Global phosphorylation analysis of β-arrestin–
mediated signaling downstream of a seven
transmembrane receptor (7TMR)
Kunhong Xiaoa, Jinpeng Suna,1, Jihee Kima,1, Sudarshan Rajagopala, Bo Zhaib, Judit Villénb, Wilhelm Haasb,
Jeffrey J. Kovacsa, Arun K. Shuklaa, Makoto R. Haraa, Marylens Hernandezc, Alexander Lachmannc, Shan Zhaoc,
Yuan Lina, Yishan Chenga, Kensaku Mizunod, Avi Ma’ayanc, Steven P. Gygib,2, and Robert J. Lefkowitza,e,f,2
Departments ofaMedicine and
Cell Biology, Harvard University Medical School, Boston, MA 02115;cDepartment of Pharmacology and Systems Therapeutics, Systems Biology Center New
York, Mount Sinai School of Medicine, New York, NY 10029; anddDepartment of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University,
Sendai, Miyagi 980-8578, Japan
eBiochemistry andfHoward Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710;bDepartment of
Contributed by Robert J. Lefkowitz, June 16, 2010 (sent for review April 21, 2010)
β-Arrestin–mediated signaling downstream of seven transmembrane
receptors (7TMRs) is a relatively new paradigm for signaling by these
receptors. We examined changes in protein phosphorylation occur-
(AT1aR) were stimulated with the β-arrestin–biased ligand Sar1, Ile4,
Ile8-angiotensin (SII), a ligand previously found to signal through
β-arrestin–dependent, G protein-independent mechanisms. Using a
phospho-antibody array containing 46 antibodies against signaling
molecules, we found that phosphorylation of 35 proteins increased
abrogated after depletion of β-arrestin 2 through siRNA-mediated
knockdown. We also performed an MS-based quantitative phospho-
proteome analysis after SII stimulation using a strategy of stable iso-
phosphoproteins (4,552 unique phosphopeptides), of which 171 pro-
teins (222 phosphopeptides) showed increased phosphorylation, and
stimulation of the AT1aR. This study identified38 protein kinases and
three phosphatases whose phosphorylation status changed upon SII
treatment. Using computational approaches, we performed system-
including construction of a kinase-substrate network for β-arrestin–
mediated AT1aR signaling.Our analysis demonstrates thatβ-arrestin–
dependent signaling processes are more diverse than previously
appreciated. Notably, our analysis identifies an AT1aR-mediated cyto-
skeletal reorganization network whereby β-arrestin regulates phos-
phorylation of several key proteins, including cofilin and slingshot.
phorylation events downstream of a 7TMR and opens avenues for
research in a rapidly evolving area of 7TMR signaling.
G protein-coupled receptor|GPCR|phosphoproteome
protein-coupled receptors,” are encoded by nearly 1,000 genes
in the human genome and regulate virtually all known physiolog-
ical processes in humans (1). In the classical paradigm, 7TMR
signaling is mediated through the activation of heterotrimeric G
proteins, generally referred to as “G-protein–mediated signaling”
(1). Dissociation of Gα and Gβγ subunits, each of which signals to
downstream effectors, leads to changes in cellular physiology.
Following activation, 7TMRs are phosphorylated by G protein-
coupled receptor kinases, and subsequently recruit cytosolic
β-arrestins. β-Arrestin binding uncouples the receptors from G
protein subunits and desensitizes G protein-mediated signaling.
desensitizing actions, β-arrestins also serve as multifunctional
adaptors and signal transducers, linking the receptors, in an acti-
vation-dependent manner, to a growing list of endocytic and sig-
naling molecules [e.g., MAPK, Src, and protein kinase B (AKT)],
initiating a newly appreciated method of signaling referred to
even transmembrane receptors (7TMRs), also known as “G
as “β-arrestin–mediated signaling” (2). Although G protein-de-
pendent signaling downstream of 7TMRs has been studied for
many years, a complete understanding of G protein-independent,
β-arrestin–mediated signaling is still emerging.
