A functional siRNA screen identifies genes modulating angiotensin II-mediated

EGFR transactivation

Amee J. George1,3,4,5, Brooke W. Purdue1, Cathryn M. Gould4, Daniel W. Thomas4, Yanny

Handoko4, Hongwei Qian6, Gregory A. Quaife-Ryan1, Kylie A. Morgan3, Kaylene J. Simpson2,4,5,

Walter G. Thomas1*# and Ross D. Hannan1,2,3,4,7,8*

1 School of Biomedical Sciences, The University of Queensland, St. Lucia, Queensland, 4072,

Australia; 2 Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville,

Victoria, 3010, Australia; 3 Oncogenic Signalling and Growth Control Program, Peter MacCallum

Cancer Centre, East Melbourne, Victoria, 3002, Australia; 4 The Victorian Centre for Functional

Genomics, Peter MacCallum Cancer, East Melbourne, Victoria, 3002, Australia; 5 Department of

Pathology, The University of Melbourne, Parkville, Victoria, 3010, Australia; 6 Baker IDI Heart and

Diabetes Institute, Melbourne, Victoria, 3004, Australia; 7 Department of Biochemistry and

Molecular Biology, The University of Melbourne, Parkville, Victoria, 3010, Australia; 8 Department

of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia.

* Both authors share senior authorship of this manuscript

# Corresponding author

Corresponding author address:

Professor Walter G. Thomas

School of Biomedical Sciences

The University of Queensland

St. Lucia QLD 4072 Australia

Telephone: +61 7 3365 3034

Facsimile: +61 7 3365 1766

Email: w.thomas@uq.edu.au

Running title: Genes modulating EGFR transactivation

© 2013. Published by The Company of Biologists Ltd.Jo

urna

l of C

ell S

cien

ceA

ccep

ted

man

uscr

ipt

JCS Advance Online Article. Posted on 17 September 2013

2

Summary

The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR)

to mediate cellular growth, although the molecular mechanisms are not resolved. To address this, we

performed a functional siRNA of the human kinome in human mammary epithelial cells that

demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes that both positively

and negatively regulate AT1R-EGFR transactivation. Many candidates comprised components of

EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, had not been

previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA

attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but not following

direct stimulation of the EGFR with EGF, indicating that these genes function between the activated

AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely

to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in

response to another GPCR ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR

crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new

signalling mediators important for cell biology related to tissue remodelling in cardiovascular disease

and cancer.

Keywords: angiotensin/EGFR/G protein-coupled receptor/siRNA/transactivation

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

3

Introduction

Apart from its well-studied roles in vasoconstriction, aldosterone release, and fluid balance,

angiotensin II (AngII), the principal effector of the renin-angiotensin system (RAS), influences a

selection of homeostatic and modulatory processes that can also serve as targets for dysregulation

and subsequent development of pathological states. These include cellular growth (hypertrophy) and

remodelling (fibrosis); dyslipidaemia; endothelial dysfunction, aneurysms and angiogenesis (Iwai

and Horiuchi, 2009; Lu et al., 2008; Mehta and Griendling, 2007; Weiss et al., 2001); pro-

inflammatory responses (Rompe et al., 2010; Schiffrin, 2010); stem cell programming and

haematopoiesis (Heringer-Walther et al., 2009; Park and Zambidis, 2009; Zambidis et al., 2008);

neuromodulation including cognition, memory and dementia (Li et al., 2010; Miners et al., 2009) and

more recently described in cancer, as reviewed by us (George et al., 2010).

AngII acts primarily on the angiotensin type 1 receptor (AT1R), a G protein-coupled receptor

(GPCR) encoded by a single gene (AGTR1) in humans and two homologous isoforms in rodents

(AT1A and AT1B) (de Gasparo et al., 2000; Hunyady and Catt, 2006; Mehta and Griendling, 2007).

Activated AT1Rs couple to the heterotrimeric G proteins Gq/11 (to stimulate phospholipase C β-

mediated calcium mobilisation), Gi/o, G12/13 and Gs, as well as the monomeric G proteins (e.g. Rho,

Ras and Rac) (de Gasparo et al., 2000; Guilluy et al., 2010). The activated AT1R also couples to

soluble and receptor tyrosine kinases, the mitogen-activated protein kinases (MAPK) (extracellular

regulated kinases, ERK1/2, p38 MAPK and Jun N-terminal kinase), the JAK-STAT pathway, the

generation of reactive oxygen species and various ion channels (de Gasparo et al., 2000; Hunyady

and Catt, 2006; Mehta and Griendling, 2007). Signal termination is mediated by receptor

phosphorylation and recruitment and binding of arrestins (Qian et al., 2001), which terminate initial

signalling, mediate receptor endocytosis and also act as scaffolds to support secondary and tertiary

signalling complexes (Shenoy and Lefkowitz, 2011).

Daub and colleagues first reported that the epidermal growth factor receptor (EGFR) and the neu

oncoprotein (ErbB2/HER2) are phosphorylated when stimulated with GPCR agonists endothelin 1,

lysophosphatidic acid (LPA) and thrombin, which could be inhibited when cells were treated with

EGFR antagonist AG1478, suggestive of a role for GPCRs in receptor tyrosine kinase signalling

cross-talk (Daub et al., 1996). Since then, we and others have demonstrated that the AT1R can

‘hijack’ EGFR familly signalling machinery and this is crucial for the downstream AT1R-mediated

growth signalling and functional effects, such as cellular hypertrophy and/or proliferation (Asakura

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

4

et al., 2002; Eguchi et al., 2001; Mifune et al., 2005; Thomas et al., 2002). Various mechanisms have

been described to explain AT1R-EGFR transactivation, including metalloproteinase-mediated EGF

ligand shedding (Mifune et al., 2005; Ohtsu et al., 2006), Ca2+-mediated Pyk2 activation (Eguchi et

al., 1999), or direct interaction of the EGFR with the activated AT1R, via its tyrosine 319 residue

(Seta and Sadoshima, 2003), though this does not appear to be the mechanism in a rat cardiomyocyte

model (Smith et al., 2011).

To date, most studies have utilised a candidate approach to identify the proteins that are involved in

AT1R-mediated EGFR transactivation in specific cell types. While this has been informative, more

often than not, they fail to give the broader mechanistic picture of the underlying biology. Thus, the

exact molecular mechanism linking the AT1R to the EGFR remains poorly defined and much detail

is lacking. To address this issue, we generated a robust and tractable human cellular model of AT1R-

EGFR transactivation, and used the model to perform functional siRNA screening to unbiasedly

identify novel genes involved in the AT1R-EGFR transactivation process.

Results

Generation and characterisation of a stable cellular model of AT1R-EGFR transactivation

In order to examine the molecular mechanisms underlying AT1R-EGFR transactivation, a human

cellular model was generated by introducing the AT1R into HMEC-LST cells (HMEC-LST-AT1R)

using retroviral delivery of a vector that co-expresses mCherry. The AT1R-expressing cell population

was FACS enriched, and a heterogeneous population of cells displaying mCherry expression (Fig.

1A) were collected and expanded for further analysis. We confirmed expression of the ectopically

expressed AT1R protein bearing an N-terminal HA epitope tag using western blot analysis (Fig. 1B).

Immunofluorescent detection of the AT1R revealed localisation of expression to the cell surface (Fig.

1C and Supplementary Fig. 1). Competition [125I]-AngII radioligand binding assays revealed a Kd for

AngII of 1.7 nM and a calculated receptor density of 1.87 pmol receptor/ mg cellular protein (Fig.

1D). The AT1R expressing cells demonstrated a robust, dose-dependent Ca2+ transient upon AngII

stimulation, with an EC50 value of 24.8 nM, indicating appropriate coupling to Gq/11 (Fig. 1E).

Stimulation of the HMEC-LST-AT1R cells with 100 nM AngII led to the robust and reproducible

phosphorylation of EGFR (pY1068) and ERK1/2 (p42/44; pERK1/2) (Fig. 2A). Quantitation of

western blot data revealed a 2-fold activation of EGFR above unstimulated (Fig. 2B) and a 2.5-fold

activation of ERK1/2 above unstimulated upon AngII treatment (Fig. 2C). Maximal activation of

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

5

EGFR and ERK1/2 occurred between 5-10 minutes (Supplementary Fig. 2), whereas activation of

AKT (Ser473), which can also be activated downstream of EGFR and PI3K was not observed,

revealing the MAP kinase pathway is a prominent pathway activated in response to AngII

stimulation. As is well described, other pathways do exist for ERK1/2 activation from GPCRs

(namely Ca2+, PKC and arrestins), therefore, to confirm that activation of the EGFR and downstream

ERK1/2 by AngII stimulation was due to transactivation of the EGFR by the AT1R, HMEC-LST-

AT1R cells were pretreated for 30 minutes with EGFR (AG1478) or AT1R (candesartan cilexetil)

antagonists prior to stimulation with AngII (Fig. 2D). The results show that phosphorylation of

EGFR and ERK1/2 was abrogated when cells were pretreated with either inhibitor, demonstrating

that activation of EGFR and ERK1/2 is due to the transactivation of the EGFR by ligand (AngII)

activation of the AT1R. These findings were also confirmed when the AT1R was introduced into

another human mammary epithelial cell line, HMEC-HMLE-AT1R (Supplementary Fig. 3).

AT1R-EGFR transactivation is inhibited by siRNAs targeting known transactivation components

We performed proof-of-concept experiments to demonstrate that the AT1R-EGFR transactivation

response can be inhibited when cells were transfected with siRNAs that target genes involved in the

process, specifically the AT1R, its cognate G protein (Gq/11) and the EGFR. We used siGENOME

SMARTpools targeting Agtr1a to knock down expression of the rat AT1R by 80% (Fig. 3A) and this

abrogated both EGFR and ERK1/2 phosphorylation upon AngII stimulation (Fig. 3B). Robust

knockdown of Gq/11 (GNAQ), essential for AT1R-mediated cardiomyocyte hypertrophy (Smith et al.,

2011) and the development of cardiac hypertrophy in mice (Wettschureck et al., 2001) also abolished

EGFR and ERK1/2 phosphorylation following AngII stimulation (Figs. 3C and D). Similarly,

knockdown of EGFR using SMARTpool siRNAs prevented EGFR and ERK1/2 phosphorylation

upon AngII stimulation (Figs. 3E and F) as well as the expected reduction in total EGFR protein.

Together, these experiments validated this system as suitable for detecting novel genes involved

AT1R-EGFR transactivation.

