MOLECULAR AND CELLULAR BIOLOGY, Feb. 2011, p. 766–782
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 4
Phosphorylation of Trask by Src Kinases Inhibits Integrin Clustering
and Functions in Exclusion with Focal Adhesion Signaling?
Danislav S. Spassov,1,4Ching Hang Wong,1,4Natalia Sergina,1,4Deepika Ahuja,1,4Michael Fried,2,4
Dean Sheppard,1,3and Mark M. Moasser1,4*
Departments of Medicine,1Cancer Research Institute,2Lung Biology Center,3and Helen Diller Family Comprehensive Cancer Center,4
University of California, San Francisco, San Francisco, California 94143
Received 21 July 2010/Returned for modification 2 September 2010/Accepted 6 December 2010
Trask is a recently described transmembrane substrate of Src kinases whose expression and phosphoryla-
tion has been correlated with the biology of some cancers. Little is known about the molecular functions of
Trask, although its phosphorylation has been associated with cell adhesion. We have studied the effects of
Trask phosphorylation on cell adhesion, integrin activation, clustering, and focal adhesion signaling. The small
hairpin RNA (shRNA) knockdown of Trask results in increased cell adhesiveness and a failure to properly
inactivate focal adhesion signaling, even in the unanchored state. On the contrary, the experimentally induced
phosphorylation of Trask results in the inhibition of cell adhesion and inhibition of focal adhesion signaling.
This is mediated through the inhibition of integrin clustering without affecting integrin affinity state or ligand
binding activity. Furthermore, Trask signaling and focal adhesion signaling inactivate each other and signal
in exclusion with each other, constituting a switch that underlies cell anchorage state. These data provide
considerable insight into how Trask functions to regulate cell adhesion and reveal a novel pathway through
which Src kinases can oppose integrin-mediated cell adhesion.
Src family kinases (SFKs) are a family of nonreceptor pro-
tein tyrosine kinases with a domain structure consisting of a
highly conserved kinase domain as well as an SH2 domain and
an SH3 domain, a C-terminal negative-regulatory tyrosine res-
idue, and an N-terminal myristoylation site. Three members of
the family, Src, Yes, and Fyn, are ubiquitously expressed, while
the expression of the other members is largely restricted to
specific hematopoietic cell lineages. SFKs participate in numer-
ous cellular pathways in association with growth factor receptors,
G protein-coupled receptors, steroid hormones, STAT transcrip-
tion factors, and integrin receptors (14, 17, 34). The role of
SFKs in regulating cell adhesion signaling at sites of adhesion
to the extracellular matrix (ECM) is particularly well estab-
lished. Upon integrin engagement with the ECM and clus-
tering of integrins at sites of cell adhesion to matrix, mac-
romolecular complexes are assembled in association with
the intracellular tails of activated integrins (30, 40, 48). Within
these focal adhesions, focal adhesion kinase (FAK) is activated
by autophosphorylation at tyrosine 397, creating a binding site
for the Src SH2 domain (30, 37). Upon binding to FAK, Src is
activated and phosphorylates a number of additional tyrosine
residues on FAK, creating additional binding sites for SFKs
and other proteins. Activated Src also phosphorylates a num-
ber of additional cytoskeletal proteins, including paxillin and
p130Casand proteins involved in regulating the RhoA, Rac1,
and Cdc42 GTPases (23). These events function to stabilize
focal adhesions, generating a force-induced mechanical link
with the actin cytoskeleton, and regulate the surrounding
SFKs are required for proper establishment of focal adhe-
sions, as fibroblasts deficient in Src kinases have significantly
reduced tyrosine phosphorylation at focal contacts and defec-
tive cell adhesion to matrix (7, 26, 47). Although this loss-of-
function model supports the current molecular models of focal
adhesion establishment, the conclusions are not reciprocated
by gain-of-function experiments. The constitutively activated
v-src oncogene product interacts with focal contacts, phosphor-
ylating target proteins within them (20, 33). However, the
activities of the v-src product are destructive to focal adhesions,
and in fact, v-src-transformed cells appear to have significantly
reduced focal adhesions (11). Therefore, the evidence suggests
that SFKs are capable of promoting both adhesive and an-
tiadhesive functions, and most current models reconcile this
by proposing that SFKs function in focal adhesion turnover
(15). The mechanisms that mediate the antiadhesive func-
tions of SFKs are less well understood. Some evidence sug-
gests that SFKs can mediate focal adhesion disassembly
through a RhoA- and mDia1-mediated pathway or through a
calpain-mediated pathway (18, 51). In this paper, we describe
a novel mechanism by which SFKs can negatively regulate
focal adhesion assembly.
