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PKA-regulated VASP phosphorylation promotes extrusion of transformed cells 1
from the epithelium 2
3
Katarzyna A. Anton1,7, John Sinclair2, Atsuko Ohoka3, Mihoko Kajita3, Susumu Ishikawa3, Peter M. 4
Benz4, Thomas Renne5,6, Maria Balda1, Claus Jorgensen2, Karl Matter1,*,‡ and Yasuyuki Fujita3,7,*,‡ 5
6
1Department of Cell Biology, UCL Institute of Ophthalmology, University College London, London, 7
United Kingdom. 8
2Division of Cancer Biology, Cell Communication Team, The Institute of Cancer Research, London, 9
United Kingdom. 10
3Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Sapporo, 11
Japan. 12
4Institute for Vascular Signaling, University of Frankfurt, Frankfurt, Germany. 13
5Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Karolinska 14
Institute, Stockholm, Sweden. 15
6Institute for Clinical Chemistry, University Hospital Hamburg-Eppendorf, Hamburg, Germany. 16
7MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, 17
London, United Kingdom. 18
19
*These authors contributed equally to this work 20
‡Authors for correspondence ([email protected] and [email protected]) 21
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Running title: Regulation of transformed cells extrusion 23
Key words: apical extrusion, Ras-transformed, VASP, PKA 24
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© 2014. Published by The Company of Biologists Ltd.Jo
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JCS Advance Online Article. Posted on 24 June 2014
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ABSTRACT 26
At the early stages of carcinogenesis, transformation occurs in single cells within tissues. In an 27
epithelial monolayer, such mutated cells are recognized by their normal neighbors and are often 28
apically extruded. The apical extrusion requires cytoskeletal reorganization and cell shape changes, but 29
it is poorly understood what molecular switches are involved in regulation of these processes. Here, 30
using SILAC-based quantitative mass spectrometry we have identified proteins that are modulated in 31
transformed cells upon their interaction with normal cells. Phosphorylation of VASP at serine 239 is 32
specifically upregulated in RasV12-transformed cells when they are surrounded by normal cells. VASP 33
phosphorylation is required for the cell shape changes and apical extrusion of Ras-transformed cells. 34
Furthermore, PKA is activated in Ras-transformed cells surrounded by normal cells, leading to VASP 35
phosphorylation. These results indicate that the PKA/VASP pathway is a crucial regulator for tumor 36
cell extrusion from the epithelium and shed light on the events occurring at the early stage of 37
carcinogenesis. 38
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INTRODUCTION 39
At the initial stage of carcinogenesis, oncogenic transformation occurs in a single cell within the 40
epithelium. We have recently reported that Ras-transformed cells are apically extruded when 41
surrounded by normal neighbors (Hogan et al., 2009). This phenomenon is shared by cells transformed 42
with other oncogenes, such as v-Src, and occurs in both invertebrates and vertebrates in vivo (Kajita et 43
al., 2010). The interaction with normal neighbors induces Ras-transformed cells to undergo cell shape 44
changes, resulting in increased cell height, and to remodel their actin cytoskeleton, leading to F-actin 45
accumulation at cell-cell contacts (Hogan et al., 2009). However, the molecular mechanisms regulating 46
these processes remain obscure. In particular, it is not clear what molecular switches are involved in the 47
morphological changes of transformed cells required for extrusion. Uncovering the mechanism of 48
apical extrusion is not only crucial for understanding early carcinogenesis, but it may shed light on the 49
mechanics of other cell sorting events that take place during development. 50
In this study, we used quantitative mass spectrometry to identify proteins modulated in 51
transformed cells interacting with normal cells. Phosphorylation of VASP at serine 239 was 52
specifically upregulated in Ras-transformed cells interacting with normal cells. VASP phosphorylation 53
was required for apical extrusion of Ras-transformed cells and occurred downstream of PKA. These 54
results reveal a novel molecular mechanism controlling the elimination of transformed cells from the 55
epithelium. 56
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RESULTS AND DISCUSSION 57
SILAC screening for phosphorylation in Ras-transformed cells interacting with normal cells 58
To reveal molecular mechanisms of apical extrusion of Ras-transformed cells surrounded by normal 59
epithelial cells, we performed a quantitative mass spectrometry analysis (Jorgensen et al., 2009; Mann, 60
2006). Using stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative 61
proteomics, we examined phosphorylated proteins in transformed cells. We used MDCK cells 62
expressing GFP-tagged constitutively active, oncogenic Ras (RasV12) controlled by a tetracycline-63
inducible promoter (hereafter called Ras cells) (Hogan et al., 2009). Three types of isotope-labeled 64
arginine and lysine were used: heavy (Arg 10, Lys 8) and medium (Arg 6, Lys 4) for labeling Ras cells, 65
and light (Arg 0, Lys 0) for normal untransfected MDCK cells (Fig. 1A). Heavy-labeled Ras cells were 66
mixed with light-labeled MDCK cells, while medium-labeled Ras cells were cultured alone (Fig. 1A). 67
Following 6 h-induction of RasV12 expression with tetracycline, the cell lysates were combined, and the 68
amounts of heavy- and medium-labeled phosphorylated peptides were compared by quantitative mass 69
spectrometry; the ratio of heavy to medium label (hereafter called H/M ratio) was calculated for each 70
peptide (Fig. 1B). For over 35% of peptides identified, we were able to calculate the H/M ratio. 71
Peptides with an H/M ratio higher than 1.5 or lower than 0.5, reproduced in at least two out of three 72
independent experiments, were considered as biologically relevant modifications (Figs 1C, S1). Over 73
80% of the H/M ratios were between 0.5 and 1.5, indicating that the phosphorylation status of most of 74
the proteins was not significantly affected. In total we identified 17 proteins that were more 75
phosphorylated and 15 that were less phosphorylated in Ras cells mixed with normal cells as compared 76
to Ras cells cultured alone. We found a number of proteins involved in cytoskeletal rearrangements and 77
cell motility, as well as proteins that function in basic cellular processes such as cell cycle, cell growth, 78
and membrane biogenesis. 79
80
Induction of VASP phosphorylation in Ras-transformed cells surrounded by normal cells 81
The actin cytoskeleton is dramatically rearranged when Ras-transformed cells are extruded (Hogan et 82
al., 2009). In the SILAC screening, we found that phosphorylation of vasodilator-stimulated 83
phosphoprotein (VASP) at serine 242 (serine 239 for the human orthologue) was upregulated (Figs 84
1B,C, S1A). VASP is a member of Ena/VASP family of actin cytoskeleton regulators. It bundles actin 85
fibers and by antagonizing the capping of elongating filaments, promotes actin polymerization (Barzik 86
et al., 2005; Bear et al., 2002; Hansen and Mullins, 2010; Huttelmaier et al., 1999; Pasic et al., 2008). 87
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The protein localizes at sites of high actin turnover including lamellipodia and filopodia where it 88