Functional siRNA screening of the human kinome reveals candidates that modulate the AT1R-

EGFR transactivation response

We used functional genomic screening to identify genes that modulate the AT1R-EGFR

transactivation response. Using the HMEC-LST-AT1R cells, we optimised an AT1R-EGFR

transactivation assay in microplate format for use in a siRNA screen. We determined that stimulating

the HMEC-LST-AT1R cells for 10 minutes with 100 nM AngII in 96 well microplates using the

AlphaScreen SureFire phospho-ERK1/2 assay (as a surrogate readout for AT1R-EGFR

transactivation) gave a robust 1.5-fold activation of ERK1/2 above unstimulated cells

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

6

(Supplementary Fig. 4). Furthermore, stimulation of cells with 100 ng/ml EGF for 10 minutes led to

a 2.8-fold increase in ERK1/2 activation above stimulated, while pretreatment of cells with 5 μM

AG1478 30 minutes prior to stimulation blocked AngII and EGF mediated ERK1/2 activation. This

method was adapted to an siRNA screening format (Supplementary Fig. 5, and Supplementary

siRNA Screen Data file).

We performed a primary siRNA screen using the human Dharmacon SMARTpool siRNA kinome

library encompassing 720 kinase genes, outlined in Fig. 4A, as we hypothesised that this gene set

was likely to encode products that modulate the signalling underlying AT1R-EGFR transactivation

and were potentially druggable. Robust Z-score analysis of the data and ranking of each gene from

the primary screen was performed (Supplementary siRNA Screen Data file). As expected, and also

to validate our screening approach, EGFR present in the siRNA library and independent of the

siRNA plate controls, was identified as being one of the top ‘hits’ in the screen.

We selected 50 highly ranking candidates from the primary screen for further target validation, and

performed a secondary siRNA screen using the Dharmacon siGENOME deconvoluted SMARTpool

siRNA library (4 siRNA duplexes per gene, which comprised the original SMARTpool used in the

primary screen). We took an unbiased approach in our candidate gene selection for secondary

screening, selecting candidates with the 20 highest and 20 lowest robust Z-scores. In addition, we

selected a further 10 highly ranked candidates for analysis based on literature that linked them with

AT1R or EGFR signalling, or had demonstrated some association with at least one of the other

highest ranked candidates identified by the primary screen.

For our secondary screen analysis, we could not use robust Z scoring to rank hits. This is because the

method relies on the assumption that the majority of the siRNAs will not have a biological effect

(Birmingham et al., 2009), whereas in the secondary screen, the expectation is that the majority of

the siRNAs identified (in the primary screen) are positive hits. Hence, we calculated the average

pERK1/2 value for the mock and siGFP transfected cells and normalised the pERK1/2 reading of

each deconvoluted siRNA duplex to the average of the mock and siGFP transfected cells (outlined in

the Supplementary siRNA Screen Data file). We classified a duplex as being a ‘hit’ if the fold

change was greater than 1.5 or less than -1.5 (equating to a 50% or more increase or decrease in

AngII-mediated ERK1/2 activation compared to mock/siGFP stimulated cells). The hit score was

identified as the maximum number of hits in one particular direction (either positive or negative; the

maximum hit score was 4, the minimum was 0). The duplexes that did not score within our cut-offs

were deemed to be ‘unchanged’ and thus did not contribute to the ‘hit’ score. From there, we used hit

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

7

scoring to identify high (4/4 and 3/4), medium (2/4) and low (1/4, 0/4) confidence targets for their

role in AT1R-EGFR transactivation (Fig. 4A). The list of genes analysed in the secondary screen and

validation score is located in Table 1.

The 50-gene list was further analysed using STRING 9.0 (http://string-db.org), an online database of

known and predicted physical and functional protein interactions (Szklarczyk et al., 2011) to

generate an AT1R-EGFR transactivation ‘interactome’, color coded to represent the high, medium

and low confidence scores for each candidate in the secondary screen analysis (Fig. 4B).

Interestingly, we identified 36/50 candidates from the secondary screen that were either functionally

or physically connected to at least one other candidate. Validated high and medium confidence

targets (30 in total) were implicated in signalling pathways linked to EGFR/ERBB2, PI3K/AKT,

MAPK and PKC signalling, which was predicted, as these pathways, by candidate analysis in the

literature have previously been implicated in EGFR transactivation.

We subsequently performed validation on a selection of genes from the high and medium hit list to

determine (i) if the SMARTpool siRNA was on target, and (ii) how knockdown of the target gene

affected transactivation of the EGFR (Supplementary Figs. 6A, B). We focused primarily on three

novel genes, TRIO (triple functional domain (PTPRF interacting), a Rho/Rac GEF protein

containing a kinase domain, CHKA (choline kinase alpha) and BMX (BMX non-receptor tyrosine

kinase), as these have not been previously implicated in GPCR-mediated EGFR transactivation.

TRIO, CHKA and BMX modulate AT1R-EGFR transactivation

We used SMARTpool siRNAs to knock down the expression of TRIO, CHKA and BMX (Figs. 5A,

C and E, respectively) and to confirm their contribution to AngII-stimulated activation of EGFR and

ERK1/2 (Figs. 5B, D and F). We observed a reduction in AngII-mediated EGFR and ERK1/2

activation with knockdown of TRIO, CHKA and BMX, and a modest reduction in basal ERK1/2

activity with knockdown of all three candidates. We also confirmed that individual siRNA duplexes

knocked down the target mRNA transcripts and assessed their impact on AT1R-EGFR

transactivation by western blot analysis (Supplementary Figs. 7A-F).

TRIO, CHKA and BMX function between the AT1R and the EGFR

Our data are consistent with TRIO, CHKA and BMX acting downstream of the AT1R, but upstream

of the EGFR. To test this, we reasoned that the phosphorylation of the EGFR and ERK1/2, by direct

stimulation with EGF ligands to bypass the AT1R (see Fig. 6A), should not be affected by the

knockdown of TRIO, CHKA and BMX if they function downstream of the AT1R, but upstream of

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

8

the EGFR. We therefore knocked down TRIO, CHKA and BMX using SMARTpool siRNAs in

HMEC-LST-AT1R cells and stimulated with either 100 nM AngII or 0.5 ng/ml EGF for 10 minutes

(Fig. 6B, C and D and Supplementary Figs. 8A-C). Knockdown of TRIO, CHKA or BMX in

HMEC-LST-AT1R cells did not prevent activation of EGFR and ERK1/2 by direct stimulation with

EGF, indicating that they are likely to sit mechanistically between the AT1R and EGFR in

transactivation. Furthermore, our analysis revealed that pre-treatment of cells with 5 μM AG1478

abrogated AngII-mediated ERK1/2 activation following TRIO, CHKA and BMX knockdown,

implying that the activation of ERK1/2 is primarily EGFR dependent, and that candidate knockdown

combined with AG1478 treatment may also eliminate EGFR independent and dependent signalling,

respectively (Supplementary Figs. 9A-C). Moreover, TRIO, CHKA and BMX knockdown did not

have a dramatic impact on Ca2+ mobilisation upon AngII stimulation (Supplementary Figs. 10A, B)

suggesting that the mechanism of action of these candidates is unlikely due to changes in AngII-

mediated intracellular Ca2+ levels.

Mechanistic insights into the function of TRIO, CHKA and BMX in GPCR-mediated EGFR

transactivation

We further investigated the mechanism(s) by which the three lead candidates identified in our siRNA

screen act to modulate AT1R-EGFR transactivation. TRIO contains two guanine nucleotide

exchange factor domains that can activate RhoA and Rac1. We took a genetic approach to determine

whether RAC1 or RHOA, downstream of TRIO, modulated the AT1R-EGFR transactivation

response. We achieved > 90% knockdown of RAC1 and RHOA mRNA transcripts at 24 hours using

SMARTpool siRNAs (Fig. 7A). Furthermore, we observed a reproducible robust reduction in EGFR

and ERK1/2 activation with RAC1 knockdown (Fig. 7B), though RAC1 knockdown also had a

modest effect on total EGFR expression. RHOA knockdown lead to a reduction in EGFR activation,

but only minimal reduction in AngII-mediated ERK1/2 activation (Fig. 7C). To further elucidate

whether RAC1 is involved in AT1R-EGFR transactivation, we pretreated the HMEC-LST-AT1R

cells for 30 minutes with 100 μM NSC-23766 compound (Fig. 7D), which lead to a reduction in

AngII-mediated EGFR and ERK1/2 activation. We also examined the requirement of CHKA activity

for AngII-mediated EGFR transactivation. Pretreatment of cells with 10 μM CK37, an inhibitor of

CHKA activity, for 30 minutes prior to AngII stimulation, also led to a reduction in EGFR and

ERK1/2 activation, with no observable effect on total CHKA (ChoK) protein expression (Fig. 7E).

We next tested the parental HMEC-LST cell line with a selection of GPCR ligands (endothelin-1,

thrombin and 17-β-oestradiol) to determine whether or not we could achieve GPCR-mediated EGFR

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

9

transactivation with endogenous GPCRs, as a prelude to determining whether our identified

screening candidates generically modulate GPCR-EGFR transactivation (Supplementary Fig. 11A).

Only thrombin was able to elicit a transactivation response. We next took advantage of our

observation that thrombin also promoted robust ERK1/2 signalling via EGFR transactivation in the

parental HMEC-LST cell line to determine whether TRIO, CHKA and BMX knockdown modulates

GPCR-EGFR transactivation (Fig. 7F). HMEC-LST cells stimulated with 10 nM of thrombin for 10

minutes robustly activated the EGFR and ERK1/2, which was blocked when the cells were

pretreated for 30 minutes with 5 μM AG1478. We next tested whether the knockdown of TRIO,

CHKA or BMX affected thrombin-mediated EGFR transactivation (Fig. 7G and Supplementary Fig.

11B). While thrombin–mediated EGFR and ERK1/2 phosphorylation was reduced with CHKA

knockdown, it remained unchanged when TRIO and BMX were silenced, indicating that CHKA may

be required for the transactivation of EGFR by GPCR receptors other than AT1R, whereas TRIO and

BMX are more selective and may be specific to AT1R-mediated EGFR transactivation. This data

provides evidence that both common and distinct mechanisms control GPCR-EGFR transactivation.

Discussion

The development of siRNA platform technologies to permit genome-wide, loss-of-function

screening has provided an unprecedented resource for delineating molecular and cellular processes,

including, for example, cell migration (Simpson et al., 2008), necrotic cell death (Hitomi et al.,

2008), and cell division (Kittler et al., 2007). In the present study, we used RNAi-based screening to

unbiasedly identify kinase genes involved in AngII-mediated activation of ERK1/2, specifically

those related to AT1R-EGFR transactivation. We developed and characterised a human cellular

model of AT1R-EGFR transactivation, then performed a primary kinome screen, comprising

SMARTpool siRNAs targeting 720 kinase genes, and a subsequent secondary validation screen of 50

of the highest-ranking candidates using deconvoluted siRNAs. We identified multiple candidates that

modulate the AT1R-EGFR transactivation response; these included molecules clearly related to the

EGFR/PKC/PI3K/MAPK signalling axis, as well as novel genes, such as TRIO, BMX and CHKA,

that appear to act mechanistically between the AT1R and the EGFR. CHKA is likely to be a general

mediator of GPCR-induced EGFR transactivation, whereas TRIO and BMX might act more

specifically in this process.