7TMR signaling and function (3). Consequently, developing
a global understanding of the signaling events downstream of
β-arrestins is of great importance. Recent advances in the techni-
ques and strategies of systems biology and MS-based quantitative
proteomics (4–7) offer great opportunities to investigate β-arrestin–
mediated signaling on a large scale. To gain insight into the in-
tricacies of β-arrestin–mediated signaling, we previously performed
a large-scale MS-based proteomic analysis of β-arrestin interacting
partners (the “β-arrestin interactome”) with and without angioten-
sin II (AngII) stimulation of the angiotensin II type 1A receptor
induced reversible phosphorylation of key targets, in the present
study we carried out an MS-based quantitative phosphoproteomics
screen downstream of AT1aR upon stimulation by Sar1, Ile4, Ile8-
angiotensin (SII). SII is a known β-arrestin “biased ligand” for
AT1aR, which is unable to activate G protein-dependent signaling
as evidenced by a lack of phosphatidylinositol hydrolysis, calcium
mobilization, or diacylglycerol activity (9, 10). However, SII can
recruit β-arrestin to the AT1aR and stimulate ERK and several
other biochemical effector mechanisms in an entirely β-arrestin–
dependent manner (9). Using this β-arrestin–biased ligand, we se-
lectively activated β-arrestin–mediated signaling and identified
multiple bioinformatic and systems biology approaches to construct
a kinase network involved in β-arrestin–dependent signaling down-
stream of the AT1aR.
Results and Discussion
β-Arrestin–Dependent Phosphorylation Events Downstream of AT1aR
Are Activated by SII. Because protein phosphorylation is funda-
mental to many aspects of cell signaling, we set out to interrogate
the mechanisms involved in β-arrestin–mediated signaling by
exploring SII-induced phosphorylation events. In an initial ap-
proach, we used a human phospho-antibody array (Human
Phospho-Kinase Array, Proteome Profiler Array Kit, ARY003;
R&D Systems) to study a subset of phosphorylation events after
A.K.S., Y.L., and Y.C. performed research; K.M. provided new reagents/analytic tools; K.X.,
S.R., J.J.K., M. Hara, M. Hernandez, A.L., S.Z., Y.C., A.M., S.P.G., and R.J.L. analyzed data; and
K.X. and R.J.L. wrote the paper.
The authors declare no conflict of interest.
1J.S. and J.K. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: email@example.com
This articlecontains supporting information onlineat www.pnas.org/lookup/suppl/doi:10.1073/
pnas.1008461107/-/DCSupplemental and http://lefko-gpcr-phosphoproteome-data.org/pdf/.
| August 24, 2010
| vol. 107
| no. 34
SII stimulation. Furthermore, to assess the degree to which these
events depend on β-arrestin, HEK293 cells transiently expressing
AT1aR were stimulated with or without SII under conditions in
which β-arrestin 2 was present or knocked down using siRNA
technology. Among 46 phospho-antibodies (most for proteins
known to play roles in cell signaling) spotted on the array, the
relative extent of phosphorylation of 35 proteins increased upon
SII stimulation relative to nonstimulated samples (Fig. 1 and Fig.
S1). This high percentage of proteins observed to undergo SII-
stimulated changes in phosphorylation may reflect the fact that
the proteins on the array are preselected kinases known to be
involved in important signaling pathways. When β-arrestin 2 was
depleted by siRNA technology, the SII-induced changes in
phosphorylation detected by all 35 antibodies were largely ab-
rogated, consistent with SII-mediated phosphorylation events
being β-arrestin dependent.
Quantitative Phosphoproteome Analysis of AT1aR Signaling upon SII
Stimulation. Findings with the phospho-antibody array prompted
activation of AT1aR signaling on a large scale using MS-based
quantitative phosphoproteomics technology (11) and a strategy of
stable isotope labeling of amino acidsin cell culture (SILAC) (Fig.
S2 A–C) (12). In three independent experiments we identified
a total of 14,328 phosphopeptides with 5,046, 5,332, and 3,950
phosphopeptides (in experiments 1, 2, and 3, respectively). Sta-
tistical analysis of the data from these three independent experi-
ments showed good correlations between the three data sets (Fig.
S3A). After removing the redundant peptides, we identified 4,552
unique phosphopeptides(unique phosphorylation events) in 1,555
phosphoproteins (Table S1). To determine phosphorylation sites
in these peptides and measure the confidence of assignment, we
applied theAscore algorithm, which computes the probability that
a difference in detected site-determining ions in a MS/MS spec-
trumbetweentwopotential site locations occurredby chance(13).