promotes actin polymerization and regulates the geometry of F-actin networks (Benz et al., 2009; 89
Lanier et al., 1999; Rottner et al., 1999; Scott et al., 2006). VASP is a well-established target of protein 90
kinases, and human VASP harbors three major phosphorylation sites: serine157 (S157), serine239 91
(S239), and threonine278 (T278) (Benz et al., 2009; Blume et al., 2007; Butt et al., 1994). The first two 92
sites are phosphorylated by the cAMP- and cGMP-dependent protein kinase (PKA and PKG), whereas 93
the AMP-activated protein kinase (AMPK) phosphorylates T278. Functionally, these serine/threonine 94
phosphorylations impair the actin polymerization and anti-capping activities of VASP and control its 95
subcellular targeting (Benz et al., 2009; Blume et al., 2007). Given the importance of the actin 96
cytoskeleton for cell shape changes and for apical extrusion of transformed cells, we examined the 97
functional significance of VASP phosphorylation. 98
As we were not able to detect S242-phosphorylation of endogenous VASP by 99
immunofluorescence, we produced Ras cells stably expressing His-tagged wild-type VASP (Ras-VASP 100
WT; Fig. S2A). When Ras-VASP WT cells were co-cultured with normal cells, we observed enhanced 101
immunostaining for phosphorylated VASP at S239 (pVASP) compared to Ras-VASP WT cells 102
cultured alone (Figs 2A, S2B). Previously, we reported that prior to apical extrusion the height of Ras 103
cells along the apico-basal axis significantly increased compared to the height of the surrounding 104
normal cells (Hogan et al., 2009). Here, we found that the enhanced pVASP was closely correlated to 105
that cell shape change in Ras cells surrounded by normal cells (Fig. 2B), suggesting that 106
phosphorylation of VASP is involved in the process of apical extrusion. 107
To quantify pVASP, we immunoprecipitated exogenous VASP proteins from Ras-VASP WT 108
cells cultured alone (Alo) or co-cultured with normal cells (Mix) using anti-His-tag antibody. We found 109
that pVASP was increased twofold under the mix culture condition (Fig. 2C,D). The difference in 110
pVASP was also detected in the total cell lysates where both endogenous and exogenous VASP 111
proteins were present (Fig. 2C,E) or when only endogenous VASP was analyzed (Fig. S2C). The 112
timing of the increase in pVASP (16-20 h after the induction of RasV12 expression) was correlated to 113
the time of extrusion (Figs 2E, S2C). When RasV12 expression was not induced in the absence of 114
tetracycline, the increase in pVASP was not observed under the mixed culture condition (Fig. S2D), 115
suggesting that the non-cell autonomous upregulation of pVASP is dependent on RasV12 expression. 116
Thus, VASP is differentially phosphorylated in Ras-transformed cells mixed with normal cells at a time 117
when the actin cytoskeleton is reorganized and the cell shape is altered. 118
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Phosphorylation of VASP promotes apical extrusion 120
To test the role of VASP, we depleted VASP in Ras cells using RNA interference (Fig. 3A,B). When 121
VASP–knockdown Ras cells were surrounded by normal cells, apical extrusion was significantly 122
promoted (Fig. 3C,D). Expression of exogenous wild-type VASP rescued the knockdown phenotype 123
(Figs 3E, S2E). In contrast, expression of the phosphomimetic mutant (S239D) of VASP did not 124
complement the knockdown phenotype, and a partial rescue was obtained by expression of the non-125
phosphorylatable mutant (S239A) (Fig. 3E). The lack of rescue was not due to the lower expression 126
levels of the mutant proteins (data not shown). No extrusion was observed when VASP was depleted in 127
non-transformed pTR-GFP cells cultured in the presence of normal MDCK cells (Fig. S2F). When 128
expression of GFP-RasV12 was not induced, the presence of VASP WT did not result in cell extrusion 129
(Fig. S2G). These data indicate that VASP plays an inhibitory role in apical extrusion of transformed 130
cells and that phosphorylation of VASP at S239 relieves its suppressive effect. The lack of a complete 131
rescue by the non-phosphorylatable mutant might be due to the necessity of spatiotemporal regulation 132
of phosphorylation. 133
It was previously reported that phosphorylation of VASP at S239 induces a round cell shape 134
and prevents formation of various types of protrusions (Lindsay et al., 2007; Zuzga et al., 2012). To 135
investigate whether VASP phosphorylation had a comparable effect in Ras cells, we cultured cells at 136
low density, allowing them to spread, and analyzed the effect of expression of VASP on cell 137
morphology. Overexpression of VASP S239D reduced the formation of lamellipodia, prevented 138
spreading, and promoted a compact cell shape (Fig. S2A,H). We next analyzed VASP-knockdown Ras 139
cells expressing the exogenous wild-type or mutant forms of VASP proteins. VASP-knockdown in Ras 140
cells inhibited cell spreading and consequently decreased the average planar surface of the cells, and 141
expression of VASP S239D in depleted cells further promoted this phenotype, suggesting that the 142
mutant form of VASP acted in a dominant negative manner and blocked the endogenous VASP still 143
expressed in the knockdown cells (Figs 3A, S2I). Conversely, expression of VASP WT or S239A 144
increased cell spreading (Figs 3A, S2I). When expression of GFP-RasV12 was not induced, the presence 145
of VASP WT did not significantly alter cell spreading (Fig. S2J). Although knockdown of VASP 146
suppressed the initial formation of cell adhesions, expression of VASP or VASP mutants 147
complemented this phenotype equally, suggesting that phosphorylation does not affect initial adhesion 148
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rates (Fig. S2K). These data indicate that VASP phosphorylation regulates the cell shape of Ras-149
transformed cells. 150
151
PKA regulates VASP phosphorylation in Ras-transformed cells in a non-cell-autonomous 152
manner 153
Expression of RasV12 induced a temporal increase in pVASP (Fig. S3A), which was further enhanced 154
when Ras cells were co-cultured with normal cells (Fig. 2A,C-E). To identify the responsible kinase, 155
we examined the involvement of PKG, which phosphorylates S239 in various cell types (Becker et al., 156
2000; Benz et al., 2009; Zuzga et al., 2012). However, PKG inhibitor Rp-8-Br-PET-cGMPS did not 157
affect phosphorylation of VASP in Ras cells (Figs 4A, S3B). Similarly, an inhibitor of nitric oxide 158
synthase (NOS), often functioning upstream of PKG/pVASP, did not suppress VASP phosphorylation 159
(data not shown). Another kinase phosphorylating VASP at serine 239 is PKA (Smolenski et al., 1998). 160
Treatment with the PKA inhibitor H89 strongly suppressed pVASP (Figs 4A, S3C,D). A different PKA 161
inhibitor KT5720 also suppressed pVASP (Fig. S3E). In contrast, the PKA activator DBcAMP 162
increased pVASP levels (Fig. 4B). Collectively, these data indicate that PKA regulates phosphorylation 163
of VASP in Ras-transformed cells. PKA has also been reported to phosphorylate VASP S157, which 164
leads to decreased mobility of VASP in SDS-PAGE gels (Smolenski et al., 1998). Two bands were 165
detected by western blotting with the anti-VASP antibody, and the upper band (with reduced mobility 166
in SDS-PAGE) was more pronounced in mixed cultures, which was diminished by the treatment with 167
the PKA inhibitor (Fig. 4A,C), suggesting that phosphorylation on S157 was likewise regulated in Ras-168