The capacity of EGFRs (and other growth factor receptors) to act as important conduits for multiple

GPCR-related stimuli that modulate cell growth and tissue remodelling is generally accepted. Many

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

10

studies, including our own, have linked the activation of the AT1R to the phosphorylation and

transactivation of the EGFR in cardiac, renal and vascular tissues (Eguchi et al., 2001; Eguchi et al.,

1998; Thomas et al., 2002; Touyz et al., 2002; Uchiyama-Tanaka et al., 2001). In support of the

“triple membrane passing signalling paradigm” proposed by Ullrich and colleagues (Daub et al.,

1996), some evidence exists for matrix metalloprotease (MMP) and A Disintegrin and

Metalloprotease (ADAM) shedding of EGF ligands as a mechanism for AT1R mediated EGFR

transactivation and tissue remodelling. In the kidney, ADAM17 shedding of TGFα has been

associated with renal disease (Shah and Catt, 2006); in heart, ADAM12 shedding of heparin-binding

EGF like growth factor (HB-EGF) reportedly mediates cardiac hypertrophy (Asakura et al., 2002);

and ADAM17 inhibition reduces AngII-mediated EGFR phosphorylation and the proliferation of

vascular smooth muscle cells (Ohtsu et al., 2006). In contrast, others have suggested alternative

mechanisms for EGFR transactivation, such as via intracellular kinases, including Pyk2 and Src, or

subsequent to direct interaction between the AT1R and the EGFR (Eguchi et al., 1999; Seta and

Sadoshima, 2003; Touyz et al., 2002). So, while many would accept the authenticity and importance

of EGFR transactivation by GPCRs, such as the AT1R, the mechanisms remain poorly understood. It

was this lack of clarity that compelled us to undertake and unbiased approach to identify novel

components involved in transactivation EGFR by AT1R.

To permit a functional siRNA screen, we developed a cellular model of AT1R-EGFR transactivation

which met our selection criteria: 1) the cells were of human origin and readily expandable to

generate large cell numbers required for screening; 2) they could be efficiently transfected with

siRNAs; and 3) they robustly transactivated the EGFR upon stimulation with AngII. Primary cell

models of cardiomyocytes, vascular and renal cells used in previous studies to examine AT1R-EGFR

transactivation were not amenable to siRNA screening. Therefore, we initially tested 14 human

vascular, endothelial and epithelial cell lines for their ability to transactivate the EGFR with either

ectopically or endogenously expressed AT1R, under the above criteria. While several of these cell

lines demonstrated AT1R-mediated EGFR transactivation, we ultimately selected an immortalised

human mammary epithelial cell line, stably expressing the AT1R (HMEC-LST-AT1R). This cell line

displayed high affinity and functional AT1R expression on the cell surface and exhibited appropriate

AT1R pharmacology and Gq/11-mediated signalling. Importantly, these cells robustly activated the

EGFR and ERK1/2 in response to AngII stimulation and showed efficient siRNA knockdown of

known targets, making it suitable for our screen. Additionally, these cells were selected for their

functional relevance, where AT1R overexpression has been implicated in breast cancer pathogenesis

(De Paepe et al., 2001; Rhodes et al., 2009; Tahmasebi et al., 2006). Furthermore, AT1R-EGFR

transactivation could be modulated by siRNA knockdown of either the AT1R or EGFR as well as by

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

11

targeting Gq/11, an absolute requirement for AT1R-EGFR transactivation in other cell types, including

cultured cardiomyocytes (Smith et al., 2011) and vascular smooth muscle cells (VSMC) (Mifune et

al., 2005; Ohtsu et al., 2008).

Using our cellular model, we adapted, optimised and developed an AT1R-EGFR transactivation

assay in microplate format using the AlphaScreen SureFire phospho-ERK1/2 kit as our readout. We

screened using the Dharmacon SMARTpool siRNA kinome library, as we reasoned that kinases

were likely to be important for AT1R-EGFR transactivation and any hits would be likely to be

druggable in subsequent studies on function. We ranked the candidates from the primary kinome

screen on the basis of robust Z-score, and pursued 50 highly-ranked candidates for a secondary

screen, where we classified ‘hits’ as those with more than a 1.5-fold change (in either direction)

compared to the mock and siGFP transfected cells. The result was a list of high, medium and low

confidence candidates, a number of which we pursued using a candidate-type approach to further

validate their role in AT1R-EGFR transactivation.

One of the major outcomes of our screen was the finding that many of the candidates related to the

EFGR/ErbB2/PKC/PI3K/MAPK signalling axis. While in a way expected, this observation provided

important confidence/validation that the screen was sufficiently powered to interrogate AT1R-EGFR

transactivation. The finding that ErbB2 (the common dimerising partner for ErbB receptors)

modulates the AT1R-EGFR transactivation response in our mammary epithelial cell model is

consistent with previous studies suggesting that ErbB2 is required for AngII-mediated EGFR

transactivation (Chan et al., 2006; Negro et al., 2006). It also corroborates data from other GPCRs,

for example, the thrombin-dependent PAR1 activation in MDA-MB-231 breast cancer cells that

transactivates EGFR/ErbB2 and increases cell invasiveness (Arora et al., 2008). In addition to

ErbB2, we also identified and validated a number of PKC isoforms (PKC-δ and PKC-ι) as

modulators of AT1R-EGFR transactivation, consistent with previous reports of AT1R-EGFR

transactivation and PKC-δ translocation in response to AngII in primary breast cancer cells (Greco et

al., 2003), the PKC-δ/Pyk2/Src-dependent AT1R-EGFR transactivation observed in hepatic C9 cells

(Shah and Catt, 2002) and thromboxane A2 receptor-induced EGFR transactivation, involving Gq/11

mediated PKC-δ and PKC-ε activation (Uchiyama et al., 2009). Moreover, it has not escaped our

attention that a number of hits from our screen are proteins that contain pleckstrin homology (PH)

domains (including AKT1, AKT3, TRIO, BMX and DGKH), which, along with PI3K and the PKC

isoforms, suggests that phosphoinositide biosynthesis, localisation, compartmentalisation and/or

signalling are critical factors for AT1R-EGFR transactivation. Interestingly, kinases previously

associated with both AT1R and GPCR-mediated EGFR transactivation, including Pyk2 and Src

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

12

(Andreev et al., 2001; Eguchi et al., 1999; Shah and Catt, 2002) were not strong hits identified by our

screen, which could suggest, at least in our breast epithelial cell model, that other signalling

candidates may play a more prominent role in AT1R-EGFR transactivation.

When considering potential hits, our primary interest was in candidates that had not been previously

(or at least ostensibly) associated with ErbB function. We were also seeking hits where a

demonstrable effect on EGFR/ERK activation was direct and not secondary to global effects on cell

viability or “non-specific” alterations in total EGFR abundance, both of which might result in

apparent reduction in AngII-induced ERK1/2. With this in mind, we assayed a number of the

high/medium confidence siRNA screening hits (14 in total) to quantify mRNA knockdown by qRT-

PCR and assess their performance in AT1R-EGFR transactivation. For most of these candidates, we

confirmed siRNA-mediated knockdown and observed a reduced ERK1/2 activation following AngII

stimulation. However, we noted for some candidates, a large, presumably non-specific effect on total

EGFR expression (e.g. CDC2L2), or alternatively, little apparent effect on the phosphorylation of the

EGFR (e.g DYRK1A, STYK1), indicating the effect on ERK signalling was probably independent

of EGFR transactivation. We dismissed such candidates in favour of others with clear and robust

effects on AngII-induced EGFR/ERK1/2 activation, without obvious changes in total ERK/EGFR

protein expression. We focused on three leading, proof-of-principle candidates that have not

previously been implicated in GPCR-EGFR transactivation, Trio (triple function domain (PTPRF

interacting), Chka (choline kinase alpha), and Bmx (bone marrow kinase X-linked, a member of the

Tec non-receptor tyrosine kinase family). Knockdown of these candidates selectively and strongly

abrogated AngII-mediated, but not EGF-mediated activation of the EGFR, locating their site of

action between the AT1R and the initiation of EGFR activation.

Trio (also known as UNC-73) is a particularly interesting target with respect to potential

AT1R/Gq/11/EGFR activity. The human full-length cDNA encodes a 2861 amino acid protein,

containing a serine/threonine kinase domain and two functional guanine nucleotide exchange factors

(GEF) domains, one specific for GDP/GTP exchange on RhoA and the other for Rac1 (Debant et al.,

1996). Rho/Rac activity has not, to our knowledge, been directly related to activation of the EGFR,

but their capacity to engage downstream kinases (e.g., ROCK) and affect cell growth and function is

well described. While few publications link Trio activity to AngII stimulation, AngII does

productively engage other RhoA GEFs, such as Arhgef1, with important roles in vascular tone and

hypertension (Guilluy et al., 2010). Importantly, the Trio homologue, UNC-73, was revealed in a

forward genetic screen in C. elegans as a major mediator of Gq signalling, growth, reproduction and

locomotion in worms (Williams et al., 2007). Mechanistically, Gq/11 has been identified to activate

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

13

the C-terminal Rho-specific DH-PH domain of Trio, and of the closely related protein, p63RhoGEF

(Rojas et al., 2007). Moreover, the crystal structure for activated Gq/11 and p63RhoGEF has been

solved, demonstrating that direct binding of activated Gq/11 to p63RhoGEF could specifically induce

Rho signalling independent of and in competition to PLCβ activation (Lutz et al., 2005).

Intriguingly, a recent publication that utilised a genome-wide RNAi screen in Drosophila cells to

identify regulators of AP-1 transcription factor complex activation (downstream of Gq) found that

Trio activation is a requirement for mitogenic signalling mediated by Gq, and is part of a ‘hard-

wired’ protein-protein-interaction based signalling circuitry’ that is required for sustained cellular

growth signalling and a regulator of normal and aberrant cellular growth (Vaque et al., 2013).

Together with our data, these studies provide robust evidence that Trio mediates GPCR activation of

downstream transcriptional events, in part by EGFR transactivation.

Interestingly, in our HMEC-LST-AT1R cell line, RAC1 knockdown and the NSC-23766 compound

(downstream of Trio) were both able to blunt the AT1R-EGFR transactivation response. An inhibitor

of Rac1 binding and activation by the Rac1-specific GEF domain of Trio (and Tiam1), NSC-23766

is suggested to have no demonstrable effect on RhoA or Cdc42 (Gao et al., 2004). This data suggest

that the AngII via AT1R/Gq/11 can enhance Trio Rac-GEF activity that contributes to activation of

signalling pathways and/or cytoskeletal remodelling that is permissive for AT1R-EGFR

transactivation.