We considered sites with Ascore ≥ 13 (P ≤ 0.05) to be confidently
localized. All calculated Ascores are listed in Table S1.
To compare quantitatively the AT1aR phosphoproteome from
untreated HEK293 cells with that of cells from stimulated for 5 min
“heavy” (SII-treated) phosphopeptide pairs were calculated using
a previously described software program (14). Each calculated
abundance ratio (Vista ratio) reflects the change in the extent of
Vista ratios of phosphopeptides in three independent experiments
(Fig. S3B and Table S1). We chose a 1.5-fold ratio change (which is
equivalent to three times the SD of the log2transformation of Vista
ratios) as a cutoff and considered an increase of more than 1.5-fold
or a decrease of more than 33.3% as a significant increase or de-
crease in phosphorylation level. We define a “β-arrestin–mediated
phosphopeptide” as one whose phosphorylation is increased by at
least 1.5-fold or decreased by at least 33.3% in at least two in-
dependent experiments. Using this criterion, we have established
phosphopeptides (“β-arrestin–mediated phosphopeptides”) from
220 phosphoproteins (“β-arrestin–mediated phosphoproteins”).
Among these 288 phosphopeptides, the phosphorylation status of
222 peptides (from 171 different phosphoproteins) increased, and
the phosphorylation status of 66 peptides (from 53 different phos-
some representative β-arrestin–mediated phosphopeptides.
Some phosphorylation sites in this β-arrestin–mediated phos-
phoproteome, such as T202/Y204 of ERK1 and T184/Y186 of
ERK2, have been characterized previously as undergoing
β-arrestin–dependent phosphorylation upon SII stimulation of
AT1aR (9). The phosphorylation of T202/Y204 of ERK1 and
T184/Y186 of ERK2 increased 1.5- to 2.4-fold using this tech-
nique. This result indicates that the 1.5-fold change threshold we
chose may reflect a physiologically significant phosphorylation
change. Furthermore, because most of the β-arrestin–mediated
phosphorylation sites we identified have not been observed pre-
viously in the context of β-arrestin–mediated 7TMR signaling, we
antibodies for Western blotting analysis. All SII-induced phos-
phorylation changes measured by Western blotting analysis con-
firmed the phosphoproteome data (Fig. S2D).
Protein Kinases and Phosphatases Are Overrepresented in the
β-Arrestin–Mediated Phosphoproteome. Protein kinases and phos-
phatases are critical for reversible phosphorylation of important
via reversible phosphorylation. It is notable that among the 220
β-arrestin–mediated phosphoproteins identified in this study, 26
are protein kinases (more than would be expected by chance; P<
with those identified in the phospho-antibody array analysis, we
identified 38 protein kinases that are regulated by SII stimulation
(Table S3). Included in this group of 38 are kinases in the MAPK
tion events induced by SII are primarily β-arrestin–dependent. (A) Normal-
ized intensities for six representative phosphoproteins from samples applied
to the phospho-antibody array. Lysates were from HEK293 cells transfected
with either control or β-arrestin 2 (βarr2) siRNA followed by 30 μM SII stim-
ulation for 5 min [SII(+)] or no stimulation [SII(−)]. The normalized intensity
for each antibody was calculated as a percentage of the corresponding SII-
stimulated sample treated with control siRNA. The experiment was repeated
at least three times, and statistical analysis was performed using a one-way
ANOVA. ***P < 0.001. (B) A list of β-arrestin–regulated phosphoproteins
revealed by human phospho-antibody array analysis. The residues indicated
in parentheses are the phosphorylation sites.
Human phospho-antibody array analysis reveals that phosphoryla-
| www.pnas.org/cgi/doi/10.1073/pnas.1008461107Xiao et al.
signaling cascades, p21 protein (Cdc42/Rac)-activated kinases, ri-
kinases involved in cell-cycle regulation. Nonreceptor tyrosine
kinases, such as Src, Yes, Lck, Hck, Lyn, Fyn, proline-rich tyrosine
mediated phosphoproteome (Fig. 2). The three phosphatases we
identified are the serine/threonine phosphatase protein phospha-
tase 1, the dual-specificity phosphatase slingshot 1, and the SH2
containing protein tyrosine phosphatase 2 (SHP2) (Table S3).
details of β-arrestin–dependent signaling networks can be eluci-
dated simply by examining the phosphorylation status of a mole-
cule and comparing it with previously published observations. For
example, dephosphorylation of Src Tyr-530 by protein tyrosine
phosphatases has been proposed as a likely mechanism for its ac-
tivation (15). Interestingly, Src was found to be dephosphorylated
at its inhibitory site Tyr-530 in our phosphoproteome, suggesting
potential involvement ofβ-arrestins inactivation ofSrc inresponse
to AT1aR activation. Activation of Src was the first β-arrestin–
dependent signaling mechanism identified more than 10 y ago.