transformed cells co-cultured with normal cells. We next asked whether PKA activity could be 169
specifically increased in Ras-transformed cells interacting with normal cells. To detect PKA activity, 170
we used an antibody against a phosphorylated consensus sequence recognized by PKA. Western 171
blotting data suggested that the amount of proteins detected by the antibody was higher in the mixed 172
culture of normal and Ras cells than in the separate culture, and was decreased by PKA inhibitor (Fig. 173
4D,E). By immunofluorescence, PKA activity was indeed elevated in Ras cells surrounded by normal 174
cells (Figs 4F, S3F). Finally, we analyzed the effect of PKA inhibition or activation on the cell 175
morphology of Ras cells. Treatment of Ras cells with a PKA inhibitor increased cell spreading and 176
decreased apico-basal cell height, whereas treatment with a PKA activator had the opposite effect (Fig. 177
S3G,H). The effect of the PKA inhibitor on cell spreading was counteracted by knockdown of VASP 178
(Fig. S3I), indicating that PKA stimulates cell shape changes of Ras cells in a VASP-dependent 179
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manner. At present, there is no evidence suggesting that the other members of the Ena/VASP family, 180
Mena and EVL, also play a role in apical extrusion, similarly to VASP. Evl does not have a serine 181
residue corresponding to VASP S239. Both Mena and EVL are expressed in MDCK cells (Sabo et al., 182
2001; Tang and Brieher, 2013), but upregulated phosphorylation of Mena or EVL was not identified in 183
our SILAC screening. In addition, knockdown of VASP substantially promoted apical extrusion. 184
Although the possibility of an overall dosage effect of these homologous proteins cannot be ruled out, 185
these data indicate that depletion of VASP alone is sufficient to promote extrusion. 186
Here, we identified a signaling pathway that drives extrusion of Ras-transformed cells from a 187
monolayer of normal epithelial cells; the non-cell-autonomously activated PKA-VASP pathway 188
stimulates cell shape changes of Ras cells, which promotes their apical extrusion. VASP is a 189
functionally important target of PKA in cell extrusion; however, it will be essential to identify other 190
targets as our results suggest that PKA phosphorylates multiple proteins in Ras-transformed MDCK 191
cells (Fig. 4D,E). Activation of PKA occurred in a non-cell-autonomous fashion, indicating that Ras-192
transformed cells recognize and respond to the presence of normal cells, resulting in the stimulation of 193
PKA in the transformed cells; thus, the molecular mechanism of how PKA is activated remains to be 194
determined. Cell sorting is thought to be crucial for tissue homeostasis and development, and has been 195
proposed to involve the difference of membrane tension between cells. Apical extrusion of transformed 196
cells can be considered to be a process of three-dimensional cell sorting, and given their effects on the 197
actin cytoskeleton and cell shape, PKA and VASP may regulate membrane tension at the boundary 198
between normal and transformed cells. Understanding how this pathway is stimulated might help to 199
understand the events occurring at the early stage of carcinogenesis and open new avenues to fight this 200
life-threatening disease. 201
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MATERIALS AND METHODS 202
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Plasmids, antibodies and materials 204
pcDNA3-VASP-WT-His, pcDNA3-VASP-S239D-His, and pcDNA3-VASP-S239A-His were as 205
described (Benz et al., 2009). The following primary antibodies were used: rabbit anti-VASP (IF 1:200; 206
WB 1:10,000), rabbit anti-pVASP S239 (IF 1:50), and rabbit anti-phospho-(Ser/Thr) PKA substrates 207
(IF 1:100; WB 1:1,000) from Cell Signaling (supplied by New England Biolabs in Hitchin, 208
Hertfordshire, UK); mouse anti-pVASP S239 (WB 1:5,000) from Santa Cruz Biotechnology 209
(Heidelberg, Germany); mouse anti-His-tag (WB 1:3,000) from Sigma-Aldrich (Gillingham, Dorset, 210
UK); mouse anti--tubulin as previously described (Kreis, 1987). Following secondary antibodies were 211
used for immunofluorescence (1:300): Alexa-647-conjugated anti-rabbit and anti-mouse antibodies 212
from Molecular Probes by Life Technologies (Paisley, Scotland, UK); Cy3-conjugated anti-rabbit and 213
anti-mouse antibodies from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania, USA). 214
Atto-647N-phalloidin from Sigma-Aldrich (Gillingham, Dorset, UK) was used at 20 nM. The 215
following inhibitors were used: PKG inhibitor Rp-8-Br-PET-cGMPS hydrate (100 nM) and NOS 216
inhibitor L-NG-Nitroarginine Methyl Ester (0.3 mM) from Santa Cruz Biotechnology (Heidelberg, 217
Germany); PKA inhibitor H89 from Cambridge Biosciences (Cambridge, Cambridgeshire, UK) at 10 218
M for sparsely plated cells, or 20 M for confluent cells; PKA inhibitor KT5720 (10 or 20 M) and 219
PKA activator Dibutyryl-cAMP (200 mM) from Enzo Life Sciences (Exeter, Devon, UK). 220
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Cell culture and RNA interference 222
MDCK cells, MDCK pTR-GFP and MDCK pTR-GFP-RasV12 cells were cultured as previously 223
described (Hogan et al., 2009). MDCK pTR-GFP-RasV12 cells expressing His-VASP WT, His-VASP 224
S239D, or His-VASP S239A were maintained in medium supplemented with 500 g ml-1 of G418 225
(Calbiochem by Merck Millipore in Darmstadt, Germany). To induce GFP-RasV12 expression, 2 g ml-226
1 tetracycline was added to the culture medium. For immunofluorescence and time-lapse experiments, 227
cells were cultured on type I collagen gels from Nitta Gelatin (Nitta Cellmatrix type 1 A; Osaka, 228
Japan) as previously described (Hogan et al., 2009). For adhesion assay, 96-well plates were coated 229
with 0.5 mg ml-1 collagen solution in 1 mM acetic acid over several hours, then dried overnight at room 230
temperature in sterile conditions, and either used immediately or stored at 4oC. For spreading assay, 231
cells were plated onto plastic-bottom dishes. To establish MDCK pTR-GFP-RasV12 cell lines stably 232
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expressing VASP proteins, MDCK pTR-GFP-RasV12 cells were transfected with pcDNA3-VASP-WT-233
His, pcDNA3-VASP-S239D-His or pcDNA3-VASP-S239A-His, together with pCB6 using 234
LipofectamineTM 2000 (Life Technologies in Paisley, Scotland, UK), followed by selection in medium 235
containing 625 g ml-1 of G418, 5 g ml-1 of blasticidin, and 400 g ml-1 of zeocin. 236
Non-targeting control siRNAs and siRNAs targeting VASP were purchased from Thermo 237
Scientific (Hemel Hempstead, Hertfordshire, UK), resuspended to a final concentration of 20 M in 1X 238
siRNA buffer (20 mM KCl, 0.2 mM MgCl2, 6 mM HEPES/NaOH pH 7.5), and stored at -80oC. The 239
siRNA sequence targeted within canine VASP is: 5’- GGAAATAAGATGAGGGAGA-3’, which does 240
not target exogenously expressed human VASP. For siRNA transfection experiments, cells were plated 241
in 48-well plates at 1.2 x 104 cells per well in 200 l of medium without selective antibiotics. Then, 0.5 242
l of 20 M siRNA and 1 l of Interferin (Polyplus-transfection, Berkeley, California, USA) were 243
sequentially added to 50 l of OptiMEM (Invitrogen by Life Technologies in Paisley, Scotland, UK). 244
The transfection mixture was incubated at room temperature for 25 min and applied onto the cells. 245
After 2 days, cells were plated on glass coverslips, collagen-coated coverslips, or collagen-coated 246
