Pseudopodium-enriched atypical kinase 1 regulates
the cytoskeleton and cancer progression
Yingchun Wanga,b,1,2, Jonathan A. Kelbera,b,1, Hop S. Tran Caoc, Greg T. Cantind, Rui Line, Wei Wanga,b,
Sharmeela Kaushalb, Jeanne M. Bristowa,b, Thomas S. Edgingtone, Robert M. Hoffmanc,f, Michael Bouvetb,c,
John R. Yates IIId, and Richard L. Klemkea,b,3
Departments of aPathology and cSurgery and bMoores Cancer Center, University of California, La Jolla, CA 92093; Departments of dChemical Physiology and
e
Immunology, The Scripps Research Institute, La Jolla, CA 92037; and fAntiCancer, Inc., San Diego, CA 92111
Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved April 27, 2010 (received for review December 24, 2009)
Regulation of the actin-myosin cytoskeleton plays a central role in cell
migration and cancer progression. Here, we report the discovery of
a cytoskeleton-associated kinase, pseudopodium-enriched atypical
kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase
that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation, regulates the p130CasCrk-paxillin and Erk signaling pathways, and operates downstream
of integrin and epidermal growth factor receptors (EGFR) to control
cell spreading, migration, and proliferation. Perturbation of PEAK1
levels in cancer cells alters anchorage-independent growth and
tumor progression in mice. Notably, primary and metastatic samples
from colon cancer patients display amplified PEAK1 levels in 81% of
the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy.
cancer
| cell migration | phosphoproteomics
A
ll cells have the fundamental ability to change shape and to
extend membrane projections from the cell surface (1). This
process is mediated by the actin-myosin cytoskeleton and is
critical for sensing and adapting to changes in the extracellular
environment. Although proper cytoskeletal regulation is important for numerous physiological processes including axon/dendrite formation, migration, differentiation, and proliferation, its
deregulation can also contribute to human diseases including
cancer metastasis (1–5). Therefore, it is crucial to understand the
mechanisms that control the cytoskeleton and how it contributes
to cancer progression.
Tyrosine phosphorylation of cytoskeleton-associated proteins
plays a central role in pseudopodium formation (6). A pseudopodium (or invadopodium in a cancer cell) is a highly specialized,
actin-rich structure that protrudes from the cell surface in migrating cells. It serves to tether the extending membrane to the
underlying substrate via the formation of focal adhesions and to
mediate traction forces that propel the cell forward (7, 8). The
pseudopodium also plays an important role in steering the cell by
degrading extracellular matrix protein barriers and by sensing
changes in chemokine and adhesive gradients that serve as guidance cues. Therefore, understanding how this structure is regulated is crucial to understanding how cells migrate and invade
tissues during cancer progression. To this end, we described
a novel method for purifying the pseudopodia from cells for signal
transduction and proteomic studies (9–11). Using this fractionation method and quantitative mass spectrometry (MS), we have
profiled the relative differences in the pseudopodium and cell
body proteomes (10). Although this approach has identified important new pseudopodial proteins, it did not provide a robust
method for detailed analysis of phosphotyrosine (pY) proteins or
kinases present in this subcellular structure (10). pY proteins are
difficult to detect because of their low abundance and typically
require an enrichment step before MS analysis (12, 13).
In this report, we use pY immunoaffinity enrichment and Multidimensional Protein Identification Technology (MudPIT) to define the pseudopodial phosphotyrosine proteome and to search for
10920–10925 | PNAS | June 15, 2010 | vol. 107 | no. 24
novel kinases involved in cell migration and cancer cell invasion
(14). This approach uncovered a unique 190-kDa nonreceptor
atypical tyrosine kinase family member KIAA2002 (sgk269) that is
enriched by 2.6-fold in the pseudopodium. We have named this
protein pseudopodium-enriched atypical kinase 1 (PEAK1). Our
biochemical and biological findings indicate that PEAK1 is a previously undescribed member of the canonical Src-p130Cas-Crk-IIPaxillin and Erk cytoskeletal signaling pathways. Furthermore, we
observed that PEAK1 localizes to focal adhesions, strongly associates with the actin cytoskeleton, and plays an important role in
cancer cell migration and proliferation in vitro and in vivo.
Results
Proteomic and Bioinformatic Analyses of the Pseudopodial pY
Proteome. The pseudopodium is highly enriched with pY pro-
teins involved in the regulation of actin polymerization and focal
adhesion dynamics (Fig. S1A) (9). To identify kinases and their
substrates that spatially regulate these processes, we used
a strategy that allows for the large-scale purification of pseudopodia actively extending toward an LPA gradient (9–11). The
relative differences in pY proteins from pseudopodia were
compared with pY proteins isolated from purified cell bodies by
using pY immunoaffinity purification followed by MudPIT (14).