Bmx is a member of the Tec family of non-receptor tyrosine kinases (Tamagnone et al., 1994) and

contains Tec homology (TH), PH, SH2, SH3 and kinase domains. Bmx is expressed in a variety of

human cell types, including bone marrow, haematopoieic and endothelial cells (Tamagnone et al.,

1994), and in the endocardium and large arteries of the mouse (Ekman et al., 1997). Bmx expression

is altered in a number of different cancers, including those of the bladder and prostate (Dai et al.,

2006; Guo et al., 2011; Jiang et al., 2007). Tec family kinases have been directly implicated in

GPCR signalling, where Gq and/or βγ subunits of the heterotrimeric G protein complex can bind to

and activate the kinase (Bence et al., 1997; Langhans-Rajasekaran et al., 1995; Tsukada et al., 1994).

Bmx, through its PH domain, binds to the thrombin activated GPCR, PAR1, and this is a

requirement for association with Shc and oncogenic activity (Cohen et al., 2010). Together, this

evidence suggests that Tec family kinases can modulate GPCR function by acting as signalling

scaffolds that allow recruitment of other binding partners and effector proteins, or as second

messengers that modulate downstream signalling activities. Indeed, with respect to our observation

that AT1R-EGFR transactivation leads to hypertrophic growth in rat cardiomyocytes (Thomas et al.,

2002), it is interesting to note that Bmx knockout mice display reduced cardiac hypertrophy in a

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

14

model of transverse aortic constriction, proposing that Bmx is required for the morphological

responses to pressure overload in the heart (Mitchell-Jordan et al., 2008). Combined with our finding

that BMX knockdown prevents AT1R-EGFR transactivation, this evidence suggests a novel and

plausible signalling mechanism underpinning AT1R-EGFR cardiomyocyte hypertrophy, which

warrants further investigation.

Choline kinase alpha (Chka, ChoK) phosphorylates choline to produce phosphocholine (PCho), an

important intermediate in the generation of the key membrane phospholipid, phosphatidylcholine

(Aoyama et al., 2004). Homozygous knockout of Chka in mice results in embryonic lethality at the

blastocyst stage (Wu et al., 2008), whereas upregulation of Chka activity, or increased abundance of

choline/PCho is commonly observed in cancer (Glunde et al., 2008; Hernando et al., 2009; Miyake

and Parsons, 2012). Chka is required for growth factor induced cellular proliferation in primary

human mammary epithelial cells (Ramirez de Molina et al., 2004), while inhibition of Chka in HeLa

cells reduces the steady-state levels of phosphatidylcholine and phosphatidic acid, affecting both

PI3K and MAPK signalling (Yalcin et al., 2010). Recent evidence suggests that in breast cancer and

immortalised mammary epithelial cells, EGFR and c-Src can synergise to regulate Chka protein

expression and activity (Miyake and Parsons, 2012). Furthermore, this group demonstrated that

EGFR and Chka form a complex in the presence of c-Src, which is required for maximal EGF-

dependent cellular growth. Attempts to determine whether Chka binds to either the HA-tagged AT1R

or the EGFR upon AngII stimulation using co-immunoprecipitation were inconclusive (data not

shown).

We demonstrate that inhibition of Chka with CK37, a competitive inhibitor of Chka activity also

blunts the AT1R-EGFR transactivation response. This is particularly interesting, as CK37 has been

shown to inhibit MAPK and PI3K/AKT signalling, along with disrupting actin cytoskeleton

organisation and reducing membrane ruffling in transformed cells (Clem et al., 2011). While no

published reports link AngII/AT1R to Chka, its role in lipid biosynthesis and its binding to the EGFR

and regulation of Src-mediated growth signalling are provocative, especially considering that its

knockdown also impeded thrombin-mediated EGFR transactivation, indicating that it affects GPCR-

EGFR transactivation more generally than just the AT1R. Furthermore, it may suggest that

spatiotemporal changes within a cell upon stimulation with GPCR ligands play an essential role in

GPCR mediated EGFR transactivation, though this will require further investigation.

In conclusion, the present study screened 720 kinase genes to identify critical signalling molecules

controlling AT1R-EGFR transactivation that were potentially “druggable”, which would facilitate

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

15

subsequent studies to test their importance in pathophysiological states associated with dysregulated

AT1R signalling. Using this approach, we have uncovered a suite of new and interesting signalling

targets involved in AT1R-EGFR transactivation and have also confirmed that the

EFGR/ErbB2/PKC/PI3K/MAPK signalling axis is important in AT1R-EGFR transactivation. In

particular, this work provides the platform for investigating the molecular basis for Trio, Bmx and

Chka action in AT1R-EGFR transactivation and provides proof of principle that genome-wide

approaches will provide powerful new insights into this process and permit the assembly of the

AT1R-EGFR transactivation ‘interactome’, which is increasingly appreciated as an important

mediator of cell and tissue function.

Materials and Methods

Cell lines and culturing

Human mammary epithelial cells immortalised with human telomerase (hTERT) and transformed

with SV40 Large T and small t antigens (HMEC-LST, HMEC-HMLE) were a gift from Professor

Robert Weinberg (Whitehead Institute for Biomedical Research, Massachusetts Institute of

Technology). HEK293T cells were obtained from Open Biosystems (Thermo Fisher Scientific,

Scoresby, Australia). Unless otherwise specified, media and tissue culture consumables were

obtained from Gibco BRL (Life Technologies, Mulgrave, Australia). HMEC-LST and HMEC-

HMLE cells were maintained in HuMEC ready medium containing HuMEC growth supplements

and bovine pituitary extract (#12752-010). For serum starvation experiments, cells were washed with

Dulbecco’s phosphate buffered saline (PBS, pH 7.4) and serum starved with HuMEC basal medium

(#12753-018). Unless otherwise indicated, HuMEC media (ready or basal) was supplemented with

50 μg/ml gentamicin. HEK293T cells were grown in DMEM with 20 mM HEPES, 10% FBS and

antibiotic-antimycotic. HMEC-LST, HMEC-HMLE and HEK293T cell lines were maintained in

vented flasks (Becton Dickinson, North Ryde, Australia) in a humidified incubator at 37oC with 5%

CO2. Unless otherwise stated, incubation steps for cell culture assays were performed at 37oC with

5% CO2. Cell number was determined using a Z2 Cell and Particle Counter (Beckman Coulter, Lane

Cove, Australia). Specific details for passaging the HMEC cell lines are located in the

Supplementary siRNA Screen Data file.

Retroviral construct generation

The cloning of pRc2A/NHA-AT1A, a vector encoding the N-terminally HA-epitope tagged rat

angiotensin type Ia receptor cDNA has been described previously (Thomas et al., 1998). The

pRc2A/NHA-AT1A plasmid was digested with HindIII and the NHA-AT1A cDNA was subcloned

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

16

into the HindIII site of the pBluescript KS- plasmid (Stratagene, Agilent Technologies, Mulgrave,

Australia). The pBluescript KS-NHA-AT1A plasmid was digested with BamHI and XhoI to liberate

the HindIII-flanked NHA-AT1A cDNA, which was inserted into the MSCV-IRES-mCherry vector (a

gift from Dr. Sarah Russell, Peter MacCallum Cancer Centre, Melbourne, Australia) to generate the

MSCV-IRES-NHA-AT1A (AT1R) plasmid.

Generation of stable cell lines

HEK293T cells were co-transfected with MSCV-IRES-mCherry (mCherry, vector control) or

receptor containing MSCV-IRES-NHA-AT1A (AT1R) plasmids in the presence of an amphotrophic

packaging vector (Dr. Phillip Darcy, Peter MacCallum Cancer Centre, Melbourne, Australia) at a 2:1

ratio. Viral supernatant was filtered through a 0.45 μm Minisart filter (Sartorius Stedim, Dandenong

South, Australia) and Sequabrene (Sigma-Aldrich, Castle Hill, Australia) was added at a final

concentration of 4 μg/ml before addition to target cells. Cells were exposed to fresh viral supernatant

three times over 24 hours, grown to ~80% confluency and passaged twice prior to fluorescence-

activated cell sorting (FACS) using the FACS Vantage SE Diva (BD Biosciences).

Antibodies, ligands and inhibitors

Angiotensin II (human) (#2078) and Endothelin-1 (#2110) was purchased from Auspep

(Tullamarine, Australia), and Human EGF (#100-15) was purchased from Peprotech (Rocky Hill,

NJ, USA). Thrombin (human plasma, high activity, #605195), CK37 (#229103) and AG1478

(#658552) were obtained from Calbiochem (Merck Millipore, Kilsyth, Australia). NSC-23766

(#2161) was obtained from Tocris Bioscience (Abacus ALS, East Brisbane, Australia). Total EGFR

(sc-03), Total ERK 1 (sc-93), ChoK (sc-376489) and RhoA (sc-418) antibodies were obtained from

Santa Cruz Biotechnology (Dallas, TX, USA). Rac1 antibody (05-389) was purchased from Merck

Millipore. Phospho-p44/42 MAPK (ERK1/2) (#9106), Total EGFR (#4267), Phospho-AKT (Ser473)

(#4058) and Total AKT (#9272) antibodies were purchased from Cell Signalling Technologies

(Danvers, MA, USA). Phospho-EGFR (pY1068) (#44-788G) and anti-HA (high affinity,

#1867423001) were purchased from Life Technologies and Roche (Castle Hill, Australia)

respectively. 17-β-oestradiol (#E8875) and anti-α-Tubulin (#T5168) were obtained from Sigma

Aldrich. Goat anti-rabbit IgG (H+L) HRP conjugate (#170-6515) and anti-mouse (#170-6516) were

purchased from Bio-Rad (Gladesville, Australia), and polyclonal rabbit anti-rat

immunoglobulin/HRP (P0450) was purchased from DAKO (Campbellfield, Australia). Alexa 488

goat anti-rat antibody (#A-11006) was obtained from Molecular Probes (Life Technologies).

Candesartan cilexetil was a gift from Astra Zeneca (North Ryde, Australia).