dishes. After overnight incubation, tetracycline was added to induce expression of GFP-RasV12. Cells 247
were then fixed with 4% PFA/PBS at 4-24 h after tetracycline addition or observed by time-lapse 248
microscopy. For Figs. 3A and S2E, I, knockdown of VASP was analyzed at 3 days after siRNA 249
transfection. For Fig. 3E, siRNA-treated cells were further transfected with the indicated plasmids 250
using LipofectamineTM 2000 (Invitrogen by Life Technologies in Paisley, Scotland, UK). About 40 h 251
after siRNA transfection, the new transfection mixtures were prepared: (1) 0.35 g plasmid DNA in 40 252
l OptiMEM; (2) 1 l Lipofectamine 2000 in 40 l OptiMEM. After 5 min, (1) and (2) were combined 253
and incubated for 25 min. The mixture was then applied onto the knockdown cells, and after 8 h cells 254
were plated on collagen-coated coverslips. After overnight incubation, tetracycline was added, and 255
after 15 h, cells were fixed with 4% PFA/PBS. 256
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Time-lapse analyses and immunofluorescence 258
For each assay, between 0.8 x 106 and 1.2 x 106 cells were plated in collagen-coated 6-well plates. 259
MDCK pTR-GFP-RasV12 cells were mixed with MDCK cells at a ratio of 1:100 or 1:50. After 260
incubation for 6–16 h, tetracycline was added to induce GFP-RasV12 expression. Cells were incubated 261
for the indicated times or observed by time-lapse microscopy. For time-lapse experiments, at 4 h of 262
tetracycline induction, cell groups smaller than four cells were chosen for observation. The behavior of 263
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each Ras cell was followed during the time-lapse observation, and the time of the respective extrusion 264
event was recorded. Extrusion rates were quantified by dividing the number of extruded cells by the 265
total number of cells present during the analyzed time course. For Fig. 2, A and B, cells were incubated 266
for 2 h after plating, followed by tetracycline addition and further incubation for 6 h (as for the SILAC 267
screen). For immunofluorescence, cells were fixed at indicated times with 4% PFA/PBS for 10 min. 268
Cells were permeabilized in 0.5% Triton X-100/0.3% BSA/PBS for 15 min, then washed once in 269
quenching solution (20 mM glycine and 0.5% BSA in PBS) and incubated in the same solution for 30 270
min. Next, cells were incubated with blocking buffer (0.5% BSA and 0.2% Triton X-100 in PBS) for 1-271
2 h. Primary antibodies diluted in blocking buffer were applied for 2-3 h or overnight (for cells on 272
collagen). Cells were washed 3 times for 10 min with blocking buffer, and incubated with fluorescent-273
conjugated secondary antibodies and/or phalloidin for 1–3 h at room temperature. Cells were then 274
washed 3 times for 10 min in blocking buffer and incubated with Hoechst 33342 (Life Technologies in 275
Paisley, Scotland, UK) in PBS for 3 min, followed by washing in PBS and mounting onto a glass slide 276
with mowiol (MERCK Millipore in Darmstadt, Germany) when cells were cultured on collagen, or 277
with Prolong Gold Antifade Reagent (Life Technologies in Paisley, Scotland, UK) when cultured on 278
glass. For immunofluorescence of phosphorylated proteins, PBS was replaced with TBS containing 279
phosphatase inhibitors (1 mM sodium orthovanadate, 0.1 mM ammonium molybdate, and 10 mM 280
sodium fluoride). 281
Time-lapse analyses were performed using a Zeiss Axiovert 200 M Microscope with a 282
Hamamatsu Orca AG camera using 20x/0.5 or 20x/0.4 air objectives (Carl Zeiss NTS in Cambridge, 283
Cambridgeshire, UK) or a Nikon Eclipse Ti-E with a CoolSNAP HQ2 camera using a 10x/0.45 air 284
objective (Nikon Instruments Europe in Kingston Upon Thames, Surrey, UK). Images were captured 285
and analyzed using Volocity 6.2.1 software (PerkinElmer in Waltham, Massachusetts, USA) or NIS-286
Elements software 4.0 (Nikon, Tokyo, Japan), respectively. Fixed immunofluorescence samples were 287
analyzed with a Leica SPE3 DM4000 with a single PTM using a 63x/1.3 oil objective (Leica 288
Microsystems in Wetzlar, Germany). Images were captured and analyzed using Leica Application Suite 289
(LAS) software. Pixel intensity was quantified using ImageJ 1.48 (Fiji) digital analysis software 290
(National Institute of Health in Bethesda, Maryland, USA): cell boundaries in xz confocal sections 291
were outlined using the actin channel, and this region was used to measure average pixel intensity in 292
the PKA-S channel. Cells were counted following 15 h of tetracycline treatment. 293
294
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Adhesion assay and spreading assay 295
For adhesion assay, cells were suspended to a concentration of 0.5 x 106 cells ml-1 in DMEM/1% BSA, 296
and 50 l of cell suspension was plated onto collagen-coated 96-well plates in a total volume of 200 l, 297
followed by incubation for 30 min. Unattached cells were then removed by three consecutive PBS 298
washes. Attached cells were fixed and stained with 1% crystal violet in 20% methanol for 15 min. 299
Excess of the dye was removed by 5 water-washes. Crystal violet trapped in the cells was extracted 300
with 300 l of 1% SDS in PBS, and absorbance was measured at 570 nm using FLUOstar OPTIMA 301
microplate reader (BMG Labtech in Ortenberg, Germany). Cell numbers were normalized to protein 302
concentrations measured using Precision Red reagent (Cytoskeleton in Denver, Colorado, USA) in the 303
microplate reader according to the manufacturer’s protocol. 304
For spreading assay, 24 h after plating, tetracycline was added with or without PKA inhibitor, 305
and cells were analyzed after 4 h or observed for 24 h by time-lapse microscopy. Cell images were 306
analyzed using ImageJ 1.48 (Fiji) digital analysis software (National Institute of Health in Bethesda, 307
Maryland, USA), and the planar cell surface was calculated. 308
309
Western blotting and immunoprecipitation 310
For western blotting, 0.1-0.2 x 106 cells were plated in 48-well plates in three conditions: (1) normal 311
MDCK cells alone, (2) MDCK pTR-GFP-RasV12 cells alone, and (3) a 1:1 mixed population. 312
Tetracycline was added either 8-10 h from plating or after overnight incubation. Inhibitors were added 313
with tetracycline for 16-24 h. Cells from each well of conditions (1) and (2) were combined for ‘Alone’ 314
fractions, whereas ‘Mixed’ fractions were from 2 wells of condition (3). Western blotting data were 315
quantified using ImageJ software. For immunoprecipitation, cells were plated in three conditions: 316
normal MDCK cells alone, MDCK pTR-GFP-RasV12 cells alone, and a 1:1 mixed population at 4.0 x 317
108 cells per a 15-cm plate. Tetracycline was added 2 h after plating. After 6 h, cells were washed with 318
ice-cold PBS, and harvested in 1 ml GB buffer (10 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 1% 319
Triton X-100, 0.5% sodium deoxycholate, and 0.2% sodium dodecylsulfate) containing 5 g ml-1 320
leupeptin, 50 M phenylmethylsulfonylfluoride, 7.2 trypsin inhibitor units of aprotinin, 50 ng/ml 321
benzamidine, 10 ng/ml pepstatin A, and phosphatases inhibitors (1 mM sodium orthovanadate, 0.1 mM 322
ammonium molybdate, 10 mM sodium fluoride, and 24 nM calyculin A). Samples were homogenized 323
with a syringe (needle of 25 G) and incubated with prewashed sepharose beads for 30 min at 4oC. After 324
centrifugation at 14,000 x g for 10 min at 4oC, 50 l of the precleared supernatants were taken as the 325