Importantly, many tyrosine-phosphorylated proteins bind
strongly to the actin cytoskeleton and focal adhesions in migratory cells and, thus, are largely insoluble in detergents like
Nonidet P-40 and TX-100 (15). Therefore, to increase the yield
of pY proteins, cellular extracts were prepared by using denaturing conditions that maximize protein solubility by boiling
the samples in 1% SDS lysis buffer. These conditions also
eliminate non-pY to pY protein–protein interactions. After dilution of the SDS/protein extract, pY proteins were then selectively purified with antiphosphotyrosine antibodies and identified
by MudPIT (14). This method significantly enhances the specific
purification and coverage of the pseudopodial pY proteome.
Using this approach, a total of 309 pY proteins were identified in
the cell body and pseudopodial fractions (Dataset S1 and Table
S1). Of these, 211 proteins were enriched by at least 1.5-fold in
the pseudopodium compared with the cell body (Fig. S1B).
Functional annotation and statistical analysis of these pseudopodial-enriched pY proteins using the Fatigo search engine (16)
and the KEGG pathway databases revealed that the highest
percentage of pY proteins mapped to signaling pathways that
Author contributions: Y.W., J.A.K., G.T.C., W.W., J.M.B., and R.L.K. designed research; Y.W.,
J.A.K., H.S.T.C., G.T.C., R.L., W.W., S.K., J.M.B., and R.M.H. performed research; Y.W., M.B.,
J.R.Y., and R.L.K. contributed new reagents/analytic tools; Y.W., J.A.K., H.S.T.C., G.T.C., R.L.,
W.W., J.M.B., T.S.E., and R.L.K. analyzed data; and Y.W., J.A.K., and R.L.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
Y.W. and J.A.K. contributed equally to this work.
2
Present address: Institute of Genetics and Developmental Biology, Chinese Academy of
Sciences, No.1 West Beichen Road, Beijing 100101, China.
3
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.0914776107/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.0914776107
regulate the actin cytoskeleton and focal adhesions (Fig. S1C).
Protein network analysis using the Ingenuity pathway analysis
software (10) also revealed a well-connected cytoskeletal network of pY proteins known to regulate the pseudopodium and
focal adhesion dynamics including paxillin, cortactin, talin, shc,
GIT1/2, p130Cas, FAK, and Src family kinases (9, 17–26) (Fig.
S1D). As expected, signaling pathways involved in regulation of
cell cycle and calcium signaling were highly represented in the
cell body fraction, which contains the nuclear compartment.
Thus, the combined methods of pseudopodium purification, pY
protein enrichment, and MudPIT provided a representative
sample of the pseudopodium pY proteome and its spatial organization in migrating cells.
Bioinformatic Analyses Indicate That PEAK1 Contains Multiple Motifs,
Domains and Consensus Phosphorylation Sites That Couple It to the
Cytoskeleton. Twelve unique phosphoproteins were found to be
enriched in the pseudopodium (Table S1). Although these proteins have not been previously studied, their function can be
inferred from bioinformatic software, which identify known protein interaction domains and consensus kinase phosphorylation
motifs that are based on amino acid sequence homologies (27). Of
these 12 proteins, PEAK1 was chosen for detailed analysis.
PEAK1’s complete domain structure, its known and predicted
phosphorylation sites, and its predicted signaling pathway are
shown schematically in Fig. 1A. Previous work has shown that
PEAK1 is ubiquitously expressed in tissues with the highest level of
expression in brain and kidney (28) and is highly conserved from
zebrafish to human. The complete gene structure and cloning
strategy for obtaining full-length PEAK1 is shown in Fig. S2.
Using the bioinformatic software Scansite (27), PEAK1 is
predicted to be phosphorylated by Abl (Y797), Src (Y665), and
ERK (S779, T783) kinases (27) (Fig. 1A). All of these enzymes are
known to regulate the cytoskeleton and contribute to cancer
progression (18, 29–31). Although we have not yet mapped the
sites of phosphorylation on PEAK1, at least 13 phosphotyrosine
sites have been identified in previous phosphoproteomic studies
including Y665 (32) (see also the phosphosites database for the
complete list of PEAK1 phosphosites: www.phosphosite.org).
There are also several important protein interaction sites present
in PEAK1, including an SH2 binding motif for Src kinase (Y665),
a proline rich motif for binding to the Crk SH3 domain (P1153),
a Shc binding site (Y1188), and an ERK binding motif (P228).
The kinase domain spans aa 1330–1664, and the ATP binding site
spans aa 1133–1140. Overall, our bioinformatic analyses predict
that PEAK1 is a phosphotyrosine protein and an atypical tyrosine
kinase that associates with the actin cytoskeleton.