Phosphorylation Motif and Kinase Enrichment Analysis Reveal Kinases
Crucial for β-Arrestin–Mediated Signaling Downstream of AT1aR. To
identify candidate kinases that may play roles in regulating the
phosphorylation status of the β-arrestin–mediated phosphopro-
teome, we used the Motif-X program (16) to identify phosphory-
lation motifs present in the β-arrestin–mediated phosphopeptides
and compared them with known consensus sequences of protein
Table 1. Representative phosphoproteins identified in phosphoproteome analysis
Protein name Phospho-peptide sequence*
Sii (+)/Sii (−)
Fold Increase% decrease
SH3 and multiple
ankyrin repeat domains 3
Protein phosphatase 1,
catalytic subunit, α
Rap guanine nucleotide
exchange factor 2
↑ 27.6 -fold
↑ 2.0 -fold
↑ 1.9 -fold
↑ 1.8 -fold
↑ 1.8 -fold
↑ 1.7 -fold
↑ 1.7 -fold
↑ 1.7 -fold
↑ 1.7 -fold
↑ 1.6 -fold
↑ 1.6 -fold
*Phosphorylation sites are indicated in bold.
†Relative abundances of phosphopeptides in samples with [SII(+)] and without [SII (−)] SII treatment. Increases of more than 1.5-fold or decreases of more than
33.3% in relative abundance were chosen as cutoffs for significant increases or decreases in phosphorylation.
downstream of AT1aR. A comparison of experimentally identified, bio-
informatically predicted, and previously reported protein kinases involved in
β-arrestin–mediated signaling. Bioinformatic predictions were performed
using Motif-X analysis and KEA as described in the text.
Protein kinases involved in β-arrestin–mediated cellular signaling
Xiao et al. PNAS
| August 24, 2010
| vol. 107
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kinases. We found that the consensus sequences for proline-
directed kinases [such as MAPKs and cyclin-dependent kinase
(CDKs)], AKT, protein kinase D (PKD), calmodulin-dependent
protein kinase (CaMKII), and casein kinase II are enriched in the
β-arrestin–mediated phosphopeptides (Fig. S4).
In another approach to identifying candidate kinases for the
quantitative phosphoproteomics dataset, we used Kinase En-
richment Analysis (KEA) (17). KEA uses a kinase-substrate da-
tabase of reported mammalian kinase–substrate interactions and
protein–protein interactions that involve kinases to compute en-
richment for kinase substrates. This analysis identified 10 protein
kinases [DNAPK, AKT1, CDK1, CDK2, glycogen synthase ki-
nase 3β (GSK3β), Src, and p90RSK3] as well as several kinases in
the MAPK pathway (Table S4), consistent with the kinases that
were found to be differentially phosphorylated and those identi-
fied by Motif-X.
that previously have been reported to be involved in β-arrestin–
mediated signaling. As shown in Fig. 2, a number of kinases
identified or predicted here have been reported previously to play
important roles in β-arrestin–mediated signaling. This comparison
demonstrates a good correlation between the phosphoproteomics
or bioinformatic data and findings using traditional approaches,
although here we have identified a number of previously un-
reported kinases that appear to be important for β-arrestin–
mediated cellular signaling.
Signaling Pathway and Subnetwork Analyses Reveal a β-Arrestin–
Dependent Cytoskeletal Reorganization Network Downstream of
AT1aR. To analyze the global extent of β-arrestin–mediated
that yield complementary information. These analyses include
(KEGG) canonical pathway, and Ingenuity subnetwork analyses.