We have been studying a novel substrate of SFKs named
Trask. Trask is a recently described 140-kDa transmembrane
protein with little homology to known families of proteins. It
has a large extracellular region containing CUB domains and a
smaller intracellular region containing five tyrosines (5). Trask
is widely expressed in epithelial cells and tissues as a variable
blend of 140-kDa and 85-kDa forms, the latter due to proteo-
lytic cleavage of its distal extracellular region by serine pro-
teases, including the membrane bound MT-SP1 (5, 42). Trask
is phosphorylated in vitro by SFKs, including Src and Yes, and
is also phosphorylated by SFKs in cells, and its phosphorylation
can be inhibited by all classes of SFK inhibitors (5). The phos-
* Corresponding author. Mailing address: Box 1387, University of
California, San Francisco, San Francisco, CA 94143-1387. Phone:
(415) 476-0158. Fax: (415) 353-9592. E-mail: email@example.com.
?Published ahead of print on 28 December 2010.
phorylation of Trask is exclusively dependent on SFKs, since it
fails to undergo phosphorylation in Src/Yes/Fyn knockout cells
(SYF cells) unless transfected with an SFK member (50).
Trask has also been independently identified as a cancer-asso-
ciated gene by other groups. In a microarray analysis of colon
cancers, it was identified as a transcript with increased expres-
sion in tumors compared with that in adjacent normal tissues
and was named CDCP1 (39). In another line of study, a sub-
tractive immunization screen designed to identify antibodies
against more metastatic variants of HEp-3 carcinoma cells
identified a surface protein that was named SIMA135, which is
identical to Trask/CDCP1 (22).
The suggestion that Trask/CDCP1 is important in cancer
progression has been further supported by correlative studies
of human tumors, although the data are mixed and the nature
of this association and the cellular role of Trask/CDCP1 in
cancer is a matter of ongoing interest and investigation. In an
extensive analysis of Trask expression and phosphorylation in
human tissues, we found that Trask is widely expressed in most
epithelial tissues; however, the SFK phosphorylation of Trask
is restricted to physiological circumstances of detachment,
such as in mitotically detached cells in the colonic crypts (42).
However, in a large survey of human tumor sections, we found
that Trask is phosphorylated in many epithelial cancers at all
stages, including preinvasive cancers such as tubular adeno-
mas, but not in their normal-tissue counterparts (50). In other
studies, the elevated expression of Trask/CDCP1 has been
linked with poorer prognosis in cancers of the lung, kidney,
and pancreas (3, 24, 31) but with better prognosis in endo-
metrioid cancer (28).
The phosphorylation of Trask is linked with cell adhesion
such that Trask is phosphorylated almost instantly when epi-
thelial cells detach from matrix and is dephosphorylated when
cells readhere to matrix (42). In the current study, we mecha-
nistically studied the link between Trask phosphorylation and
cell adhesion through loss-of-function and gain-of-function
studies, looking at cell adhesiveness, at the affinity, binding,
and clustering of integrins, and at focal adhesion assembly and
signaling. We report that the SFK phosphorylation of Trask
inhibits cell adhesion through the inhibition of integrin binding
activity. This is mediated through the inhibition of clustering,
but not regulation of affinity state, and consequent inhibition of
focal adhesion assembly and signaling. We also found that the
SFK phosphorylation of Trask functions in opposition to and
in exclusion with focal adhesion signaling. These two signaling
pathways oppose and inactivate each other, defining a switch
that regulates cell anchorage state.