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‘TOT’ fraction (total cell lysate). The rest of the supernatants were subjected to immunoprecipitation 326
for 2 h with 50 l of prewashed protein G beads conjugated with 2 g of control mouse-IgG or anti-327
His-tag antibody. The beads were subsequently washed twice with the GB buffer and once with PBS. 328
Bound proteins were eluted with 100 l SDS-PAGE sample buffer for 10 min at 99oC, followed by 329
western blotting. 330
331
Data analyses 332
Two-tailed Student’s t tests were used to determine P values. 333
334
SILAC labeling combined with Mass Spectrometry 335
Medium preparation - Medium for SILAC labeling was prepared as follows: 13.26 g of arginine- and 336
lysine-deficient DMEM powder (Caissons Laboratories in North Logan, Utah, USA) was mixed with 337
3.7 g of sodium bicarbonate, and dissolved in 1 L ultrapure autoclaved water. pH was adjusted to 7.1 338
and medium was filtered. Before culturing MDCK cells, the medium was supplemented with dialyzed 339
FBS (30 MWCO) (Live Technologies in Paisley, Scotland, UK), as well as penicillin/streptomycin and 340
GlutaMax (Live Technologies in Paisley, Scotland, UK). Labeled amino acids (Sigma-Aldrich in 341
Gillingham, Dorset, UK) were added at a final concentration of 50 g/L as follows: for a “light” label, 342
lysine-0 (unlabeled) and arginine-0 (unlabeled); for a “medium” label, lysine-4 ([2D4]-labeled) and 343
arginine-6 ([13C6]-labeled); for a “heavy” label, lysine-8 ([13C6], [15N4]-labeled) and arginine-10 ([13C6], 344
[15N4]-labeled). 345
Labeling of Ras and MDCK cells - MDCK and two sets of Ras cells were labeled for five 346
generations in SILAC medium with “light”, “heavy” and “medium” amino acids, respectively. The 347
labeling efficiency was calculated from MS analysis of labeled lysates. In short, cell lysates were 348
mixed (heavy to light in a 1:1 ratio or medium to light in a 1:1 ratio) and separated by SDS-PAGE. 349
Two gel bands of high and low molecular weight were excised, digested and analyzed by LC-MS. 350
Relative quantification of heavy to light ratio and medium to light ratio in these proteins revealed a 351
labeling efficiency of 98% for both arginine and lysine isotopic labels. 352
Cell culture and lysis - Cells were plated at 4 x 107 cells per a 15-cm plate in two conditions: 353
“medium” Ras cells were plated alone in 2 plates, while “heavy” Ras cells were mixed in a 1:1 ratio 354
with “light” MDCK cells in 4 plates. The high cell density ensured that cells reattached and 355
reestablished junctions rapidly. All the cells were then cultured in “light” medium for 2 h, and after 356
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tetracycline addition, cells were further incubated in “light” medium for 6 h. We confirmed that during 357
the 8 h incubation in “light” medium, label exchange was not higher than 10-20%. Cells were washed 358
with TBS and lysed in 10 ml of 100 mM Tris/Cl pH 8.0, 9 M Urea, and 0.2 mM sodium vanadate. The 359
cell lysates were combined in a tube and sonicated (3 x 15 s each). After spinning down for 10 min at 360
10,000 g, the supernatant was collected in a fresh tube. To reduce disulfide bonds, DTT was added to a 361
final concentration of 5 mM, followed by incubation at 55oC for 15 min. After cooling down, 362
alkylating agent iodoacetamide was added up to a final concentration of 10 mM, and the tube was 363
incubated for further 15 min in the dark. Finally, the lysates were topped up to 45 ml with 100 mM 364
Tris/Cl pH 8.0, and 20 g trypsin was added at a 1:100 ratio. After digestion for 48 h, the obtained 365
peptides were kept at 4oC. 366
Purification of peptides and Isolation of phospho-peptides - Sep-Pak C18 Plus Short Cartridges 367
(Waters in Milford, Massachusetts, USA) were used to purify peptides. The column was activated by 368
application of 5 ml acetonitrile (MeCN) and washed twice with 7 ml of 0.1% trifluoroacetic acid 369
(TFA). TFA was added to the peptide solution at a final concentration of 1%. The lysate was then spun 370
down at 5,000 g for 5 min, and the supernatant was loaded onto the column. The column with bound 371
peptides was subsequently washed twice with 10 ml of 0.1% TFA. Peptides were eluted with 8 ml of 372
50% MeCN/0.1% TFA, and the eluates were frozen for at least 30 min on dry ice and finally dried in a 373
Savant SC250EXP SpeedVac concentrator (Thermo Scientific in Hemel Hempstead, Hertfordshire, 374
UK). For isolation of phosphopeptides, dried peptide pellets were resuspended in 25% MeCN/0.1% 375
TFA to a concentration of 20 mg/ml. Phosphorylated peptides were purified using Phos-Select Iron 376
Affinity gel (Sigma-Aldrich in Gillingham, Dorset, UK). 300 l of the beads were washed with 0.1% 377
TFA, followed by two washes with 25% MeCN/0.1% TFA. 100 l of the peptide solution (2 mg of 378
peptides) was diluted to a concentration of 5 mg/ml in 150 l of 25% MeCN/0.1% TFA and applied 379
onto the gel for 2 h-rotation at room temperature. The gel with bound phosphorylated peptides was then 380
spun down for 30 s at 800 g and washed with the following buffers: twice with 0.1% TFA, once with 381
0.1% TFA/50% MeCN, twice with 1% TFA/50% MeCN, and finally once with ultrapure water. The 382
elution was performed twice with 250 l of 100 mM ammonium bicarbonate pH 9.5 for 10 min at room 383
temperature. Eluted fractions were combined, and acidified with 20 l of 20% TFA, frozen on dry ice 384
for 30 min, and dried in a vacuum centrifuge. 385
Hydrophilic-interaction liquid chromatography (HILIC) - Phosphorylated peptides isolated 386
from 10 mg of total peptides were resuspended in 500 l of 80% ACN prior to injection, and 387
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fractionated using HILIC chromatography (Ultimate 3000 HPLC system, Thermo Scientific in Hemel 388
Hempstead, Hertfordshire, UK). The following solvents were used: A (0.005% TFA), B (90% 389
CAN/0.005% TFA), and C (0.04% TFA). Samples were loaded in 90% solvent B on a 2.1 x 150 mm 390
TSKgel Amide 80 column (Hichrom in Theale, Berkshire, UK). The following gradient steps were 391
performed: 100% B for 3 min, to 90% B in 2 min, to 70% B in 20 min, up to 20% in 10 min while 392
solvent C increased from 0 to 20%. Between 12 and 44 min, 16 fractions (300 l each) were collected 393
and lyophilized for LC-MS analyses. 394
Liquid chromatography tandem mass spectrometry (LC-MS/MS) - Phosphorylated peptides 395
were identified by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) using nanoflow 396
capillary reversed-phase LC on an Eksigent NanoLC-Ultra 2D with a cHiPLC-nanoflex system 397
(Eksigent, Dublin, California) coupled to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific 398
in Hemel Hempstead, Hertfordshire, UK). ‘Trap and elute’ configuration was chosen on the NanoFlex 399
system, and two type of columns were used: a trap column (200 m x 0.5 mm) and an analytical 400
column (200 m x 15 cm), both packed with ChromXP C18-CL 3 m 120 Å. Samples dried after 401
HILIC were resuspended in 10 l of 0.1% FA and loaded at a flow rate of 5 l/min for 5 min. Elution 402
was performed at a flow rate of 300 nl/min in a linear gradient of 5% to 30% of ACN, 0.1% FA 403
solution in 120 min. Data-dependent mode switching automatically between Orbitrap MS and MS/MS 404
acquisition was used. Acquisition of survey full scan MS spectra (from m/z 400-2000) was performed 405
in the Orbitrap Velos using a resolution of 30,000 at m/z 400. Isolation and fragmentation of top 10 406
