B) WT

Tiam1-HA and cSrcYF were transfected together or by themselves in H293T cells. Endogenous

Grb2 was isolated by IP from lysates and exogenous Tiam1 that co-precipitated was detected by

IB. Levels of Src, Tiam1 and a loading control in the lysates are shown. (C) H293T cells treated

with control or Grb2-specific RNAi were transfected with WT Sos1-Myc. Also H293T cells

were co-transfected with WT Sos1-Myc and WT Grb2-Myc. Lysates were incubated with

immobilised phospho-Y384-Tiam1 peptide. Associated Sos1 and Grb2 were detected by IB, with

levels in the lysates also shown. Tubulin levels are shown as a loading control. (D) Model for the

recruitment of the Grb2-Sos1 complex to Tiam1 in response to phosphorylation of Y384 by Src.

Figure S6. Inhibition of Src, Grb2, MEK, or Calpain Impairs the Removal of Tiam1 from

Cell–Cell Adhesions by vSrc

Pools of ts-vSrc MDCK cells stably expressing wild-type Tiam1-HA were cultured at the

restrictive temperature (41°C) for 2 days and either fixed (A, vSrc – off), shifted to the

permissive temperature (35°C) for 1 hour to activate vSrc and fixed (B, vSrc – on), or pre-treated

for 2 hours with SU6656 (20 µM, C), TB03 (100 µM, D), UO126 (50 µM, E) or ALLN (50 µM,

F) before being shifted to the permissive temperature for 1 hour and fixed. Fixed cells were

stained with anti-HA. Images in the x/y and x/z planes are shown. The x/y plane shown is 2.4 µm

above basal. Scale bars, 20 µm.

Figure S7. Localization of Calpain Activity

ts-vSrc MDCK cells were cultured at the restrictive temperature for 2 days and either shifted to

the permissive temperature for 1 hour (vSrc – on) or pre-treated with calpain inhibitor (ALLN,

50 µM) for 2 hours before being shifted to the permissive temperature for 1 hour. Localisation of

calpain activity was visualised by fluorescence microscopy after the incubation of cells with the

cell-permeable fluorogenic calpain substrate BOC-LM-CMAC (25 µM). Outlines of cells are

marked by dashed lines. Scale bars, 10 µm.