MATERIALS AND METHODS
Cell culture and immunoblotting. Cell lines were obtained from the American
Type Culture Collection. To force cells into suspension, cells were washed in
phosphate-buffered saline (PBS), exposed to a 2 mM solution of EDTA in Hanks
buffer, and when fully detached, cultured in growth media in ULC plates (Corn-
ing) for 2 h. ULC plates are not permissive to cell adhesion.
Antiphosphotyrosine antibodies (PY99), anti-FAK, anti-p-Y397 FAK, anti-
paxillin, and anti-p130Casantibodies were from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Generation of polyclonal and monoclonal anti-Trask and
anti-p-Y743 Trask antibodies were previously described (50). PP1 and PP2 were
from EMD-Calbiochem (San Diego, CA). Anti-integrin ?1 antibodies were
mouse monoclonal anti-integrin ?1 (BD Biosciences) for immunoprecipitation
and immunoblotting studies and anti-integrin ?1 clone P5D2 for integrin-acti-
vating and flow cytometry studies (6, 13, 52).
For wound healing experiments, near-confluent MCF10A cells were mechan-
ically scraped across the center of the well to generate a cell-free lane and
observed over the next 24 to 48 h for wound closure by cell migration. For
migration assays, the Transwell migration assay (Millipore) was used accord-
ing to manufacturer’s procedure. To the bottom wells, Dulbecco’s modified
Eagle’s medium (DMEM)–10% fetal bovine serum (FBS) containing 20 ng/ml
epidermal growth factor (EGF) was added. Cells were harvested, resuspended in
serum-free medium at densities of 5 ? 105cells/ml, and added to the top well.
When necessary, the cells were preincubated with anti-Trask or control antibod-
ies (80 ?g/ml) for 30 min at room temperature. After overnight incubation, the
cells were stained with crystal violet and enumerated.
Total cellular lysates were harvested in modified radioimmunoprecipitation
assay (RIPA) buffer (10 mM Na phosphate [pH 7.2], 150 mM NaCl, 0.1% SDS,
1% NP-40, 1% Na deoxycholate, protease inhibitors, 1 mM sodium orthovana-
date). For Western blotting, 50 ?g of each lysate was separated by SDS-PAGE,
transferred to membrane, and immunoblotted using appropriate primary and
secondary antibodies and enhanced chemiluminescence visualization. For immu-
noprecipitation studies, 300 ?g of lysate was incubated overnight with specific
antibodies, immune complexes were collected by protein G-Sepharose beads and
washed, and the denatured complexes were immunoblotted as described above.
Quantitative analysis of cell attachment. Wells of 96-well plates (not tissue
culture treated) were incubated overnight at 4°C with 100 ?l of matrix proteins
(5 ?g/ml fibronectin, 10 ?g/ml collagen type IV [Sigma], and 10 ?g/ml laminin).
MDA-468TR/WT Trask MDA-468TR/pcDNA4-TO cells were induced for 16 h
with or without doxycycline (Dox) and were plated at a density of 5 ? 105cells/ml
in 100 ?l per well. Cells were incubated at 37°C with 5% CO2for 1 h, followed
by three washes with PBS to remove unattached cells. Adhesion was assessed by
a colorimetric assay using crystal violet (Sigma), a cytochemical stain that binds
to chromatin. Cells were fixed in cold methanol for 15 min, and plates were then
air dried and stained with 0.1% crystal violet in PBS for 5 min at room temper-
ature. After removal of the crystal violet solution, the plates were washed ex-
tensively and dried, and the stain was released by using 2% SDS in PBS. Stain
intensity was quantified by spectrophotometry (at 570 nm) using a plate reader.
Generation of Trask-overexpressing cells. Generation of MDA-468 cells ex-
pressing doxycycline-inducible Trask was previously described (5). Several clones
were analyzed, all of which showed identical adhesion phenotypes when treated
with doxycycline. These cells were named MDA-468TR/Trask. Tyrosines 707,
734, and 743 in Trask were mutated to phenylalanine in the pcDNA4-Trask
vector by using the Stratagene Quik Exchange kit. The mutated insert was
sequenced for confirmation and stably transfected into MDA-468TR cells. Sev-
eral clones were expanded, and the doxycycline-inducible expression of mutant
Trask was confirmed by myc immunoblotting. All clones had similar phenotypes.