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

17

GPCR-EGFR transactivation assays

HMEC-LST cells were seeded into cell culture or microplates and incubated for 24 hours at 37oC

with 5% CO2, then serum starved in HuMEC basal media for 24-48 hours prior to stimulation. For

experiments involving inhibitors, cells were pre-treated for 30 minutes at 37oC with either AG1478

(5 μM), candesartan cilexetil (1 μM), CK37 (10 μM) or NSC-23766 (100 μM). Cells were

stimulated with 100 nM AngII (or 10 nM thrombin) for specified timepoints at 37oC prior to

harvesting. The AlphaScreen SureFire phospho-ERK 1/2 assay (TGR Biosciences #TGRES10K,

Thebarton, Australia; Perkin Elmer #658552, Glen Waverley, Australia), a high-throughput

microplate-based ERK1/2 activation assay described in the literature (Osmond et al., 2005) was

performed as per the standard protocol, details of which can be found in the MIARE (Minimum

Information About an RNAi Experiment) located in the Supplementary siRNA Screen Data file.

siRNA screening and siRNA transfections

A detailed methodology for siRNA screening is outlined in the MIARE, and primary and secondary

siRNA screen data is located in the Supplementary siRNA Screen Data file, and is also publically

available on PubChem (http://pubchem.ncbi.nlm.nih.gov). siGENOME SMARTpool siRNAs to rat

Agtr1a (M-093349-00), human GNAQ (M-008562-00), EGFR (M-003114-03), BMX (M-003106-

04), CHKA, (M-006704-01), TRIO (M-005047-00), RAC1 (M-003560-06), RHOA (M-003860-03)

and the GFP duplex (D-001300-01) were obtained from Dharmacon RNAi Technologies

(ThermoFisher Scientific) and resuspended in 1x Dharmacon siRNA buffer prior to use. For siRNA

validation (quantifying mRNA knockdown and determining the effect of knockdown on

transactivation), conditions outlined from the siRNA screen were scaled up to a 12-well plate format

to harvest protein and/or RNA. Briefly, 40 nM of Dharmacon SMARTpool siRNA or 25 nM of

Dharmacon SMARTpool deconvoluted siRNA (individual duplex) were complexed for 20 minutes

at ambient temperature in a 200 μl volume of HuMEC basal media with a final concentration of 1 μl

per well of DharmaFECT 1 transfection lipid (ThermoFisher Scientific). HMEC-LST-AT1R cells

(~100,000) in antibiotic-free HuMEC complete media (800 μl volume) were seeded into each well

along with the complexed siRNA and incubated for 24 hours. For RNA experiments, RNA was

extracted from cells at a 24-hour knockdown (see RNA extraction and cDNA synthesis section for

details). For protein analysis, after a 24-hour knockdown, wells were washed once with Dulbecco’s

PBS (pH 7.4) serum starved for 48 hours in HuMEC basal media (total 72 hour knockdown) prior to

stimulation with AngII for 10 minutes at 37oC, and protein harvested.

Protein extraction, SDS-PAGE and western blot analysis

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

18

Cells were washed twice with ice cold PBS and harvested in ice cold RIPA buffer (50 mM Tris HCl

pH 7.5, 100 mM NaCl, 2 mM EDTA, 50 mM sodium fluoride, 0.1% (w/v) SDS, 0.5% (w/v) sodium

deoxycholate, 1% (v/v) Triton X-100, 10 mM sodium pyrophosphate, 10 mM sodium orthovanadate)

containing Complete EDTA-free protease inhibitor cocktail (Roche). Cells were gently lysed for 1

hour at 4oC and centrifuged at 15,000 x g for 15 minutes at 4oC. Protein concentration was

determined using the DC protein assay kit (Bio-Rad) in microplates as per manufacturers’

instructions. For the resolution of all proteins (apart from the AT1R), protein samples were mixed

with Laemmli sample buffer (Laemmli, 1970) containing 8% β-mercaptoethanol and heated at 95oC

for 5 minutes. To resolve the AT1R, freshly prepared lysate was mixed at a 1:1 ratio with 62.5 mM

Tris-HCl (pH 6.8), 2% (w/v) SDS, 6 M urea, 10% β-mercaptoethanol and 20% glycerol and heated

to 60oC for 15 minutes. Samples were electrophoresed on Tris-Glycine SDS-PAGE gels, and protein

transferred to PVDF membrane (Immobilon-P, Merck Millipore). Membranes were blocked with 5%

skim milk (Diploma, Fonterra Foodservices, Mount Waverley, Australia) or 1% BSA (Sigma-

Aldrich) in Tris buffered saline (TBS) pH 7.6 containing 0.05% (v/v) Tween 20 (TBST). Antibodies

were prepared in either 5% skim milk or 1% BSA in TBST. Membranes were washed with TBST

and treated with the relevant secondary antibody in 5% skim milk/TBST. Membranes were

developed using Western Lightning ECL Plus (Perkin Elmer) to Hyperfilm (GE Healthcare,

Rydalmere, Australia).

Immunofluorescence to detect AT1R expression

HMEC-LST cells (150,000) in HuMEC complete media were seeded into chamber slides (Nunc

Lab-Tek II, 4 chamber, BD Biosciences) and incubated for 24 hours. Chambers were washed with

PBS, fixed with 4% PFA, blocked in 1% BSA and treated with 40 ng/ml of anti-HA high-affinity

antibody in 1% BSA. Chambers were washed with PBS containing 0.05% Tween 20 (PBST). Alexa

488 goat anti-rat antibody (Molecular Probes) in 1% BSA was added to each well (protected from

light). Nuclei and cell membranes were counterstained with DAPI (Sigma-Aldrich) and Cell Mask-

Alexa Fluor 647 (Life Technologies), respectively. Slides were mounted in Vectashield (Vector

Labs, Burlingame, CA, USA) then coverslipped and sealed prior to use. Confocal images (Z stacks at

2 μm intervals) were obtained with the Nikon C2 confocal microscope using the NIS elements

AR.3.2 program (Nikon Instruments, Melville, NY, USA) at a 40X objective. Z stack images were

imported into NIH ImageJ (version 1.44o, available online at http://imagej.nih.gov/ij) (Abramoff et

al., 2004). Maximum intensity of each stack for each channel (3 channels in total) was obtained,

images were smoothed and despeckled and a scale bar added. RGB channel merge was used to

merge the respective channels. Individual images and a three channel merged image can be observed

in Supplementary Fig. 1.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

19

Radioligand competition binding assay

For competition radioligand binding assays, 500,000 HMEC-LST-AT1R or mCherry cells were

seeded into 24 well plates, and allowed to adhere for 24 hours. Culture media was replaced with

unlabelled AngII and 330,000 cpm of [125I]-AngII (Prosearch International, Malvern, Australia)

diluted in OptiMEM (Life Technologies) supplemented with 1% BSA was added and incubated at

ambient temperature for 1 hour. Wells were washed with PBS and cells solubilised with 500 μl 0.1

M NaOH. Radioactivity was counted using a 2470 Wizard 2 Automatic Gamma Counter (Perkin

Elmer). Data for the displacement of bound radiolabelled [125I]-AngII by unlabelled AngII was

collected. Concentrations of unlabelled AngII were assayed in triplicate wells and averaged over 4

independent experiments. Relative protein concentration in each well was determined using the DC

protein assay kit (Bio-Rad) as per manufacturers’ instructions, and relative number of binding sites

per amount of protein (pmol receptor/ mg cellular protein) calculated.

Calcium mobilisation assay

Cells were seeded at a density of 100,000 cells/well into 96-well plates pre-coated with poly-l-lysine,

and incubated for approximately 5 hours. Cells were washed with PBS and serum starved overnight.

The following day, cells were loaded with 2.9 μg/ml of Fluo-4AM (0.3 μg/well) in assay buffer

(HBSS, 20 mM HEPES, 2.5 mM probenecid, pH 7.4) for 45 minutes at 37oC, protected from light.

Wells were washed once with assay buffer and de-esterified for 30 minutes at ambient temperature,

protected from light. Prior to stimulation with AngII, background fluorescence of cells was imaged

for 10 seconds on the FLIPR Tetra (Molecular Devices, Sunnyvale, CA, USA). AngII (diluted in the

assay buffer at various concentrations) was added using the FLIPR Tetra where fluorescence was

measured for a total of 250 seconds/well. Data was analysed using the FLIPR Tetra Software

(Screenworks 3.1.0.3, Molecular Devices) to calculate Max-Min (10-250 seconds), then data plotted

into GraphPad Prism 5.0d (Graphpad Software, La Jolla, CA, USA) to fit curves using non-linear

regression. Concentrations of AngII were assayed in triplicate wells in each independent experiment.

To perform calcium mobilisation with siRNA knockdown, the protocol was performed as described

above with the following modifications: HMEC-LST-AT1R cells (50,000 cells/well) were reverse

transfected with 40 nM Dharmacon SMARTpool siRNA and 0.1 μl DharmaFECT 1 (per well;

duplicate plates for each condition) and incubated for 24 hours. At 24 hours, RNA was extracted

from 8 wells of one plate and pooled to assess target knockdown (described below). Th second plate

was serum starved for 48 hours (72 hour knockdown) prior to Fluo-4AM loading and proceeding

with the assay as described. Data is displayed as the percentage change in fluorescence (ΔF) over

baseline fluorescence (F0).

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

20

RNA extraction and cDNA synthesis

Cell monolayers were washed with ice-cold PBS, and RNA extracted using the Bioline Isolate RNA

mini kit (Bioline, Alexandria, Australia) as per manufacturers’ instructions. RNA was eluted from

columns with 40 μl of RNase-free H2O and the concentration determined using the Nanodrop ND-

1000 spectrophotometer (Thermo Scientific). RNA was stored at -80oC until use. For cDNA

synthesis, up to 400 ng of RNA was treated with 1U of RQ1 RNase-free DNase (Promega,

Alexandria, Australia) for 30 minutes at 37oC, then reverse transcribed for 1 hour at 50oC using the

Superscript III kit (Invitrogen, Life Technologies) with random primers (Promega). cDNA was

stored at -20oC prior to use.

Primers and real-time quantitative PCR

Primers for real-time quantitative PCR were designed over exon-exon junctions (where possible)

using NCBI Primer Blast (Primer3) (http://www.ncbi.nlm.nih.gov/tools/primer-blast) (Rozen and

Skaletsky, 2000) with predicted amplicon sizes ranging from 110-160bp. Sequences are located in

Supplementary Table 1. Primers were obtained from Geneworks (Hindmarsh, Australia),

resuspended in sterile H2O and stored at -20oC prior to use. For real-time PCR analysis, Fast SYBR

Green Master Mix (Applied Biosystems, Life Technologies), cDNA template and 300 nM of forward

and reverse primers in a final reaction volume of 20 μl was assayed. Samples were run on the

Applied Biosystems StepOne Real-Time PCR System for 40 cycles using the default machine

settings with a melt curve ramp of 0.7oC. Data was analysed using the 7000 SDS 1.1 RQ Software

(Applied Biosystems) where relative quantification of gene expression was performed and gene

expression was normalised to GAPD expression.

STRING analysis

STRING 9.0 (http://string-db.org), a publically available online database of functional protein

interactions (Szklarczyk et al., 2011) was used to generate an AT1R-EGFR transactivation

‘interactome’. The secondary siRNA screen gene list was submitted to the database using the ‘high

confidence interactions’ option. Data was redrawn in Adobe Illustrator CS5 (Adobe Systems, San

Jose, CA, USA) to incorporate the confidence level of each target identified from the siRNA screen.