These analyses are described in detail in Figs. S5 and S6 A–H and
Tables S5 and S6. Recent work has shown that β-arrestins play an
of a number of proteins downstream of 7TMRs, including LIM ki-
nase, cofilin, RhoA, and myosin light-chain kinase (18). We found
both pathway and subnetwork analyses, with a number of key reg-
ulators of cytoskeletal reorganization showing changes in phos-
phorylation status upon SII stimulation of AT1aR. With this
phosphoproteomics data, in combination with the previously
published β-arrestin interactome (8) and the β-arrestin signaling
partners characterized in this study, we identified a β-arrestin–
dependent cytoskeletal reorganization subnetwork downstream of
AT1aR (Fig. 3).
To test the validity of this putative subnetwork, we examined the
on cytoskeletal reorganization. Slingshot is a phosphatase that
regulates actin filament dynamics via dephosphorylation and acti-
vationoftheactindepolymerizing protein cofilin. The phosphatase
Ser-937 and Ser-978 (19). Our phosphoproteomedata showed that
phosphorylation of slingshot Ser-937 and -940 decreased 2-fold
uponSII stimulation,indicating activation ofslingshot downstream
of AT1aR in a β-arrestin–dependent manner. We did not detect
phosphorylation of Ser-978, a well-characterized phosphorylation
site of slingshot that is associated with its activation. This site
probably was not observed because of the current technical limi-
tationsof phosphoproteomicscreensthatcapture onlya fractionof
cytoskeletal reorganization net-
work downstream of AT1aR.
proteome and β-arrestin inter-
actome revealed that many
proteins in cytoskeletal reorga-
nization pathways were regu-
lated downstream of AT1aR/
β-arrestin, suggesting a critical
reorganization. The β-arrestin–
interacting proteins were iden-
tified in the previous β-arrestin
interactome study (8) or in bio-
chemical experiments in the
slingshot and cofilin downstream of AT1aR. HEK293 cells stably overexpressing
AT1aR were stimulated with 30 μM SII for the indicated time periods. (A)
β-Arrestin–dependent dephosphorylation of slingshot. The cell lysates were
probed with the slingshot pSer-978 antibody in the presence of control,
total slingshot was calculated for each data point and plotted as fold decrease
from the nonstimulated sample. (B) β-Arrestin–dependent dephosphorylation
phospho-cofilin/total cofilin was calculated for each data point and plotted as
fold decrease from the nonstimulated sample. (C) Cofilin pSer-3 de-
phosphorylation downstream of AT1aR is mediated via slingshot and not via
chronophin. HEK293 cells stably overexpressing AT1aRs were transfected with
control, chronophin, or slingshot1/2 siRNA. Cofilin dephosphorylation was pro-
bed with phospho-cofilin Ser-3. The phospho-cofilin/total cofilin ratio was cal-
sample. The data presented here are from at least three independent experi-
SII stimulation leads to β-arrestin–mediated dephosphorylation of
| www.pnas.org/cgi/doi/10.1073/pnas.1008461107 Xiao et al.
relevant peptides. We then used a slingshot phospho-antibody
targeting pSer-978 (the only phospho-slingshot antibody available)
to demonstrate a time-dependent activity profile. Upon SII stim-
ulation ofAT1aR,thephosphorylation level ofslingshot decreased
more than twofold at 10 and 15 min (Fig. 4A and Fig. S7A). In
addition, either β-arrestin 1 or 2 siRNA ablated slingshot de-
phosphorylation and activation, confirming its β-arrestin–mediated
activation (Fig. 4A and Fig. S7 A and D).
Activated slingshot dephosphorylates cofilin at Ser-3 (Fig. S8),
thereby activating its actin-binding/depolymerizing ability and
promoting actin reorganization and lamellipodia formation (20).
In the AT1aR system, we used a phospho-antibody against pSer-3
and demonstrated SII stimulation-dependent dephosphorylation
of cofilin (Fig. 4B and Fig. S7B). Knockdown of β-arrestin 1 or 2
significantly blocked SII-mediated cofilin dephosphorylation, in-
dicating β-arrestin–dependent activation of cofilin. Because pre-
vious studies on the proteinase-activated receptor 2 suggested
that β-arrestin–mediated cofilin dephosphorylation was regulated
by the phosphatase chronophin (21), we next studied cofilin de-
phosphorylation when chronophin or slingshot was individually
depleted from cells by siRNA. Knockdown of chronophin had no
effect on cofilin dephosphorylation (Fig.4C and Fig. S7 C and D).