An individual clone (with tyrosine-to-phenylalanine [Y?F] mutations) was fur-
ther studied and named MDA-468TR/Y?F Trask.
Generation of Trask knockdown cells. Small hairpin RNA (shRNA) sequences
were cloned into pSico-RGFP and pSico-RNeo vectors. pSico-RGFP expresses
green fluorescent protein (GFP) as a selectable marker, and pSico-RNeo con-
tains a neomycin resistance cassette.
To create the shTrask-1 construct, the oligonucleotide 5?-TGAATGTTGCT
TTGGATCC-3? was annealed to 5?-TCGAGGATCCAAAAAAAATGTTGCT
and cloned into HpaI and XhoI sites of the pSicoR vector. To create the
shTrask-2 construct, the oligonucleotide 5?-TGATAGATGAGCGGTTTGCAA
GGCGCGCC-3? was annealed to 5?-TCGAGGCGCGCCAAAAAAAATAGA
TCATCTATCA-3? and cloned into HpaI and XhoI sites of the vector. For
generation of the nonsilencing construct, the oligonucleotides 5?-TGTCTCGCT
TTTTTGGCGCGCC-3? and 5?-TCGAGGCGCGCCAAAAAAATCTCGCTT
A-3? were similarly annealed and cloned. The shRNA constructs were
transfected into 293T cells along with the appropriate packaging vectors. The
resulting lentiviral particles were used to infect cells. Stably integrated GFP-
expressing MCF10A cell infectants were purified by flow sorting, and Trask
knockdown was confirmed in the GFP-expressing cell population by Western
blotting. MDA-468 cells were infected with pSico-RNeo shRNA constructs and
selected with G418 (300 ?g/ml).
Bead assays. To measure the integrin signaling in suspended cells in the
absence of cell spreading, polystyrene beads (Polysciences) were used. These
beads were precoated with 125 ?g/ml fibronectin (Sigma) or 50 ?g/ml anti-?1
integrin antibody (P5D2) (Santa Cruz Biotechnology) by rotation for 1 h at room
VOL. 31, 2011p-Trask INHIBITS INTEGRIN CLUSTERING 767
temperature in PBS. A total of 106beads were incubated with 106cells in a 2-ml
suspension in ULC plates for 3 h. At this time, aggregation between beads and
cells was observed under a microscope, and the cells remained with rounded
morphology. The cell-bead suspensions were pelleted and cells lysed in RIPA
To measure bead-cell aggregation, fluorescent beads and cells were used. The
beads were FluoSphere polystyrene microspheres, which had yellow-green fluo-
rescence (505/515), from Molecular Probes. Beads were precoated with 125
?g/ml fibronectin (Sigma) or anti-?1 integrin antibody (P5D2) at 50 ?g/ml
(Santa Cruz Biotechnology) by rotation for 1 h at room temperature in PBS.
Cells were fluorescently labeled with Vybrant DiD cell-labeling solution (Mo-
lecular Probes) according to the manufacturer’s instructions. For aggrega-
tion, 1 ? 106beads were incubated with 1 ? 106cells in a 2-ml suspension in
ULC plates for 3 h. The percent aggregation was determined by flow cytometry.
FACS analysis of cell surface ?1 integrin expression. Cell surface ?1 integrin
expression was assayed by flow cytometry. MDA-468TR/Trask cells were left
uninduced or induced with doxycycline overnight and subsequently stained with
anti-?1 integrin antibodies (conformation independent) and secondary fluores-
cent antibodies and quantified by fluorescence-activated cell sorter (FACS) anal-
Integrin conformation assays. Recombinant human fibronectin fragment 3,
containing type III domains 8 to 13 and the amino acid sequence between
Glu1266 and Pro1908 (R&D Systems), was fluorescently labeled with the Alexa
Fluor 488 microscale protein labeling kit (Invitrogen) according to the manu-
facturer’s instructions. For binding, cells were detached with EDTA, washed with
serum-free medium, and incubated with the fluorescent fibronectin fragment for
30 min at room temperature. Cells were washed with serum-free medium, fixed
with paraformaldehyde, and immediately analyzed by FACS analysis. Where
indicated, samples were incubated and washed in the presence of 2 mM MnCl2.