Thicker connecting lines (linking the nodes) represent increasing confidence of interactions.

Densitometry analysis

Densitometry analysis of western blot data (scanned to TIFF format) was performed using NIH

ImageJ 1.44o software (Abramoff et al., 2004). The density of phospho-EGFR (pY1068) or

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

21

phospho-ERK1/2 bands were normalised to tubulin band density for each sample. Data was imported

into Microsoft Excel (Microsoft, Redmond, WA, USA), and graphed in GraphPad Prism 5.0d.

Statistical analyses

The statistical analyses performed for the siRNA screen are outlined in the Supplementary siRNA

Screen Data file. Data was analysed using paired two-tailed Student t-tests within the GraphPad

Prism 5.0d program. Unless otherwise described, data presented graphically is the mean ± standard

error of the mean (SEM), with statistical significance set at p < 0.05.

Acknowledgements

Our thanks to Mr Ralph Rossi and Ms Viki Milovak (FACS), Dr Jillian Danne (confocal

microscopy), Ms Alison Boast, Ms Analia Lesmana and Ms Anna-Kristen Szubert (technical

assistance), Associate Professor Phillip Darcy and Dr Sarah Russell (plasmid constructs) and Dr

Katherine Hannan (proofreading manuscript) (all from Peter MacCallum Cancer Centre, Melbourne,

Australia). Thanks to Professor Robert Weinberg (Whitehead Institute for Biomedical Research,

Massachusetts Institute of Technology, USA) for providing HMEC cell lines. The Victorian Centre

for Functional Genomics is funded by the Australian Cancer Research Foundation (ACRF), the

Victorian Department of Industry, Innovation and Regional Development (DIIRD), the Australian

Phenomics Network supported by funding from the Australian Government’s Education Investment

Fund through the Super Science Initiative, the Australasian Genomics Technologies Association

(AMATA) and the Brockoff Foundation. Conflict(s) of interest: none declared. This work was

supported by project grants from the Australian National Health and Medical Research Council

(NHMRC) [#472640, #1024726] awarded to W.G.T and R.D.H. R.D.H also holds an NHMRC

research fellowship.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

22

References

Abramoff, M. D., Magalhaes, P. J. and Ram, S. J. (2004). Image Processing with ImageJ. Biophotonics International 11, 36-42.

Andreev, J., Galisteo, M. L., Kranenburg, O., Logan, S. K., Chiu, E. S., Okigaki, M., Cary, L. A., Moolenaar, W. H. and Schlessinger, J. (2001). Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade. J Biol Chem 276, 20130-5.

Aoyama, C., Liao, H. and Ishidate, K. (2004). Structure and function of choline kinase isoforms in mammalian cells. Prog Lipid Res 43, 266-81.

Arora, P., Cuevas, B. D., Russo, A., Johnson, G. L. and Trejo, J. (2008). Persistent transactivation of EGFR and ErbB2/HER2 by protease-activated receptor-1 promotes breast carcinoma cell invasion. Oncogene 27, 4434-45.

Asakura, M., Kitakaze, M., Takashima, S., Liao, Y., Ishikura, F., Yoshinaka, T., Ohmoto, H., Node, K., Yoshino, K., Ishiguro, H. et al. (2002). Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy. Nat Med 8, 35-40.

Bence, K., Ma, W., Kozasa, T. and Huang, X. Y. (1997). Direct stimulation of Bruton's tyrosine kinase by G(q)-protein alpha-subunit. Nature 389, 296-9.

Birmingham, A., Selfors, L. M., Forster, T., Wrobel, D., Kennedy, C. J., Shanks, E., Santoyo-Lopez, J., Dunican, D. J., Long, A., Kelleher, D. et al. (2009). Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6, 569-75.

Chan, H. W., Jenkins, A., Pipolo, L., Hannan, R. D., Thomas, W. G. and Smith, N. J. (2006). Effect of dominant-negative epidermal growth factor receptors on cardiomyocyte hypertrophy. J Recept Signal Transduct Res 26, 659-77.

Clem, B. F., Clem, A. L., Yalcin, A., Goswami, U., Arumugam, S., Telang, S., Trent, J. O. and Chesney, J. (2011). A novel small molecule antagonist of choline kinase-alpha that simultaneously suppresses MAPK and PI3K/AKT signaling. Oncogene 30, 3370-80.

Cohen, I., Maoz, M., Turm, H., Grisaru-Granovsky, S., Maly, B., Uziely, B., Weiss, E., Abramovitch, R., Gross, E., Barzilay, O. et al. (2010). Etk/Bmx regulates proteinase-activated-receptor1 (PAR1) in breast cancer invasion: signaling partners, hierarchy and physiological significance. PLoS One 5, e11135.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

23

Dai, B., Kim, O., Xie, Y., Guo, Z., Xu, K., Wang, B., Kong, X., Melamed, J., Chen, H., Bieberich, C. J. et al. (2006). Tyrosine kinase Etk/BMX is up-regulated in human prostate cancer and its overexpression induces prostate intraepithelial neoplasia in mouse. Cancer Res 66, 8058-64.

Daub, H., Weiss, F. U., Wallasch, C. and Ullrich, A. (1996). Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature 379, 557-60.

de Gasparo, M., Catt, K. J., Inagami, T., Wright, J. W. and Unger, T. (2000). International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 52, 415-72.

De Paepe, B., Verstraeten, V. L., De Potter, C. R., Vakaet, L. A. and Bullock, G. R. (2001). Growth stimulatory angiotensin II type-1 receptor is upregulated in breast hyperplasia and in situ carcinoma but not in invasive carcinoma. Histochem Cell Biol 116, 247-54.

Debant, A., Serra-Pages, C., Seipel, K., O'Brien, S., Tang, M., Park, S. H. and Streuli, M. (1996). The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains. Proc Natl Acad Sci U S A 93, 5466-71.

Eguchi, S., Dempsey, P. J., Frank, G. D., Motley, E. D. and Inagami, T. (2001). Activation of MAPKs by angiotensin II in vascular smooth muscle cells. Metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK. J Biol Chem 276, 7957-62.

Eguchi, S., Iwasaki, H., Inagami, T., Numaguchi, K., Yamakawa, T., Motley, E. D., Owada, K. M., Marumo, F. and Hirata, Y. (1999). Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells. Hypertension 33, 201-6.

Eguchi, S., Numaguchi, K., Iwasaki, H., Matsumoto, T., Yamakawa, T., Utsunomiya, H., Motley, E. D., Kawakatsu, H., Owada, K. M., Hirata, Y. et al. (1998). Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem 273, 8890-6.

Ekman, N., Lymboussaki, A., Vastrik, I., Sarvas, K., Kaipainen, A. and Alitalo, K. (1997). Bmx tyrosine kinase is specifically expressed in the endocardium and the endothelium of large arteries. Circulation 96, 1729-32.

Elenbaas, B., Spirio, L., Koerner, F., Fleming, M. D., Zimonjic, D. B., Donaher, J. L., Popescu, N. C., Hahn, W. C. and Weinberg, R. A. (2001). Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev 15, 50-65.

Gao, Y., Dickerson, J. B., Guo, F., Zheng, J. and Zheng, Y. (2004). Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A 101, 7618-23.

George, A. J., Thomas, W. G. and Hannan, R. D. (2010). The renin-angiotensin system and cancer: old dog, new tricks. Nat Rev Cancer 10, 745-59.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

24

Glunde, K., Shah, T., Winnard, P. T., Jr., Raman, V., Takagi, T., Vesuna, F., Artemov, D. and Bhujwalla, Z. M. (2008). Hypoxia regulates choline kinase expression through hypoxia-inducible factor-1 alpha signaling in a human prostate cancer model. Cancer Res 68, 172-80.

Greco, S., Muscella, A., Elia, M. G., Salvatore, P., Storelli, C., Mazzotta, A., Manca, C. and Marsigliante, S. (2003). Angiotensin II activates extracellular signal regulated kinases via protein kinase C and epidermal growth factor receptor in breast cancer cells. J Cell Physiol 196, 370-7.

Guilluy, C., Bregeon, J., Toumaniantz, G., Rolli-Derkinderen, M., Retailleau, K., Loufrani, L., Henrion, D., Scalbert, E., Bril, A., Torres, R. M. et al. (2010). The Rho exchange factor Arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressure. Nat Med 16, 183-90.

Guo, S., Sun, F., Guo, Z., Li, W., Alfano, A., Chen, H., Magyar, C. E., Huang, J., Chai, T. C., Qiu, S. et al. (2011). Tyrosine kinase ETK/BMX is up-regulated in bladder cancer and predicts poor prognosis in patients with cystectomy. PLoS One 6, e17778.

Heringer-Walther, S., Eckert, K., Schumacher, S. M., Uharek, L., Wulf-Goldenberg, A., Gembardt, F., Fichtner, I., Schultheiss, H. P., Rodgers, K. and Walther, T. (2009). Angiotensin-(1-7) stimulates hematopoietic progenitor cells in vitro and in vivo. Haematologica 94, 857-60.

Hernando, E., Sarmentero-Estrada, J., Koppie, T., Belda-Iniesta, C., Ramirez de Molina, V., Cejas, P., Ozu, C., Le, C., Sanchez, J. J., Gonzalez-Baron, M. et al. (2009). A critical role for choline kinase-alpha in the aggressiveness of bladder carcinomas. Oncogene 28, 2425-35.

Hitomi, J., Christofferson, D. E., Ng, A., Yao, J., Degterev, A., Xavier, R. J. and Yuan, J. (2008). Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 135, 1311-23.

Hunyady, L. and Catt, K. J. (2006). Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. Mol Endocrinol 20, 953-70.

Iwai, M. and Horiuchi, M. (2009). Role of renin-angiotensin system in adipose tissue dysfunction. Hypertens Res 32, 425-7.

Jiang, X., Borgesi, R. A., McKnight, N. C., Kaur, R., Carpenter, C. L. and Balk, S. P. (2007). Activation of nonreceptor tyrosine kinase Bmx/Etk mediated by phosphoinositide 3-kinase, epidermal growth factor receptor, and ErbB3 in prostate cancer cells. J Biol Chem 282, 32689-98.

Kittler, R., Pelletier, L., Heninger, A. K., Slabicki, M., Theis, M., Miroslaw, L., Poser, I., Lawo, S., Grabner, H., Kozak, K. et al. (2007). Genome-scale RNAi profiling of cell division in human tissue culture cells. Nat Cell Biol 9, 1401-12.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-5.