most intense ions with charge states greater than 2 was performed to a value of 3e4 in a higher energy 407
collision dissociation (HCD) cell. Normalized collision energy was set at 42%. Fragments were 408
detected in the Orbitrap with a resolution of 7,500. Ions were selected up to 2,000 counts while ion 409
accumulation times reached a maximum of 500 ms for full scans and 400 ms for HCD. Fragmented 410
peptides were excluded for duration of 60 s. 411
Data analyses - Peptide identification was performed using raw data obtained in Xcalibur 412
software (Thermo Scientific in Waltham, Massachusetts, USA) through processing in Proteome 413
Discover v1.3 software (Thermo Scientific in Waltham, Massachusetts, USA) with Mascot v2.2 search 414
engine (Matrix Science in Boston, Massachusetts, USA) and Ensembl dog database. The following 415
Mascot search parameters were used: precursor mass tolerance – 10 ppm; fragment mass tolerance – 416
0.6 Da; trypsin missed cleavage – 2; static modifications: carbamidomethylation (C); variable 417
modifications: oxidation (M), deamidated (NQ), phosphorylation (STY), arginine 13C6 (R6), arginine 418
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13C615N4 (R10), lysine 2HD4 (K4), and lysine 13C6
15N4 (K8). Filters applied to obtained peptide 419
sequence matches (PSMs) were as follows: a mascot significance threshold of at least 0.05, peptide 420
score of at least 20, peptide maximum rank of 1 and pRS phosphorylation site probabilities ≥ 85 % 421
(PhosphoRS1.0). The false discovery rate (FDR) was set to a q-value ≤ 0.01 (Percolator) for peptides. 422
Identified peptides with a “heavy” to “medium” ratio higher than 1.5 or lower than 0.5 in any of the 423
three performed experiments were chosen for preliminary search, and the ratios were compared 424
between experiments. Phosphorylated peptides for which a “heavy” to “medium” ratio was reproduced 425
in two repeats were chosen for further analysis. The identity of chosen peptides was confirmed by 426
comparing them against the dog protein database with Basic Local Alignment Search Tool (BLAST, 427
NCBI in Bethesda, Maryland, USA). Identified phosphorylation sites were aligned to sites in human, 428
mouse, and rat proteins. Current knowledge concerning characterization of these sites (regulators, 429
function) was obtained from the PhosphoSitePlus database (Cell Signaling in Boston, Massachusetts, 430
USA) website and literature in Pubmed database (NCBI in Bethesda, Maryland, USA).431
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Acknowledgements 432
We thank Patric Turowski, John Garthwaite, and Catherine Hogan for advice and contribution of 433
reagents. 434
435
Competing interests 436
The authors declare that they have no conflict of interest. 437
438
Funding 439
K.A. was supported by a fellowship from the Medial Research Council. T.R. received supports from 440
Vetenskapsrådet (21462), German Research Society (SFB 841) and European Research Council grant 441
(ERC-StG-2012-311575_F-12). C.J. is supported by a CR-UK Carrere Establishment Award 442
(A12905). K.M. and M.S.B. were supported by the The Wellcome Trust ([084678/Z/08/Z] and 443
[099173/Z/12/Z]) and the Biotechnology and Biological Sciences Research Council. Y.F. is supported 444
by Next Generation World-Leading Researchers (NEXT Program), the Takeda Science Foundation, the 445
Uehara Memorial Foundation, Daiichi-Sankyo Foundation of Life Science, and Naito Foundation. 446
447
Author contributions 448
K.A. performed the experiments and analyzed the results. K.A., K.M. and Y.F. conceived the 449
experiments and wrote the manuscript. K.A., C.J. and Y.F. designed the SILAC screen. J.S. performed 450
HILIC and LC-MS/MS. A.O., M.K., S.I., and M.B contributed to different aspects of experimental 451
work and data interpretation. P.B. and T.R. contributed to VASP constructs. 452
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Bear, J.E., T.M. Svitkina, M. Krause, D.A. Schafer, J.J. Loureiro, G.A. Strasser, I.V. Maly, O.Y. 457 Chaga, J.A. Cooper, G.G. Borisy, and F.B. Gertler. (2002). Antagonism between Ena/VASP 458 proteins and actin filament capping regulates fibroblast motility. Cell. 109, 509‐521. 459
Becker, E.M., P. Schmidt, M. Schramm, H. Schroder, U. Walter, M. Hoenicka, R. Gerzer, and J.P. 460 Stasch. (2000). The vasodilator‐stimulated phosphoprotein (VASP): target of YC‐1 and nitric 461 oxide effects in human and rat platelets. Journal of cardiovascular pharmacology. 35, 390‐397. 462
Benz, P.M., C. Blume, S. Seifert, S. Wilhelm, J. Waschke, K. Schuh, F. Gertler, T. Munzel, and T. 463 Renne. (2009). Differential VASP phosphorylation controls remodeling of the actin 464 cytoskeleton. J Cell Sci. 122, 3954‐3965. 465
Blume, C., P.M. Benz, U. Walter, J. Ha, B.E. Kemp, and T. Renne. (2007). AMP‐activated protein 466 kinase impairs endothelial actin cytoskeleton assembly by phosphorylating vasodilator‐467 stimulated phosphoprotein. The Journal of biological chemistry. 282, 4601‐4612. 468
Butt, E., K. Abel, M. Krieger, D. Palm, V. Hoppe, J. Hoppe, and U. Walter. (1994). cAMP‐ and 469 cGMP‐dependent protein kinase phosphorylation sites of the focal adhesion vasodilator‐470 stimulated phosphoprotein (VASP) in vitro and in intact human platelets. The Journal of 471 biological chemistry. 269, 14509‐14517. 472
Hansen, S.D., and R.D. Mullins. (2010). VASP is a processive actin polymerase that requires 473 monomeric actin for barbed end association. J Cell Biol. 191, 571‐584. 474
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Lindsay, S.L., S. Ramsey, M. Aitchison, T. Renne, and T.J. Evans. (2007). Modulation of 493 lamellipodial structure and dynamics by NO‐dependent phosphorylation of VASP Ser239. J 494 Cell Sci. 120, 3011‐3021. 495
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Mann, M. (2006). Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol. 7, 952‐496 958. 497
Pasic, L., T. Kotova, and D.A. Schafer. (2008). Ena/VASP proteins capture actin filament barbed 498 ends. The Journal of biological chemistry. 283, 9814‐9819. 499
Rottner, K., B. Behrendt, J.V. Small, and J. Wehland. (1999). VASP dynamics during lamellipodia 500 protrusion. Nat Cell Biol. 1, 321‐322. 501
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Scott, J.A., A.M. Shewan, N.R. den Elzen, J.J. Loureiro, F.B. Gertler, and A.S. Yap. (2006). 505 Ena/VASP proteins can regulate distinct modes of actin organization at cadherin‐adhesive 506 contacts. Mol Biol Cell. 17, 1085‐1095. 507
Smolenski, A., C. Bachmann, K. Reinhard, P. Honig‐Liedl, T. Jarchau, H. Hoschuetzky, and U. 508 Walter. (1998). Analysis and regulation of vasodilator‐stimulated phosphoprotein serine 239 509 phosphorylation in vitro and in intact cells using a phosphospecific monoclonal antibody. The 510 Journal of biological chemistry. 273, 20029‐20035. 511
Tang, V.W., and W.M. Brieher. (2013). FSGS3/CD2AP is a barbed‐end capping protein that stabilizes 512 actin and strengthens adherens junctions. J Cell Biol. 203, 815‐833. 513
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517 518
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Figure legends 519
Fig. 1. Experimental outline of the SILAC screening. (A) MDCK pTR-GFP-RasV12 cells were 520
labeled with medium (Arg 6, Lys 4) or heavy (Arg 10, Lys 8) arginine and lysine, and normal MDCK 521
cells with light (Arg 0, Lys 0) arginine and lysine. Cells were plated under two conditions: Ras cells 522
alone or 1:1 mixed culture. After 2 h, cells were incubated with tetracycline for 6 h to induce RasV12 523