PEAK1 Shows in Vitro Kinase Activity. All active kinases are predicted to contain three motifs, VAIK, HRD, and DFG, within
the kinase domain (33). Each of these motifs contain one highly
conserved residue (VAIK: K, HRD: D, DFG: D) that is predicted
to be important for full catalytic activity. Sequence analysis
revealed that PEAK1 contains all of the three motifs YAVK,
HCD, and NFS. The YAVK and HCD motif are highly conserved on the critical K and D residues, but the D residue in the
NFS motif has been replaced by N, which classifies it as an
atypical kinase (33). However, it is not yet known whether this
amino acid substitution can affect kinase catalytic activity or
whether this site may be mutated in human cancers to confer
full catalytic activity (33). Nonetheless, to investigate whether
PEAK1 displays kinase activity, we first determined whether
PEAK1 can autophosphorylate in vitro because many kinases
phosphorylate themselves (34, 35). For these experiments, GFPPEAK1 was purified from HEK 293T cells as described in Experimental Procedures and its purity was confirmed by silver
staining (Fig. S3A). Autophosphorylation was determined by
using an in vitro kinase assay (35) and antiphosphotyrosine
Western blotting. PEAK1 displayed significant ability to autophosphorylate on tyrosine residues (Fig. S3A). To determine
whether PEAK1 can phosphorylate an exogenous substrate, we
performed an in-gel kinase assay by using myelin basic protein
(MBP) as a generic substrate and γ-[32P]ATP (36). Under these
conditions PEAK1 was able to weakly, but consistently, phosphorylate MBP (Fig. S3B). Finally, to rule out the possibility
that a coprecipitating kinase or cofactor from mammalian cell
extracts may account for the observed kinase activity of PEAK1
in these experiments, we purified PEAK1’s kinase domain (aa
1289–1746) from Escherichia coli by using a Smt3 tag and size
exclusion chromatography (Fig. S3C) and then tested it for tyrosine kinase activity toward MBP in vitro. MBP was phosphorylated by the purified kinase domain under these conditions
(Fig. S3D). Together, these results demonstrate that PEAK1 has
tyrosine kinase activity.
Fig. 1. PEAK1 localizes to the actin cytoskeleton and focal adhesions. (A)
Schematic showing predicted PEAK1 protein domains, motifs, and known pY
sites. (B and C) Immunofluorescence images showing GFP-PEAK1 colocalization with the actin cytoskeleton and vinculin-positive focal adhesions (red)
in NIH 3T3 fibroblast cells. White boxes (Inset) show the respective zoomed
images. The fluorescence intensity of GFP-PEAK1 and vinculin along the indicated line were scanned by using MetaMorph imaging software, and their
colocalization was determined by using the Pearson correlation coefficient
(r = 0.91) method. (Scale bars: 15 μm.)
Wang et al.
PEAK1 subcellular localization was determined using fluorescence microscopy in NIH 3T3 cells transfected with full-length
PEAK1 fused with GFP (GFP-PEAK1). PEAK1 strongly colocalizes with the F-actin cytoskeleton and vinculin-positive focal
adhesions, whereas control cells expressing GFP showed only
diffuse cytoplasmic staining (Fig. 1 B and C). In the majority of
cells (>80%), PEAK1 displayed punctate staining along actin
cables and cortical actin structures. To map which region of
PEAK1 is responsible for its F-actin localization, we expressed
a series of truncated forms of PEAK1 fused to GFP at the N
terminus in NIH 3T3 cells (Fig. S4A). Using this mutagenesis
approach, the actin targeting region was mapped to residues 338–
727 (Fig. S4B). Interestingly, this region of PEAK1 also displays
the majority of known and predicted tyrosine phosphorylation
sites, suggesting that upstream kinases and phosphatases may
regulate cytoskeletal localization of PEAK1 (Fig. 1A). In support
of this notion, serum starvation reduced PEAK1 localization to Factin, whereas stimulation of cells with serum or PDGF-BB induced strong PEAK1 colocalization with F-actin, and this response required residues 338–727 (Fig. S4C). In addition, using
immunofluorescence staining and a monoclonal antibody to
PEAK1, we assessed whether endogenous PEAK1 protein localized to these cytoskeletal structures. Endogenous PEAK1 was
observed to localize to actin and focal adhesions (Fig. S4D),
whereas cells depleted of PEAK1 by siRNA did not show PEAK1
immunostaining (data not shown). Although the focal adhesion
targeting domain in PEAK1 has not yet been ellucidated, our
preliminary data suggest that this process will be complex and
PNAS | June 15, 2010 | vol. 107 | no. 24 | 10921
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PEAK1 Localizes to the Actin Cytoskeleton and Focal Adhesions.
multifaceted, requiring phosphorylation of PEAK1 and binding of
multiple proteins that target PEAK1 to these structures. Nevertheless, these findings indicate that PEAK1 associates with the
actin cytoskeleton and localizes to cell-matrix focal adhesions.