Langhans-Rajasekaran, S. A., Wan, Y. and Huang, X. Y. (1995). Activation of Tsk and Btk tyrosine kinases by G protein beta gamma subunits. Proc Natl Acad Sci U S A 92, 8601-5.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

25

Li, N. C., Lee, A., Whitmer, R. A., Kivipelto, M., Lawler, E., Kazis, L. E. and Wolozin, B. (2010). Use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis. BMJ 340, b5465.

Lu, H., Rateri, D. L., Cassis, L. A. and Daugherty, A. (2008). The role of the renin-angiotensin system in aortic aneurysmal diseases. Curr Hypertens Rep 10, 99-106.

Lutz, S., Freichel-Blomquist, A., Yang, Y., Rumenapp, U., Jakobs, K. H., Schmidt, M. and Wieland, T. (2005). The guanine nucleotide exchange factor p63RhoGEF, a specific link between Gq/11-coupled receptor signaling and RhoA. J Biol Chem 280, 11134-9.

Mehta, P. K. and Griendling, K. K. (2007). Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 292, C82-97.

Mifune, M., Ohtsu, H., Suzuki, H., Nakashima, H., Brailoiu, E., Dun, N. J., Frank, G. D., Inagami, T., Higashiyama, S., Thomas, W. G. et al. (2005). G protein coupling and second messenger generation are indispensable for metalloprotease-dependent, heparin-binding epidermal growth factor shedding through angiotensin II type-1 receptor. J Biol Chem 280, 26592-9.

Miners, S., Ashby, E., Baig, S., Harrison, R., Tayler, H., Speedy, E., Prince, J. A., Love, S. and Kehoe, P. G. (2009). Angiotensin-converting enzyme levels and activity in Alzheimer's disease: differences in brain and CSF ACE and association with ACE1 genotypes. Am J Transl Res 1, 163-77.

Mitchell-Jordan, S. A., Holopainen, T., Ren, S., Wang, S., Warburton, S., Zhang, M. J., Alitalo, K., Wang, Y. and Vondriska, T. M. (2008). Loss of Bmx nonreceptor tyrosine kinase prevents pressure overload-induced cardiac hypertrophy. Circ Res 103, 1359-62.

Miyake, T. and Parsons, S. J. (2012). Functional interactions between Choline kinase alpha, epidermal growth factor receptor and c-Src in breast cancer cell proliferation. Oncogene 31, 1431-41.

Negro, A., Brar, B. K., Gu, Y., Peterson, K. L., Vale, W. and Lee, K. F. (2006). erbB2 is required for G protein-coupled receptor signaling in the heart. Proc Natl Acad Sci U S A 103, 15889-93.

Ohtsu, H., Dempsey, P. J., Frank, G. D., Brailoiu, E., Higuchi, S., Suzuki, H., Nakashima, H., Eguchi, K. and Eguchi, S. (2006). ADAM17 mediates epidermal growth factor receptor transactivation and vascular smooth muscle cell hypertrophy induced by angiotensin II. Arterioscler Thromb Vasc Biol 26, e133-7.

Ohtsu, H., Higuchi, S., Shirai, H., Eguchi, K., Suzuki, H., Hinoki, A., Brailoiu, E., Eckhart, A. D., Frank, G. D. and Eguchi, S. (2008). Central role of Gq in the hypertrophic signal transduction of angiotensin II in vascular smooth muscle cells. Endocrinology 149, 3569-75.

Osmond, R. I., Sheehan, A., Borowicz, R., Barnett, E., Harvey, G., Turner, C., Brown, A., Crouch, M. F. and Dyer, A. R. (2005). GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors. J Biomol Screen 10, 730-7.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

26

Park, T. S. and Zambidis, E. T. (2009). A role for the renin-angiotensin system in hematopoiesis. Haematologica 94, 745-7.

Qian, H., Pipolo, L. and Thomas, W. G. (2001). Association of beta-Arrestin 1 with the type 1A angiotensin II receptor involves phosphorylation of the receptor carboxyl terminus and correlates with receptor internalization. Mol Endocrinol 15, 1706-19.

Ramirez de Molina, A., Banez-Coronel, M., Gutierrez, R., Rodriguez-Gonzalez, A., Olmeda, D., Megias, D. and Lacal, J. C. (2004). Choline kinase activation is a critical requirement for the proliferation of primary human mammary epithelial cells and breast tumor progression. Cancer Res 64, 6732-9.

Rhodes, D. R., Ateeq, B., Cao, Q., Tomlins, S. A., Mehra, R., Laxman, B., Kalyana-Sundaram, S., Lonigro, R. J., Helgeson, B. E., Bhojani, M. S. et al. (2009). AGTR1 overexpression defines a subset of breast cancer and confers sensitivity to losartan, an AGTR1 antagonist. Proc Natl Acad Sci U S A 106, 10284-9.

Rojas, R. J., Yohe, M. E., Gershburg, S., Kawano, T., Kozasa, T. and Sondek, J. (2007). Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology-associated pleckstrin homology domain. J Biol Chem 282, 29201-10.

Rompe, F., Unger, T. and Steckelings, U. M. (2010). The angiotensin AT2 receptor in inflammation. Drug News Perspect 23, 104-11.

Rozen, S. and Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132, 365-86.

Schiffrin, E. L. (2010). T lymphocytes: a role in hypertension? Curr Opin Nephrol Hypertens 19, 181-6.

Seta, K. and Sadoshima, J. (2003). Phosphorylation of tyrosine 319 of the angiotensin II type 1 receptor mediates angiotensin II-induced trans-activation of the epidermal growth factor receptor. J Biol Chem 278, 9019-26.

Shah, B. H. and Catt, K. J. (2002). Calcium-independent activation of extracellularly regulated kinases 1 and 2 by angiotensin II in hepatic C9 cells: roles of protein kinase Cdelta, Src/proline-rich tyrosine kinase 2, and epidermal growth receptor trans-activation. Mol Pharmacol 61, 343-51.

Shah, B. H. and Catt, K. J. (2006). TACE-dependent EGF receptor activation in angiotensin-II-induced kidney disease. Trends Pharmacol Sci 27, 235-7.

Shenoy, S. K. and Lefkowitz, R. J. (2011). beta-Arrestin-mediated receptor trafficking and signal transduction. Trends Pharmacol Sci 32, 521-33.

Simpson, K. J., Selfors, L. M., Bui, J., Reynolds, A., Leake, D., Khvorova, A. and Brugge, J. S. (2008). Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nat Cell Biol 10, 1027-38.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

27

Smith, N. J., Chan, H. W., Qian, H., Bourne, A. M., Hannan, K. M., Warner, F. J., Ritchie, R. H., Pearson, R. B., Hannan, R. D. and Thomas, W. G. (2011). Determination of the exact molecular requirements for type 1 angiotensin receptor epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy. Hypertension 57, 973-80.

Szklarczyk, D., Franceschini, A., Kuhn, M., Simonovic, M., Roth, A., Minguez, P., Doerks, T., Stark, M., Muller, J., Bork, P. et al. (2011). The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res 39, D561-8.

Tahmasebi, M., Barker, S., Puddefoot, J. R. and Vinson, G. P. (2006). Localisation of renin-angiotensin system (RAS) components in breast. Br J Cancer 95, 67-74.

Tamagnone, L., Lahtinen, I., Mustonen, T., Virtaneva, K., Francis, F., Muscatelli, F., Alitalo, R., Smith, C. I., Larsson, C. and Alitalo, K. (1994). BMX, a novel nonreceptor tyrosine kinase gene of the BTK/ITK/TEC/TXK family located in chromosome Xp22.2. Oncogene 9, 3683-8.

Thomas, W. G., Brandenburger, Y., Autelitano, D. J., Pham, T., Qian, H. and Hannan, R. D. (2002). Adenoviral-directed expression of the type 1A angiotensin receptor promotes cardiomyocyte hypertrophy via transactivation of the epidermal growth factor receptor. Circ Res 90, 135-42.

Thomas, W. G., Motel, T. J., Kule, C. E., Karoor, V. and Baker, K. M. (1998). Phosphorylation of the angiotensin II (AT1A) receptor carboxyl terminus: a role in receptor endocytosis. Mol Endocrinol 12, 1513-24.

Touyz, R. M., Wu, X. H., He, G., Salomon, S. and Schiffrin, E. L. (2002). Increased angiotensin II-mediated Src signaling via epidermal growth factor receptor transactivation is associated with decreased C-terminal Src kinase activity in vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension 39, 479-85.

Tsukada, S., Simon, M. I., Witte, O. N. and Katz, A. (1994). Binding of beta gamma subunits of heterotrimeric G proteins to the PH domain of Bruton tyrosine kinase. Proc Natl Acad Sci U S A 91, 11256-60.

Uchiyama, K., Saito, M., Sasaki, M., Obara, Y., Higashiyama, S. and Nakahata, N. (2009). Thromboxane A2 receptor-mediated epidermal growth factor receptor transactivation: involvement of PKC-delta and PKC-epsilon in the shedding of epidermal growth factor receptor ligands. Eur J Pharm Sci 38, 504-11.

Uchiyama-Tanaka, Y., Matsubara, H., Nozawa, Y., Murasawa, S., Mori, Y., Kosaki, A., Maruyama, K., Masaki, H., Shibasaki, Y., Fujiyama, S. et al. (2001). Angiotensin II signaling and HB-EGF shedding via metalloproteinase in glomerular mesangial cells. Kidney Int 60, 2153-63.

Vaque, J. P., Dorsam, R. T., Feng, X., Iglesias-Bartolome, R., Forsthoefel, D. J., Chen, Q., Debant, A., Seeger, M. A., Ksander, B. R., Teramoto, H. et al. (2013). A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors. Mol Cell 49, 94-108.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

28

Weiss, D., Sorescu, D. and Taylor, W. R. (2001). Angiotensin II and atherosclerosis. Am J Cardiol 87, 25C-32C.

Wettschureck, N., Rutten, H., Zywietz, A., Gehring, D., Wilkie, T. M., Chen, J., Chien, K. R. and Offermanns, S. (2001). Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Galphaq/Galpha11 in cardiomyocytes. Nat Med 7, 1236-40.

Williams, S. L., Lutz, S., Charlie, N. K., Vettel, C., Ailion, M., Coco, C., Tesmer, J. J., Jorgensen, E. M., Wieland, T. and Miller, K. G. (2007). Trio's Rho-specific GEF domain is the missing Galpha q effector in C. elegans. Genes Dev 21, 2731-46.

Wu, G., Aoyama, C., Young, S. G. and Vance, D. E. (2008). Early embryonic lethality caused by disruption of the gene for choline kinase alpha, the first enzyme in phosphatidylcholine biosynthesis. J Biol Chem 283, 1456-62.

Yalcin, A., Clem, B., Makoni, S., Clem, A., Nelson, K., Thornburg, J., Siow, D., Lane, A. N., Brock, S. E., Goswami, U. et al. (2010). Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling. Oncogene 29, 139-49.