However, knockdown of slingshot 1 blocked SII stimulation-induced
cofilin dephosphorylation. This result suggests that slingshot, and
not chronophin, regulates β-arrestin–dependent cofilin dephos-
phorylation downstream of the AT1aR.
To test the hypothesis that β-arrestins scaffold slingshot to
cofilin, we overexpressed FLAG-tagged β-arrestin 1 or 2 in
HEK293 cells overexpressing the AT1aR and stimulated the cells
for 5 min with SII or vehicle control. Immunoprecipitation with
an anti-FLAG antibody demonstrated an association between
β-arrestins and slingshot that increased upon SII stimulation (Fig.
S7E, Upper). This interaction is direct, as shown by in vitro GST
pull-down experiment (Fig. S7E, Lower). Cofilin is known to in-
teract with β-arrestin (8), so it is likely that β-arrestin scaffolds
a protein complex that contains both slingshot and cofilin, thereby
promoting slingshot-dependent cofilin dephosphorylation. These
findings verify part of our signaling subnetwork and demonstrate
the hypothesis-generating potential of this combined proteomics
(8) and phosphoproteomics dataset. Furthermore, these findings,
coupled with our previous observation that PP2A and β-arrestin
form a complex (8, 22), suggest that β-arrestin may activate
slingshot through the recruitment and scaffolding of PP2A which
dephosphorylates slingshot to activate its phosphatase activity.
A β-Arrestin–Dependent Kinase Network Downstream of the AT1aR
Revealed by the β-Arrestin–Mediated Phosphoproteome. Signalsthat
are transmitted inside cells do not go through linear pathway
cascades but rather through highly interconnected networks (23).
The identification of the β-arrestin–mediated phosphoproteome
offers a system wide view of the phosphorylation signature of the
entire β-arrestin signaling network and provides an opportunity to
network was generated by an inference algorithm (24) and literature-based kinome network (17) as described in the text and SI Text. This network covers
a broad range of cellular functions including MAPK and PI3K/AKT signaling, cytoskeletal reorganization, cellular adhesion, cell-to-cell communication, and
cell cycle and development. Green rectangle, identified in β-arrestin–mediated phosphoproteome; orange ellipse, β-arrestin–interacting proteins; pink
hexagon, identified in β-arrestin–mediated phosphoproteome and β-arrestin interactome; gray rectangle, ”hub proteins” important for the signaling net-
work but not identified in either β-arrestin–mediated phosphoproteome or β-arrestin interactome; red arrow, regulation of phosphorylation/de-
phosphorylation; black line, interaction with β-arrestin; dashed line, regulation of hub proteins. Several known β-arrestin–interacting proteins (ARAF, MEK1,
p38, and JNK) that were not in our β-arrestin interactome are indicated also.
β-arrestin–dependent kinase network downstream of AT1aR revealed by the β-arrestin–mediated phosphoproteome and β-arrestin interactome. The
Xiao et al.PNAS
| August 24, 2010
| vol. 107
| no. 34
connect β-arrestin–mediated phosphoproteins to kinases and
other proteins that have been reported to regulate them using an
inference algorithm (24) and a previously constructed literature-
based kinome network (17). Using these approaches (SI Materials
and Methods), we were able to infer the activation or inhibition of
phosphosites detected on kinases in our study (24). These results
then were compared with 38 other phosphoproteomics experi-
ments reported for mammalian cells in the literature, validating
kinase phosphosites (further details are given in SI Materials and
Methods and ref. 24). By combining this kinase-substrate sub-
network and the β-arrestin interactome, along with additional
curation, we constructed a cell-signaling network specific for
β-arrestin–mediated AT1aR signaling (Fig. 5).
This β-arrestin–mediated AT1aR network provides a global
picture of how β-arrestins function to relay messages from
AT1aRs at the cell surface, through protein phosphorylation
mechanisms, to a wide variety of cellular effectors. This network
covers a broad range of cellular functions and processes such as
MAPK and PI3K/AKT signaling, cytoskeletal reorganization,
cellular adhesion, cell-to-cell communication, and cell cycle and
development. For example, this network identifies several key
molecules involved in cell-cycle regulation that are subject to
β-arrestin–dependent signaling, including CDK1, CDK2, Fyn,
SHP2, HSF1, and Lyn. All these proteins are important in
keeping the cell cycle from progressing until repairs to damaged
DNA have been completed (25) and are linked to a subnetwork
for DNA repair in the β-arrestin–mediated kinase network.