PEAK1 Expression in Cells Alters Phosphorylation of CytoskeletonAssociated Proteins. Activation of integrin and growth factor
receptors promotes tyrosine phosphorylation and the molecular
scaffolding of cytoskeleton effector proteins including Src/
p130Cas/Crk/Paxillin/Erk (9, 17, 18). Interestingly, exogenous
expression of PEAK1 in cells increased the phosphorylation of
paxillin (Y31), p130Cas (Y249), and the Erk activation sites
(T185/Y187) in response to cell adhesion to fibronectin (Fig. 2A).
Paxillin Y31 phosphorylation was shown to regulate the assembly
and formation of cell-matrix adhesions, and p130Cas Y249
phosphorylation by Src provides a docking site for the Crk cytoskeleton adaptor protein (37), whereas Erk phosphorylation on
T185/Y187 is necessary for its kinase activity. Importantly, the
phosphorylation of these proteins at these specific amino acid
residues are known to play important roles in modulation of the
cytoskeleton and cell migration (17, 18, 30, 38–40). In contrast,
depletion of endogenous PEAK1 using a specific siRNA
(siPEAK1) decreased the phosphorylation of paxillin (Y31) and
Erk under these conditions (Fig. 2B). No obvious change was
observed in the phosphorylation of p130Cas (Y249), indicating
that PEAK1 can enhance phosphorylation, but is not necessary
for phosphorylation at this specific site. Furthermore, we found
that p130Cas and Crk coimmunoprecipitated with PEAK1. Under these conditions, Crk coprecipitated only with full-length
PEAK1 or the C-terminal truncated forms of PEAK1 (C1 and C2)
that contain the predicted Crk-SH3 binding motif (P-X-L-P-X-K)
(41) and not the N-terminal truncations that lack this domain
(Fig. 2C). On the other hand, p130Cas coprecipitated with wildtype and all mutant forms of PEAK1, suggesting that it may interact with PEAK1 at multiple sites or may associate with other
proteins that interact at multiple sites with PEAK1. Taken together, these data indicate that the modulation of PEAK1 protein
levels altered the phosphorylation/activation of known cytoskeleton-associated proteins.
Src Kinase Activity Is Necessary for PEAK1 Tyrosine Phosphorylation
Induced by Integrin and Growth Factor Receptor Activation. We next
tested whether PEAK1 is phosphorylated in response to growth
factor stimulation or integrin engagement and the role of Src kinase in this response. PEAK1 was maximally tyrosine phosphorylated in HEK 293T cells exposed to EGF for 10 min, and this
response was inhibited by the Src kinase inhibitor, PP2 (Fig. 2 D
and E). Similar findings were obtained in Src/Yes/Fyn-deficient
MEF cells exposed to EGF (Fig. 2H). Cell adhesion to fibronectin
also induced PEAK1 tyrosine phosphorylation, and this was
inhibited by PP2 (Fig. 2 F and G). Together, these findings indicate that cell adhesion and growth factor stimulation induce
PEAK1 tyrosine phosphorylation in a Src-dependent manner.
PEAK1 Expression Modulates Cell Spreading and Migration. PEAK1
localization to the cytoskeleton and its ability to modulate known
focal adhesion proteins suggest that it regulates spreading and
migration. To investigate this possibility, Cos-7 or HEK 293T cells
were depleted of PEAK1 using siRNA and tested for their ability
to spread and migrate in vitro on fibronectin. Knockdown of
PEAK1 in Cos-7 cells (Fig. S5C) delayed the initial stages of cell
spreading (<90 min) as indicated by a decrease in cell area (Fig.