expression. Phosphopeptides were isolated and analyzed by mass spectrometry. (B) Relative abundance 524
of the VASP peptide from each experimental condition. The small ‘s’ represents the detected 525
phosphorylation site. (C) Overview of identified proteins of which phosphorylation was upregulated 526
(red star) or downregulated (blue star) in Ras cells upon co-incubation with normal cells. The key 527
known functions of the selected proteins based on Gene (NCBI), UniProt and PhosphoSitePlus 528
databases are color-coded. Abbreviations of protein names are in agreement with the Gene NCBI 529
database. PM – plasma membrane, NE – nuclear envelope. 530
531
Fig. 2. Phosphorylation of VASP at S239 is increased in RasV12-transformed cells interacting 532
with normal cells. (A) Immunofluorescence of phosphorylated S239 of VASP in MDCK pTR-GFP-533
RasV12-VASP WT cells co-cultured with normal cells (top panels) or cultured alone (bottom panels) on 534
collagen gels. Cells were stained with anti-phospho-S239 VASP antibody (red) and Hoechst 33342 535
(white). Scale bars: 10 m. (B) Correlation between the levels of phospho-S239 VASP and cell height 536
of Ras-VASP WT cells surrounded by normal cells. Cells were divided into two categories according 537
to their cell shape: tall and normal height (52% and 48% of all quantified cells, respectively). Within 538
each category, cells were classified according to their phospho-S239 VASP level as: ‘-‘ (no visible 539
increase), ‘+’ (slight increase), ‘++’ (considerable increase), and ‘+++’ (strong increase). (C) 540
Immunoprecipitation of VASP in Ras-VASP WT cells. Ras-VASP WT cells and normal cells were co-541
cultured (Mix) or cultured alone and the respective cell lysate were mixed (Alo). Western blotting was 542
performed with the indicated antibodies. (D) Quantification of the ratio of pVASP/VASP in the total 543
cell lysates. Values are expressed as a ratio relative to Mix. Data are means ± s.d. from three 544
independent experiments; **P<0.005. (E) Time course of pVASP induction. Cells were cultured as 545
described in (C), and RasV12 expression was induced for the indicated times. Cell lysates were 546
examined by western blotting. After plating, the cells were cultured for 2 h (A-D) (in the same way as 547
the SILAC screening) or 20-24 h (E) prior to tetracycline addition. 548
549
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Fig. 3. Phosphorylation of VASP at S239 promotes apical extrusion. (A) Knockdown of VASP in 550
MDCK pTR-GFP-RasV12 cells. Ras cells were transfected with control siRNA (Ctrl KD) or VASP 551
siRNA (VASP KD). Cell lysates were examined by western blotting with the indicated antibodies. (B) 552
Immunofluorescence of VASP-knockdown Ras cells. Ras cells transfected with control siRNA (Ctrl 553
KD) or anti-VASP siRNA (VASP KD) were co-cultured with normal cells on glass. After induction of 554
GFP-RasV12 expression with tetracycline for 8 h, cells were stained with anti-VASP antibody (red) and 555
Alexa-Fluor-647-phalloidin (purple). Scale bars: 25 m. (C) Time-lapse observation of apical extrusion 556
of Ras cells. Ras cells were transfected with control siRNA (Ctrl KD) or VASP siRNA (VASP KD) 557
and co-cultured with normal cells on collagen gels. Images were extracted from the representative 558
time-lapse movies, and the indicated time reflects the duration of the tetracycline treatment. Red arrows 559
indicate extruded Ras cells. Scale bars: 50 m. (D) Quantification of apical extrusion of Ras cells from 560
time-lapse analyses (25 h). Data are means ± s.d. from two independent experiments (n = 174 Ctrl KD 561
cells, n = 163 VASP KD cells); *P<0.05. (E) Effect of expression of VASP proteins in VASP-562
knockdown RasV12-transformed cells on apical extrusion. Ras cells were transfected with siRNAs 563
followed by transfection with the VASP WT, VASP S239D, or VASP S239A construct. Note that 564
exogenously expressed human VASP is resistant to the siRNA targeting canine VASP. The transfected 565
Ras cells were co-cultured with normal cells, followed by tetracycline treatment for 15 h. Data are 566
means ± s.d. from four independent experiments (n = 390 Ctrl KD cells, n = 388 VASP KD cells, n = 567
373 VASP KD + VASP WT cells, n = 343 VASP KD + VASP S239D cells, n = 324 VASP KD + 568
VASP S239A cells); *P<0.05. 569
570
Fig. 4. PKA is a crucial regulator for phosphorylation of VASP and cell shape of Ras-571
transformed cells. (A) Effect of PKA inhibitor treatment on phospho-S239 VASP in mixed cultures of 572
normal and Ras cells. Ras-VASP WT cells and normal cells were co-cultured (Mix) or cultured alone 573
and the respective cell lysate were mixed (Alo). Cells were incubated with tetracycline for 16 h in the 574
presence of PKA inhibitor H89 (PKAi) and/or PKG inhibitor Rp-8-Br-PET-cGMPS (PKGi). Cell 575
lysates were examined by western blotting with the indicated antibodies. (B) Effect of PKA inhibitor or 576
activator on phospho-S239 VASP in Ras cells. Ras cells were cultured for 4 h with PKA activator 577
dibutyryl-cAMP (DBcAMP) at the indicated concentrations or with PKA inhibitor H89. Cell lysates 578
were examined by western blotting with the indicated antibodies. (C) Quantification of the effect of co-579
culturing Ras and normal cells on phosphorylation of S157 VASP. Cell lysates after 20 h incubation 580
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with tetracycline were analyzed by western blotting using anti-VASP antibody. Data are means ± s.d. 581
from three independent experiments. **P<0.005. (D, E) Analyses of phosphorylated substrates of PKA 582
in normal cells and Ras cells co-cultured (Mix) or cultured alone and the respective cell lysate were 583
mixed (Alo). Cells were incubated with tetracycline for 20 h. In panel E, during the tetracycline 584
treatment, cells were incubated in the absence (Ctrl) or presence (PKAi) of PKA inhibitor H89. Cell 585
lysates were examined by western blotting with the indicated antibodies. (F) Immunofluorescence 586
analyses of phosphorylated substrates of PKA. Ras cells were co-cultured with normal cells (top 587
panels) or cultured alone (bottom panels) on collagen gels. After 20 h incubation with tetracycline in 588
the absence or presence of H89 (PKAi), cells were stained with anti-phosphorylated substrates of PKA 589
(PKA-S) antibody and Hoechst 33342 (white). Scale bars: 10 m. 590
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Fig. S1. Non-cell-autonomously regulated phosphorylations. A list of proteins in which 591
phosphorylation was upregulated (A) or downregulated (B) in Ras cells co-cultured with normal cells, 592
compared with Ras cells cultured alone. Abbreviations of protein names are in agreement with the 593
Gene NCBI database. Phosphorylated amino acid residues are marked with bold letters within the 594
peptide sequences. ‘(-)’ – the phosphorylated residue does not have an equivalent one in the human 595
orthologue. ‘H/M ratio’ – heavy to medium ratio of the isotopic labels for a given peptide. ‘N/A’ – not 596
applicable (peptide was not identified or the obtained data were insufficient for quantification). 597
598
Fig. S2. Analyses of VASP function. (A) Establishment of MDCK pTR-GFP-RasV12 cells stably 599
expressing His-VASP WT, -VASP S239D, or -VASP S239A. Cell lysates were examined by western 600
blotting with the indicated antibodies. (B) Immunofluorescence of phosphorylated S239 of VASP in 601