A very recent study also used a SILAC-based approach to ex-
HEK 293 cells (26). Christensen et al. (26) used a TiO2phospho-
peptide enrichment approach and performed analysis after stimu-
lation with SII or Ang II. However, in their study no attempt was
made to relate any of the SII-mediated events to β-arrestin–
mediated signaling. As would be expected, there was significant
overlap (∼30%) in these two studies in the phosphoproteins reg-
ulated by SII stimulation of the AT1aR; however, a significant
number of proteins are unique to one study or the other, probably
because the different phosphopeptide enrichment methods, TiO2
and immobilized metal ion affinity chromatography (IMAC), de-
tect different, partially overlapping fractions of the phosphopro-
teome (27). In their study, Christensen et al. identified PKD as an
important kinase regulating downstream signaling after SII stimu-
lation, a finding that was confirmed in our analysis. However, al-
though Christensen et al. reported more phosphoproteins that
changed because of stimulation, with our more extensive bio-
informatic analysis, we have constructed a much broader and
more specific β-arrestin–mediated kinase-substrate network that
includes additional kinases that, like PKD, regulate many phos-
signaling network identifies roles for β-arrestins in a number of cel-
lular pathways, such as cytoskeletal reorganization, DNA damage
repair, and MAPK signaling. We also validated the specific role of
slingshot in β-arrestin–mediated signaling. Taken together, these
complementary studies point the way toward a more global under-
standing of signaling downstream of 7TMRs.
Materials and Methods
A SILAC strategy was used for quantitative phosphoproteome analysis.
Phosphopeptides were prepared by strong cation exchange fractionation
followed by IMAC enrichment. Peptide samples were analyzed on a LTQ
Orbitrap XL mass spectrometer (Thermo Scientific) or a LTQ-FT mass spec-
trometer (Thermo Scientific). MS/MS spectra were searched via the SEQUEST
algorithm (28) against a composite database containing the human In-
ternational Protein Index protein database and its reversed complement.
Further detailsofthese andother procedures usedin this workare given in
SI Materials and Methods.
ACKNOWLEDGMENTS. We thank members of the R.J.L. laboratory for
stimulating discussions and critical comments, Drs. Noah Dephoure, Sean
Beausoleil, and Ramin Rad of the S.P.G. laboratory for their help with data
processing, Donna Addison and Elizabeth Hall for secretarial assistance, and
Drs. Sudha K. Shenoy and Seungirl Ahn for their comments on the kinase
network. This work was supported in part by National Institutes of Health
Grants HL16037 and HL70631 (to R.J.L.), DK088541 and GM071558-01A27398
(to A.M.), and HG3456 and GM67945 (to S.P.G.). R.J.L. is an Investigator with
the Howard Hughes Medical Institute.
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| www.pnas.org/cgi/doi/10.1073/pnas.1008461107Xiao et al.
Correction Download full-text
Correction for “Global phosphorylation analysis of β-arrestin–
mediated signaling downstream of a seven transmembrane re-
ceptor (7TMR),” by Kunhong Xiao, Jinpeng Sun, Jihee Kim,
Sudarshan Rajagopal, Bo Zhai, Judit Villén, Wilhelm Haas,
Jeffrey J. Kovacs, Arun K. Shukla, Makoto R. Hara, Marylens
Hernandez, Alexander Lachmann, Shan Zhao, Yuan Lin, Yishan
Cheng, Kensaku Mizuno, Avi Ma’ayan, Steven P. Gygi, and
Robert J. Lefkowitz, which appeared in issue 34, August 24, 2010,
of Proc Natl Acad Sci USA (107:15299–15304; first published
August 4, 2010; 10.1073/pnas.1008461107).
The authors note that within the footnotes, the line “This
article contains supporting information online at www.pnas.org/
and http://www.lefkolab.org/fileadmin/lefko/pdf/” should instead
appear as “This article contains supporting information online
DCSupplemental and http://lefko-gpcr-phosphoproteome-data.
org/pdf/.” The online version has been corrected.
| August 14, 2012
| vol. 109
| no. 33www.pnas.org