3A). However, by 180 min there was no difference in the size of
control and PEAK1-depleted cells, indicating that other spreading signals can compensate for the loss of PEAK1 (Fig. 3A). On
the other hand, exogenous expression of PEAK1 increased cell
spreading at an early stage (<30 min) (Fig. 3B). Also, cells depleted of PEAK1 showed significantly reduced chemotaxis compared to control cells (Fig. 3C). Importantly, PEAK1 depletion
did not alter cell attachment to fibronectin (Fig. S6A Left). Conversely, Cos-7 cells expressing exogenous PEAK1 showed increased cell chemotaxis toward LPA (Fig. 3D), and this did not
alter cell attachment to fibronectin Fig. S6A Center). We further
tested whether PEAK1 is necessary for haptotactic migration
10922 | www.pnas.org/cgi/doi/10.1073/pnas.0914776107
Fig. 2. PEAK1 regulates cytoskeletal and focal adhesion proteins and
undergoes Src kinase-dependent tyrosine phosphorylation in response to
cell adhesion or EGF stimulation. (A) Protein lysates of 293T cells expressing
GFP-PEAK1 (PEAK1) in suspension (Sus) or attached (Atta) to fibronectin (FN)
were immunoblotted with indicated total protein and pY site-specific antibodies. (B) Lysates from PEAK1-specific siRNA (siPEAK1) or scrambled control
siRNA (siCtrl)-treated cells in suspension or attached to FN for 30 min were
immunoblotted as in A. (C) GFP, GFP-PEAK1, and GFP-PEAK1 truncated
forms (N1–N3, C1, and C2; Fig. S4A) were immunoprecipitated then immunoblotted for associated Crk and Cas proteins. Whole-cell lysates were also
immunoblotted for the indicated proteins. (D and E) pY Western blots of
GFP-PEAK1 immunoprecipitated from cells stimulated or not stimulated with
EGF for 10 min (D) or stimulated with EGF and in the absence or presence of
the Src kinase inhibitor PP2 (E). (F and G) pY Western blots of GFP-PEAK1
immunoprecipitated from cells in suspension (Sus) or reattached to 5 μg/mL
of poly-L-lysine (PLL), FN, or laminin (LN), respectively, (F) or cells reattached
to FN in the absence or presence of PP2 (G). Total PEAK1 levels in E and F
were detected by Ponceau staining. (H) pY immunoprecipitation of total
lysates from wild-type (+/+) or Src/Fyn/Yes (SYF) knockout (−/−) MEFs and
Western blots for PEAK1 after EGF stimulation for 10 min.
toward a fibronectin gradient. For these experiments, we used
a specific shRNA to stably deplete PEAK1 from XPA-1 pancreatic cancer cells (Fig. S5D). These cells showed significantly reduced migration toward fibronectin compared with the control
cells stably expressing a nontargeting shRNA (Fig. 3E). Again,
depletion of PEAK1 in XPA-1 cells did not significantly change
adhesion to the ECM (Fig. S6A Right). Additionally, we analyzed
the migration tracks of these cells over 24 h using time-lapse video
microscopy in combination with Metamorph software. This test
revealed that PEAK1 promotes a more persistent type of migration of cancer cells (Fig. 3 F and G). Finally, because Src can
regulate PEAK1 phosphorylation (Fig. 2 E, G, and H), we wanted
to determine whether PEAK1 played a role in Src-induced cell
migration. In this case, exogenous expression of Src-enhanced
detectable Src activity (Fig. S5E), leading to an increase in the
velocity of cell migration that depended on endogenous PEAK1
protein (Fig. 3H). Taken together, these data show that PEAK1 is
sufficient and necessary for proper cell spreading and migration in
response to fibronectin and growth factors as well as Src kinase
activity. Although the mechanism through which PEAK1 modulates cell spreading and migration is not yet understood, PEAK1
expression in cells was observed to significantly increase focal
adhesion length (Fig. S4E), suggesting that it may play a role in
regulating adhesion dynamics.
PEAK1 Promotes Anchorage-Independent Cancer Cell Growth in Vitro
and Tumor Progression in Mice. The deregulation of kinases and
cytoskeletal proteins often contribute to cancer progression (42).
Therefore, we wanted to determine whether PEAK1 plays a role in
cancer progression. The ability of cells to grow in soft agar in the
Wang et al.
Fig. 3. PEAK1 regulates cell spreading and directional cell migration. Cos-7
cells treated with siPEAK1 or siCtrl (A) or HEK 293T cells expressing exogenous GFP-PEAK1 or GFP (B) only were allowed to attach and spread on 5 μg/
mL fibronectin for the indicated times. Cell areas were measured by using
MetaMorph. Bars indicate mean ± SD in all figures unless indicated otherwise. *P < 0.01; **P < 0.001. Cos-7 cell chemotaxis toward LPA after treatment with siPEAK1 or siCtrl (C) or overexpression of GFP-PEAK1 or GFP (D).
(E) Cell migration toward FN of XPA-1 pancreatic cancer cells treated with
shRNA specific for PEAK1 (shPEAK1) or a scrambled control shRNA (shCtrl). (F
and G) Cells (5 × 104) as in E were plated onto FN-coated six-well plates, and
>70 cells were tracked in each population by using phase-contrast microscopy and MetaMorph software during the subsequent 24 h. The resulting
endpoint displacement (F) and cell migration persistance (G) were plotted.
(H) After cell migration tracking as in F and G, cell velocity was quantified for
shCtrl and shPEAK1 cell populations that were transiently transfected with
empty vector or Src overexpression vector. ***P < 0.0001.
absence of integrin adhesions to the ECM is a hallmark of cancer
(43–45). To determine whether PEAK1 modulates cancer cell
growth, human MDA-MB-435 cancer cells stably expressing exogenous PEAK1 were cultured in soft agar and the number and
size of colonies measured after 14 d. Exogenous expression of
PEAK1 increased the number and size of soft agar colonies and
was associated with increased Erk kinase activity (Fig. 4 A and C).