Zambidis, E. T., Park, T. S., Yu, W., Tam, A., Levine, M., Yuan, X., Pryzhkova, M. and Peault, B. (2008). Expression of angiotensin-converting enzyme (CD143) identifies and regulates primitive hemangioblasts derived from human pluripotent stem cells. Blood 112, 3601-14.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

29

Figure Legends

Figure 1: Generation and characterisation of the AT1R-EGFR transactivation model. We

stably introduced an N-terminally HA-tagged angiotensin type I receptor (AT1R) into a human

mammary epithelial cell line (HMEC-LST) (Elenbaas et al., 2001), and used mCherry expression to

FACS enrich a heterogeneous population of cells expressing similar amounts of AT1R (A). HA-

tagged AT1R expression in HMEC-LST cells was determined by western blot analysis of cell lysate

(B). The ectopically expressed AT1R was expressed on the cell surface of the AT1R cells, but not the

mCherry cells as determined by immunofluorescence when stained with a α-HA antibody (green) to

detect the HA-tagged AT1R and DAPI (blue) to detect nuclei (C) (scale bars represent 50 μm;

individual confocal images and merged images located in Supplementary Fig. 1). Cells expressing

the AT1R (�) readily bound [125I]-AngII (EC50=1.7 nM) in a radioligand-binding assay; [125I]-AngII

was not observed to bind to the mCherry cells (�) (n=4 experiments) (D). When the HMEC-LST-

AT1R expressing cells (�) were stimulated with AngII, we observed intracellular Ca2+ mobilisation

to occur (EC50= 24.8 nM), which was not observed in the mCherry cell line (�) (n=5 experiments)

(E).

Figure 2: AT1R-EGFR transactivation occurs in HMEC-LST-AT1R cells. HMEC-LST cells

(mCherry or AT1R transduced) were serum starved then stimulated with 100 nM AngII for 5-10

minutes. Robust activation of the EGFR (pY1068) and ERK1/2 was observed in the AT1R

transduced cells (A). Quantitation of western blot data using densitometry analysis (ImageJ)

illustrated increases in phospho-EGFR (pY1068) (~2 fold) (B) and phospho-ERK1/2 (~2.5 fold) (C)

upon stimulation with AngII (n=4 experiments, paired two-tailed Student t test, * p=0.0137, **

p=0.0028). HMEC-LST-AT1R cells were treated with 5 μM AG1478 (EGFR inhibitor) or 1 μM

candesartan cilexetil (AT1R antagonist) for 30 minutes prior to stimulation with 100 nM AngII for 5-

10 minutes (D). Analysis revealed that inhibition of either EGFR or AT1R activity blocks AngII-

mediated AT1R-EGFR transactivation (n=3 experiments, data presented is a representative western

blot from the analysis).

Figure 3: AT1R-EGFR transactivation is reduced when cells are transfected with siRNAs

targeting AT1R, Gq/11 and EGFR expression. Cells were transfected with Dharmacon siGENOME

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

30

SMARTpool siRNAs targeting the ectopically expressed AT1R (siAgtr1a), Gq/11 (GNAQ, siGNAQ)

or EGFR (siEGFR) mRNA transcripts. Cells transfected with siAgtr1a demonstrated mRNA

knockdown of ~80% at 24 hours (A), and the AT1R-EGFR transactivation at 72 hour knockdown

was abolished (B) (n=3 experiments, paired two-tailed Student t test, *** p=0.0009). Cells

transfected with siGNAQ resulted in over an 85% knockdown of GNAQ mRNA at 24 hours (C), and

a reduction in EGFR and ERK1/2 phosphorylation post AngII stimulation (72 hour knockdown) (D)

(n=3 experiments, paired two-tailed Student t test, *** p=0.0002). Similarly, when cells were

transfected with siEGFR, ~75% knockdown of EGFR mRNA was observed at 24 hours post-

transfection (E) and the AT1R-EGFR transactivation response was blunted at 72 hours (F) (n=3

experiments, paired two-tailed Student t test, *** p=0.0004). Western blot data presented is

representative of all of the experiments carried out (n=3).

Figure 4: Kinome siRNA screen to determine genes involved in AT1R-EGFR transactivation.

Using the HMEC-LST-AT1R cell line, we performed a primary siRNA screen using the Dharmacon

siGENOME SMARTpool siRNA library targeting kinase genes (720 in total, 40 nM siRNA) (A).

Phospho-ERK1/2 data (SureFire) was normalised to cellular confluency (IncuCyte) for each well,

and robust Z-score analysis carried out (individual gene data and descriptions located in the

Supplementary siRNA Screen Data file). Secondary siRNA screening analysis was performed on 50

high-ranking candidates identified from the primary screen using the Dharmacon siGENOME

SMARTpool deconvoluted siRNA library (4 individual duplexes comprising the SMARTpool; 25

nM siRNA). High (4/4 and 3/4), medium (2/4) and low (1/4, 0/4) confidence candidates were then

identified. STRING analysis was performed on the AT1R-EGFR transactivation secondary screen

gene set to identify physical and functional associations of the genes within the list (B). Genes that

remained as unconnected “nodes” included FN3KRP, ASK, BMX, CDC2L2, DGKH, FRK, PSKH2,

SNRK, STYK1 and TRIO.

Figure 5: Further validation of TRIO, CHKA and BMX involvement in AT1R-EGFR

transactivation. HMEC-LST-AT1R cells were reverse transfected with 40 nM Dharmacon

siGENOME SMARTpool siRNAs targeting TRIO, CHKA or BMX expression. HMEC-LST-AT1R

cells transfected with TRIO siRNA (siTRIO) demonstrated an approximate 45% knockdown of

mRNA transcript at 24 hours by qRT-PCR (A) and at 72 hours post-transfection, knockdown of

TRIO reduced AT1R-EGFR transactivation upon stimulation with AngII (B). When cells were

transfected with CHKA siRNA (siCHKA), an approximate 90% knockdown of CHKA transcript

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

31

was observed at 24 hours (C), coupled with a reduction in ChoK (Chka) protein expression and

AT1R-EGFR transactivation at 72 hours (D). Similarly, cells transfected with BMX siRNA (siBMX)

demonstrate an approximate 90% knockdown of mRNA transcript at 24 hours (E), and a reduction in

AT1R-EGFR transactivation (as determined by phospho-EGFR and phospho-ERK1/2 abundance)

was observed after 72-hour siRNA knockdown (F). (n=3-4 experiments for both mRNA and protein

analysis, for mRNA analysis, paired two-tailed Student t test * p=0.0144 (TRIO), *** p=0.0002

(CHKA) and p=0.0008 (BMX). Western blots are representative of all experiments.

Figure 6: The mechanism of action for TRIO, CHKA and BMX lies between the AT1R and

EGFR. Our hypothesis is that TRIO, CHKA and BMX lie between the activated AT1R and EGFR

and thus modulate the AT1R-EGFR transactivation response. A proposed model for this theory is

illustrated in (A). HMEC-LST-AT1R cells were transfected with siGFP, siTRIO, siCHKA or siBMX

SMARTpool siRNAs for 72 hours and stimulated with either AngII (100 nM) or EGF ligand (0.5

ng/ml) for 10 minutes. Knockdown of TRIO (B), BMX (C) or CHKA (D) did not alter the response

of the cells to 0.5ng/ml EGF ligand. Data presented is representative of n=3 independent

experiments.

Figure 7: Elucidating the function of TRIO, CHKA and BMX in GPCR-mediated EGFR

transactivation. RHOA and RAC1, downstream of TRIO, were knocked down in HMEC-LST-

AT1R cells to assess their role in AT1R-EGFR transactivation. Greater than 90% knockdown of the

RAC1 and RHOA mRNA transcripts were observed at 24 hours (A). At 72 hours post-transfection,

knockdown of RAC1 reduced EGFR and ERK1/2 activity upon AngII stimulation, and led to a small

reduction in total EGFR protein expression (B), while RHOA knockdown affected EGFR but not

ERK1/2 activity (C). Pre-treatment of cells with NSC-23766 (D) or CK37 (E) revealed a blunting in

AT1R-EGFR transactivation upon AngII stimulation. To identify whether TRIO, CHKA and BMX

were specific to AT1R-EGFR transactivation, we stimulated the parental HMEC-LST cell line (not

containing the ectopically expressed AT1R) with 10 nM of thrombin for 10 minutes in the presence

or absence of 5 μM AG1478 and evaluated EGFR and ERK1/2 phosphorylation (F). Knockdown of

BMX and TRIO, but not CHKA in the HMEC-LST cells revealed little change in EGFR and

ERK1/2 activation post thrombin stimulation (G) (ERK1/2 activation data for three independent

experiments where CHKA is knocked down is located in Supplementary Fig. 11C). N=3-4

experiments for both mRNA and protein analysis; for mRNA analysis, paired two-tailed Student t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

32

test ** p=0.0014 (RHOA), *** p=0.0009 (RAC1). Western blots are representative of all

experiments performed.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Tables

Table 1: Genes assayed in the secondary siRNA screen and duplex scores

Entrez ID Gene name Duplex Hits

Hig

h c

on

fid

ence

1119 CHKA 4/4 2064 ERBB2 4/4

79672 FN3KRP 4/4 207 AKT1 3/4

10926 ASK 3/4

660 BMX 3/4

801 CALM1 3/4

728642 CDC2L2 3/4

1859 DYRK1A 3/4

1956 EGFR 3/4

2242 FES 3/4

2322 FLT3 3/4

5584 PRKCI 3/4

Med

ium

co

nfi

den

ce

10000 AKT3 2/4 64768 C9ORF12 2/4

1020 CDK5 2/4

1460 CSNK2B 2/4

160851 DGKH 2/4

2261 FGFR3 2/4

2444 FRK 2/4

5594 MAPK1 2/4

5290 PIK3CA 2/4

5297 PIK4CA 2/4

200576 PIP5K3 2/4

5562 PRKAA1 2/4

5580 PRKCD 2/4

85481 PSKH2 2/4

54861 SNRK 2/4

55359 STYK1 2/4

7204 TRIO 2/4

Lo

w c

on

fid

ence

657 BMPR1A 1/4 8851 CDK5R1 1/4

80347 COASY 1/4

1399 CRKL 1/4

53944 CSNK1G1 1/4

1739 DLG1 1/4

2049 EPHB3 1/4

10114 HIPK3 1/4

51347 JIK 1/4

92335 LYK5 1/4

1326 MAP3K8 1/4

9833 MELK 1/4

55750 MULK 1/4

8395 PIP5K1B 1/4

1263 PLK3 1/4

5583 PRKCH 1/4

5592 PRKG1 1/4

8986 RPS6KA4 1/4

8576 STK16 1/4

6011 GRK1 0/4

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t