MDCK pTR-GFP-RasV12-VASP WT cells co-cultured with normal cells (left panels) or cultured alone 602
(right panels) on glass. Cells were stained with anti-phospho-S239 VASP antibody (red). Scale bars: 25 603
m. We have noticed that subcellular localization of p-VASP is slightly different depending on the 604
experimental condition; p-VASP is mainly localized at the cell cortex when cells are cultured on glass 605
(Fig. S2B), whereas a more cytosolic staining pattern is observed in cells cultured on collagen gels 606
(Fig. 2A), though non cell-autonomous upregulation of p-VASP staining is observed under both 607
experimental conditions. The nature of this discrepancy is not known at present, but it may result from 608
the different immunofluorescence procedure between the two conditions (e.g. fixation time). (C) 609
Analyses of phospho-S239 levels in endogenous VASP proteins in normal cells and Ras cells co-610
cultured (Mix) or cultured alone and the respective cell lysate were mixed (Alo). Expression of RasV12 611
was induced by tetracycline treatment for the indicated times, and cell lysates were examined by 612
western blotting with the indicated antibodies. (D) Analyses of phospho-S239 VASP without induction 613
of GFP-RasV12 expression in normal cells and Ras-VASP WT cells co-cultured (Mix) or cultured alone 614
and the respective cell lysate were mixed (Alo). Cells were cultured for 30 h from plating, and cell 615
lysates were examined by western blotting with the indicated antibodies. (E) Comparison of VASP 616
expression between different experimental conditions. Ras cells and Ras-His-VASP WT cells were 617
transfected with control siRNA (Ctrl KD) or VASP siRNA (VASP KD). Cell lysates were analyzed by 618
western blotting with the indicated antibodies. (F) Immunofluorescence of VASP-knockdown MDCK 619
pTR-GFP cells. GFP cells transfected with control siRNA (Ctrl KD) or anti-VASP siRNA (VASP KD) 620
were co-cultured with normal cells on collagen gels. After induction of GFP expression with 621
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tetracycline for 24 h, cells were stained with Alexa-Fluor-647-phalloidin (purple). Scale bars: 25 m 622
(xy sections), 10 m (xz sections). (G) Immunofluorescence of VASP-WT MDCK cells. Ras-VASP 623
WT cells were co-cultured with normal cells or cultured alone for 30 h without induction of GFP-624
RasV12 expression on collagen gels. Cells were stained with anti-His-tag antibody (green) and Alexa-625
Fluor-647-phalloidin (purple). Scale bars: 10 m. (H) Effect of overexpression of VASP proteins on 626
morphology of Ras cells. Ras cells stably expressing VASP WT, VASP S239D, or VASP S239A were 627
examined after 15 h of tetracycline treatment. Scale bars: 50 m. (I) Effect of expression of VASP 628
proteins in VASP-knockdown Ras cells on cell morphology. Ras cells, Ras-VASP WT cells, Ras-629
VASP S239D cells, or Ras- VASP S239A cells were transfected with control siRNA (Ctrl KD) or 630
VASP siRNA (VASP KD). Planar cell surface was analyzed after 15 h of tetracycline treatment. Data 631
are means ± s.d. (cells from three independent experiments; n = 1,324 Ras + Ctrl KD cells, n = 1,293 632
Ras + VASP KD cells, n = 1,148 Ras-VASP WT + VASP KD cells, n = 533 Ras-VASP S239D + 633
VASP KD cells, n = 530 Ras-VASP S239A + VASP KD cells); *P<0.05, **P<0.005, ***P<0.001. (J) 634
Effect of expression of VASP protein in MDCK cells on cell morphology. Planar cell surface was 635
analyzed in MDCK cells, Ras cells, and Ras-VASP WT cells cultured without induction of GFP-RasV12 636
expression. Data are means ± s.d. (cells from three independent experiments; n = 1,887 MDCK cells, n 637
= 2,158 Ras cells, n = 2,037 Ras-VASP WT cells. (K) Effect of VASP-knockdown and overexpression 638
of VASP proteins on the initial cell attachment to the substratum. The indicated cell lines were 639
transfected with control siRNA (Ctrl KD) or anti-VASP siRNA (VASP KD) and cultured for 3 days. 640
Cells were then trypsinized and plated on type-I collagen-coated dishes. Following 30 min incubation, 641
unattached cells were washed off, and the remaining cells were stained with crystal violet. Cellular 642
attachment values were obtained from absorbance of the dye trapped in the remaining cells measured in 643
a plate reader at 570 nm. Data are means ± s.d. from three independent experiments. *P<0.05. Values 644
are expressed as a ratio relative to Ras + CTRL KD. 645
646
Fig. S3. Analyses of PKA signaling. (A) Analyses of phospho-S239 VASP upon induction of RasV12 647
expression. Normal or Ras cells were incubated with tetracycline for the indicated times. Cell lysates 648
were examined by western blotting with the indicated antibodies. (B-D) Effect of inhibitors for PKG 649
and/or PKA on phospho-S239 VASP in Ras cells. Ras cells were incubated with tetracycline in the 650
absence (Ctrl) or presence of PKG inhibitor Rp-8-Br-PET-cGMPS (PKGi) (B), PKA inhibitor H89 651
(PKAi) (C), or both inhibitors (PKGi/PKAi) (D) for the indicated times. Cell lysates were examined by 652
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western blotting with the indicated antibodies. (E) Effect of PKA inhibitors on phospho-S239 VASP in 653
Ras cells. Ras cells were cultured for 4 h with PKA inhibitor KT5720 at the indicated concentrations or 654
with PKA inhibitor H89. Cell lysates were examined by western blotting with the indicated antibodies. 655
(F) Quantification of immunofluorescence intensity of phosphorylated substrates of PKA in Ras cells 656
surrounded by normal cells (RM) or cultured alone (RR) (representative images are shown in Fig. 4F). 657
Cells were incubated with tetracycline for 20 h in the absence (Ctrl) or presence (PKAi) of PKA 658
inhibitor H89. Data are means ± s.d. (n = 9 RM cells, n = 5 RR cells, n = 5 RM PKAi-treated cells, n = 659
6 RR PKAi-treated cells). (G) Quantification of the effect of PKA inhibitor and activator on spreading 660
of Ras cells. Ras cells were cultured with tetracycline for 4 h in the presence of H89 (PKAi) and/or 661
DBcAMP. Average planar cell surface was measured. Data are means ± s.d. (cells from three 662
independent experiments; n = 1,589 Ctrl cells, n = 1,276 PKAi-treated cells, n = 1,680 DBcAMP-663
treated cells, n = 1,405 PKAi/DBcAMP-treated cells); *P<0.05, **P<0.005, ***P<0.001. (H) Effect of 664
PKA inhibitor or PKA activator on the height of the intercellular lateral membrane between Ras cells. 665
Ras cells were incubated with tetracycline for 4 h in the absence (Ctrl) or presence of PKA inhibitor 666
H89 (PKAi) or PKA activator DBcAMP (DBcAMP), followed by staining with Alexa-Fluor-647-667
phalloidin. The length of the lateral membrane was measured in xz confocal sections. Data are means ± 668
s.d. (cells from three independent experiments; n = 40 contacts of Ctrl cells, n = 29 contacts of PKAi-669
treated cells, n = 43 contacts of DBcAMP-treated cells); ***P<0.001. (I) Effect of PKA inhibitor on 670
spreading of VASP-knockdown Ras cells. Ras cells were transfected with control siRNA (Ctrl KD) or 671
VASP siRNA (VASP KD) and cultured for 2 days. Cells were then plated and incubated with 672
tetracycline for 4 h in the absence or presence of H89 (PKAi). Data are means ± s.d. (cells from three 673
independent experiments; n = 1,373 Ras + Ctrl KD cells, n = 1,188 Ras + VASP KD cells, n = 819 Ras 674
+ Ctrl KD PKAi-treated cells, n = 1224 Ras + VASP KD PKAi-treated cells); *P<0.05, **P<0.005, 675
***P<0.001. 676
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