In contrast, depletion of exogenous PEAK1 protein with an
shRNA construct inhibited this response (Fig. 4 B and C and Fig.
S5 A and B). These findings indicate that PEAK1 provides a growth
advantage to tumor cells independent of integrin adhesion signals.
Having established that PEAK1 is important for anchorage-independent tumor cell growth, we wanted to determine whether
PEAK1 could contribute to tumor formation in vivo. MDA-MB435 cells stably expressing GFP (MDA-435-GFP) or GFP-PEAK1
(MDA-435-PEAK1) were injected s.c. into the flanks of nude
mice. Tumor progression was monitored weekly by using wholebody fluorescence imaging as described (46). MDA-435-PEAK1
showed a significant advantage in tumor formation as indicated by
the increase in tumor size over the 9-wk period compared with
MDA-435-GFP (Fig. 4D). At the end of the 9 wk, the animals were
sacrificed; the tumors were removed and weighed. Consistent with
our in vitro data, cells overexpressing PEAK1 formed tumors that
showed increased area and weight compared with GFP control
tumors (Fig. 4 D and E). We also used shRNA to stably knockdown endogenous PEAK1 expression in human XPA-1 pancreatic
cancer cells and determined their ability to form tumors when
transplanted orthotopically into the pancreas of nude mice. In this
case, PEAK1-depleted tumor cells showed a reduction in tumor
growth as indicated by reduced tumor size compared with control
cells (Fig. 4F). These findings indicate that PEAK1 is necessary for
tumor growth in vitro and in vivo.
PEAK1 Expression Is Up-Regulated in Human Colon Cancer and Liver
Metastases. The observation that modulation of PEAK1 protein
Wang et al.
Discussion
Our ability to affinity purify pY proteins from isolated pseudopodia proved to be a robust system to identify proteins involved
in cell migration. Also, the solubilization of pseudopodial proteins in SDS buffer significantly improved our yield of pY proteins, which can be tightly associated with the insoluble
cytoskeleton (15). This approach allowed us to identify many low
abundant pY proteins involved in cell migration and led to the
discovery of PEAK1. Collectively, our findings demonstrate that
PEAK1 is a unique nonreceptor tyrosine kinase that operates
within the Src-p130Cas-Crk-Paxillin signaling pathway to regulate cell spreading, migration, and cancer progression.
Modulation of PEAK1 affected the phosphorylation level of
several known cytoskeleton regulatory proteins including paxillin,
p130Cas, and Erk, and it was found to associate with the Crk
adaptor protein. PEAK1 has a proline-rich sequence (P-X-L-P-XK) that conforms to the predicted binding site for the N-terminal
SH3 domain of Crk (41). Our finding that Crk coprecipitates with
the C-terminal region of PEAK1 has important implications for
how PEAK1 could regulate the cytoskeleton. Crk is an adaptor
protein that regulates cells spreading and migration by coupling
critical signaling proteins such as EGFR, PDGFR, and C-Abl to
the cytoskeleton and focal adhesions including the scaffolding
protein p130Cas (47). p130Cas and its family members are necessary for cell migration and cancer cell invasion and have been
associated with cancer progression in patients (48–51). Interestingly, p130Cas coprecipitates with PEAK1 and PEAK1
modulates p130Cas-Y249 phosphorylation (Fig. 2 B and C). Y249
is known to be phosphorylated by Src family kinases and to
provide a binding site for the Crk SH2 domain (38, 47, 52–55).
The Src/p130Cas/Crk complex has been shown to modulate Rac
activity, pseudopodium protrusion, cell migration, and cancer
progression (9, 17, 48, 56–59). Together, these findings suggest
a possible scenario in which integrins and growth factors activate
Src that, in turn, phosphorylates p130Cas Y249, leading to Crk
binding through its SH2 domain. PEAK1 is bound to Crk’s SH3
domain and, thus, is recruited to the p130Cas/Crk scaffold, where
it is phosphorylated by Src at Y665, the Src consensus site (Fig.
1A and Fig. S5E). Consistent with this idea, Src kinase activity is
necessary for PEAK1 tyrosine phosphorylation in response to
growth factor and integrin receptor activation (Fig. 2 D–H).