Integrin conformation was also assayed using the conformation state-specific
antibody 9EG7 (BD Biosciences) as follows. EDTA-detached cells were washed
with 1? Tris-buffered saline (TBS)–5% bovine serum albumin (BSA) and incu-
bated with 9EG7 in the presence of 1 mM calcium or 2 mM manganese in 1?
TBS–5% BSA for 1 h on ice. Cells were washed with 1? TBS–1% BSA and
consequently incubated with fluorescein isothiocyanate (FITC)-conjugated anti-
rat secondary antibodies for 30 min on ice. Cells were washed with 1? TBS,
resuspended, and immediately analyzed by FACS.
Microscopy. Tissue cultured cells were viewed and imaged by phase-contrast
microscopy using a Nikon TS-100F inverted microscope equipped with a Nikon
D100 digital camera attached to the photo port. Images were imported into
Photoshop software, converted to grayscale, and gamma adjusted for optimal
representation. Fluorophore-stained cells were imaged by fluorescence micros-
copy using an Axioplan 2 Zeiss microscope at the appropriate excitation wave-
lengths. Zeiss Fluar objective lenses were used with Immersol 518N as the
imaging medium. Pictures were taken at room temperature with an Axiocam
MRm camera and Axiovision 4.5 acquisition software, imported into Photoshop
software, and gamma adjusted for optimal representation.
TIRF microscopy. Cells were grown on number 1.5 coverslips, and induced
with or without Dox for 12 h and fixed with 4% paraformaldehyde. Cells were
stained with anti-integrin ?1, anti-FAK, or antipaxillin antibodies (Santa Cruz
Biotechnology) and secondary Alexa Fluor 546-conjugated antibodies (Invitro-
gen). Samples were mounted with water, illuminated with a Nikon laser total
internal reflection (TIRF) illuminator, and observed under a Nikon TE2000E
inverted microscope. Images were taken with the NIS-Elements Advanced Re-
search software and a 100? objective.
Trask phosphorylation is linked with the state of anchorage
in vitro and in vivo. To interrogate changes in signaling asso-
ciated with the anchorage state, we compared adherent epi-
thelial cells with nonadherent (suspended) epithelial cells. The
unanchored state can be induced by brief exposure to EDTA
and subsequent culture in nonadherent plates. Trask was phos-
phorylated upon loss of anchorage in MCF10A immortalized
epithelial cells (Fig. 1A). Trask phosphorylation was similarly
induced if adhesion is severed by brief exposure to trypsin (Fig.
1B). Trypsin also cleaves p140Traskto its smaller 85-kDa form.
The cleavage is not linked with the state of anchorage or with
the phosphorylation of Trask, since EDTA disrupts cell adhe-
sion, inducing Trask phosphorylation without cleavage. The
phosphorylation of Trask is tightly linked with the state of
anchorage, occurring immediately upon loss of anchorage and
continuing for as long as the cells are maintained in suspension
and rapidly reversing upon respreading and adhesion (Fig.
1B). The association between Trask phosphorylation and the
loss of adhesion is not unique to these cells and was repro-
ducible in other epithelial cell lines, including immortalized
keratinocytes (Fig. 1C) and epithelial cancer cell lines (Fig.
1D). Primary keratinocytes also showed no phosphorylation
of Trask when cultured in the adherent state but immediately
phosphorylated Trask when deprived of anchorage (data not
shown). The SFK phosphorylation of Trask in these cells is not
an artifactual finding unique to circumstances of tissue culture
detachment, but rather it experimentally reproduces physio-
logical conditions of cell detachment in vivo. In human tissue
sections, the phosphorylation of Trask is rarely seen, since
almost the entire normal epithelium is anchored. However,
Trask phosphorylation can be seen in physiologically detached
mitotic cells (Fig. 1E). To look at a physiologic circumstance of
experimentally induced adhesion disruption in vivo, we studied
skin wounding in mice. Traumatic scalpel-inflicted wounding
and disruption of the dermal tissue architecture induced the
phosphorylation of Trask in mouse skin to levels comparable
to those in detached MCF10A cells (Fig. 1F). Trask phosphor-
ylation is also frequently seen in human cancers at all stages in
vivo (50). Therefore, the SFK phosphorylation of Trask is
inversely linked with the state of anchorage in all in vivo and in
vitro circumstances that we have observed. Anchorage depri-
vation in cultured cells provides a highly relevant experimental
model system for mechanistic exploration of this physiologic
finding, which we have pursued in the following studies.