Under these conditions, PEAK1 could serve several important
purposes. First, it may modulate protein–protein interactions by
directly phosphorylating components of the p130Cas/Crk/Paxillin
scaffold via its tyrosine kinase activity (Fig. S3). Second, given
that PEAK1 translocates to focal adhesions and the actin cytoskeleton after growth factor stimulation (Fig. 1 and Fig. S4), it
could provide a mechanism to transport the Src/p130Cas/Crk
scaffold to these structures. Third, its strong association with the
actin cytoskeleton suggests that it may tether the Src/p130Cas/Crk
complex to the cytoskeleton. Finally, it may deliver unique effector proteins to focal adhesions and the cytoskeleton that, in
turn, modulate cell migration and/or proliferation. It is intriguing
that the majority of the known and predicted phosphorylation
sites cluster in the actin localization region of PEAK1. It seems
plausible then that PEAK1’s association with the cytoskeleton is
regulated by phosphorylation of one or more of these sites. In any
case, our observations that PEAK1 can interact with and moduPNAS | June 15, 2010 | vol. 107 | no. 24 | 10923
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levels in cancer cells alters tumor growth prompted us to examine whether expression of PEAK1 mRNA is altered in human
cancer. In situ hybridization was performed to analyze expression
of PEAK1 mRNA in healthy colon tissues and the corresponding
colon tumors and liver metastases by using human colon cancer
tissue array that included samples from 22 patients. Of these, 18
patients (81.8%; P < 0.001) showed elevated PEAK1 staining in
the primary tumor compared with the normal colon tissue (Fig.
4G and Fig. S6B). Twelve of these patients (54.5%) showed
positive staining in their corresponding liver metastases (P <
0.05) (Fig. 4G and Fig. S6B). In contrast, only 5 patients (22.7%)
showed positive PEAK1 staining in normal colon tissues. Taken
together these data indicate that PEAK1 is a unique cytoskeleton-associated kinase and a member of the Src/p130Cas/Crk/
paxillin/Erk signaling pathways that regulate cell migration and
promote cancer progression.
Fig. 4. PEAK1 promotes oncogenic growth of cancer cells in vitro and in vivo and is up-regulated in human colon cancer. Comparison of soft agar colony size and
number in MDA-435 cells expressing GFP-PEAK1 or GFP only (A) or depleted of GFP-PEAK1 by shRNA (B) (see also Fig. S5). The area of the colonies is shown as mean ±
SEM. (C) Cells from A and B were cultured under suspension conditions for 30 min followed by lysis and Western blot analysis of P-Erk and total Erk proteins. (D)
Whole-animal fluorescent images of MDA-435-GFP (GFP) and MDA-435-PEAK1 (PEAK1) cancer cells allowed to grow s.c. in nude mice for 9 wk. (E) Tumor data from
mice shown in D: Upper, average tumor weight after tumor excision; Lower, average tumor area as measured by whole-body fluorescence imaging throughout the
course of the experiment. (F) Average tumor size of XPA-1 human pancreatic cancer cells stably expressing control scrambled or PEAK1 shRNA and growing
orthotopically in the pancreas of nude mice for 4 and 5 wk. The data are shown as mean ± SEM for E and F. (G) Representative in situ hybridization images of PEAK1
expression levels (red stain) in normal colon tissue (NC), primary colon cancer tissue (CC), and a liver metastasis (LM) taken from the same patient.
late the Src/p130Cas/Crk/paxillin and Erk signaling pathways
point to a central role for this unique protein in mediating cell
migration and proliferation in normal and cancer cells.
Although we directly study the function of PEAK1, several independent lines of evidence also suggested that PEAK1 and its only
family member sgk223 (33% overall homology to PEAK1) play an
integral role in regulation of cell motility and tumor progression
(60, 61). For example, sgk223 has been reported to be a unique
effector of Rnd2 GTPase. It has also been shown to stimulate RhoA
activity in HeLa cells and mediated cancer cell invasion in a Srcdependent manner (61). More recently, sgk223 and PEAK1 were
both identified as the potential targets that mediate Src invasive
activity in advanced colon carcinoma cells in a quantitative phosphoproteomics study (60). Here, we demonstrate that PEAK1
modulates anchorage-independent growth in soft agar and tumor
formation in nude mice. Similar to sgk223, Src mediates PEAK1
tyrosine phosphorylation (Fig. 2 D–H). Finally, large shRNA ge10924 | www.pnas.org/cgi/doi/10.1073/pnas.0914776107
nomic screens have also revealed that PEAK1 is involved in cancer
cell proliferation (62). Together, these findings suggest that PEAK1
and sgk223 are cytoskeleton-associated kinases that regulate cell
migration and cancer progression in response to Src kinase activity.
The fact that Src is a major contributor to human cancers and our
findings that PEAK1 levels are amplified in >80% of colon cancer
patients underscore the importance of this protein in human cancer
and warrants further investigation.
Experimental Procedures
Protein Sample Preparation and Identification. Pseudopodia and cell bodies were
purified from Cos-7 cells as described (9–11). PY proteins were purified by immunoprecipitation and identified by MudPIT and mass spectrometry (see SI Experimental Procedures for details).
SiRNA and shRNA Assay. The siRNA pool specific for PEAK1 was purchased
from Dharmacon, and transfection was performed according to manufacturer’s
Wang et al.
Tumor Progression Assays. The soft agar assay was performed as described
(64) by using lentivirus infected, and FACS sorted MDA-MB-435 cells that
stably express GFP or GFP/PEAK1. An aliquot of these cells were injected s.c.
in nude mice for tumor formation. A second in vivo experiment consisted of
establishing orthotopic human pancreatic cancer xenografts in nude mice by
direct injection of XPA-1-GFP shCtrl and XPA-1-GFP shPEAK-1 into their
pancreas (65) (see SI Experimental Procedures for further details).
ACKNOWLEDGMENTS. We thank Drs. Olivier Pertz, and Hisashi Kato for
assistance in lentivirus production, Spencer Wei for imaging assistance, and
Tiffany Taylor, Ryan Matson, and Elizabeth Hampton for assisting with the
molecular biology and biochemistry work. We also thank Dr. Stephen K.
Burley and his research team at New York SGX Research Center for
Structural Genomics (J. Michael Sauder, Shawn S. Chang, Kevin Bain,
Jacqueline Freeman, and Tarun Gheyi) for the construct design, cloning,
expression, purification, and MS analysis of the PEAK1 kinase domain (a.a.
1289–1746) for our in vitro kinase assays. Finally, we thank Drs. Beverley
Emerson and Matthias Kaeser (Salk Institute for Biological Studies) for their
kind gift of the CMV-MCS lentiviral expression vector. This work was supported by the Susan G. Komen Foundation Grant PDF0503999 (to Y.W.),
National Institutes of Health Grants GM068487 (to R.L.K.) and CA097022
(to R.L.K.), and Cell Migration Consortium Grant GM064346 (to R.L.K.).
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PNAS | June 15, 2010 | vol. 107 | no. 24 | 10925
CELL BIOLOGY
instructions. The shRNA was designed using the software RNAi OligoRetriever
(63) and cloned into the lentiviral vector FG12. The depletion of PEAK1 was
confirmed by measuring the mRNA level using RT-PCR and Western blot analysis. See Table S2 for the sequence information of all siRNAs and shRNAs.
Corrections
ANTHROPOLOGY
Correction for “Y chromosome diversity, human expansion, drift,
and cultural evolution,” by Jacques Chiaroni, Peter A. Underhill,
and Luca L. Cavalli-Sforza, which appeared in issue 48, December
1, 2009, of Proc Natl Acad Sci USA (106:20174–20179; first published November 17, 2009; 10.1073/pnas.0910803106).
The authors note the following statement should be added to
the Acknowledgments: “This work was supported by ANR Program AFGHAPOP N° BLAN07-3_222301.”
www.pnas.org/cgi/doi/10.1073/pnas.1008738107
CELL BIOLOGY
Correction for “Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progession,” by Yingchun Wang,
Jonathan A. Kelber, Hop S. Tran Cao, Greg T. Cantin, Rui Lin,
Wei Wang, Sharmeela Kaushal, Jeanne M. Bristow, Thomas S.
Edgington, Robert M. Hoffman, Michael Bouvet, John R. Yates
III, and Richard L. Klemke, which appeared in issue 24, June 15,
2010, of Proc Natl Acad Sci USA (107:10920–10925; first published
June 1, 2010; 10.1073/pnas.0914776107).
The authors note that the title of their manuscript appeared
incorrectly. The title should appear as “Pseudopodium-enriched
atypical kinase 1 regulates the cytoskeleton and cancer progression.” The title has been corrected online.
www.pnas.org/cgi/doi/10.1073/pnas.1008849107
DEVELOPMENTAL BIOLOGY
Correction for “TIF1β regulates the pluripotency of embryonic
stem cells in a phosphorylation-dependent manner,” by Yasuhiro
Seki, Akira Kurisaki, Kanako Watanabe-Susaki, Yoshiro Nakajima,
Mio Nakanishi, Yoshikazu Arai, Kunio Shiota, Hiromu Sugino, and
Makoto Asashima, which appeared in issue 24, June 15, 2010, of
Proc Natl Acad Sci USA (107:10926–10931; first published May 27,
2010; 10.1073/pnas.0907601107).
The authors note that the gene name “Smarcad1” appeared
incorrectly throughout the article and supporting information.
The correct spelling of the gene is “Smarcd1.”
www.pnas.org/cgi/doi/10.1073/pnas.1008651107
13556 | PNAS | July 27, 2010 | vol. 107 | no. 30
www.pnas.org