Experimental Trask knockdown promotes adhesive signal-
ing. The loss of anchorage is associated with the phosphoryla-
tion of Trask as well as the concomitant dephosphorylation of
focal adhesion proteins, consistent with the dismantling of
focal adhesions (Fig. 2A). To study the function of Trask in cell
detachment, we generated MCF10A cells lacking Trask ex-
pression due to shRNA knockdown (MCF10A/shTrask) along
with control cells (MCF10A/shControl) (Fig. 2B). While con-
trol cells fully inactivated integrin signaling in the unanchored
state, MCF10A/shTrask cells showed persistent phosphoryla-
tion of FAK when detached, indicating a failure to properly
inactivate integrin signaling in the unanchored state (Fig. 2C).
The phenotypic consequence of this is evident during the pro-
cess of cell detachment. Treatment of control MCF10A cells
with trypsin resulted in the rapid loss of cell adhesions with
consequent cell retraction and rounding (Fig. 2D). However,
Trask knockdown cells (MCF10A/shTrask) were considerably
more persistent in adhesion to matrix than were controls and
were significantly retarded in their rates of cell detachment,
retraction, and rounding (Fig. 2D). Dissociation from the un-
derlying matrix was impaired in MCF10A/shTrask cells despite
the effective disruption of cell-cell associations (Fig. 2E). With
prolonged exposure to trypsin, MCF10A/shTrask cells eventu-
ally did go into suspension, but the process was much slower
than that for control cells. Similar results were seen with
MDA-468 breast cancer cells and when cell detachment was
induced by EDTA (discussed below; see Fig. 4D). Similar
768SPASSOV ET AL.MOL. CELL. BIOL.
proteins preferentially in its unphosphorylated state (29). The
tyrosine kinases that phosphorylate these sites have not been
well established. v-Src has been shown to phosphorylate the
NpxY motif within the ? integrin c-tail, disrupting certain
interactions and partly explaining the adhesion defects in v-src-
transformed cells (36). However, this appears to be one of
many promiscuous functions of v-src, and an analogous phys-
iological role for c-Src or other SFKs has not been described.
It remains possible that this phosphorylation activity of cellular
SFKs is induced specifically in the unanchored state, facilitated
by the appearance of p-Trask. The inhibitory effects of p-Trask
on integrin clustering and binding activity are almost surely
mediated through the phosphorylation of its intracellular ty-
rosines and not through any functions attributed to its ECD,
since Trask truncation mutants lacking the entire ECD inhibit
cell adhesion when overexpressed and phosphorylated identi-
cally to what is seen with full-length Trask (unpublished data).
Of particular interest is the fact that the inhibitory activities
of p-Trask and focal adhesion signaling are reciprocal in na-
ture, as each of these two signaling mechanisms turns off the
other, and they function in a mutually exclusive fashion. The
SFK phosphorylation of Trask, whether induced physiologi-
cally by loss of anchorage or experimentally by Trask overex-
pression, inactivates focal adhesion signaling. The reverse also
holds true, as the activation of integrin signaling, whether in-
duced physiologically by cell adhesion to matrix or experimen-
tally by fibronectin-induced activation of integrin receptors,
promotes the dephosphorylation of Trask. It is apparent from
all our experimental model systems and from all observed
physiological states that Trask phosphorylation and focal ad-
hesion signaling inactivate each other. Therefore, integrin sig-
naling and Trask signaling inhibit each other and constitute
mutually exclusive and opposing signaling programs, defining a
switch that determines the cell anchorage state. Although there
are five tyrosines within the intracellular domain of Trask, in
extensive tyrosine mutation studies, we have found that the
phosphorylation of Trask occurs as an all-or-none event con-
sistent with its function as a switch (unpublished data). The
mechanisms that mediate the dephosphorylation of Trask al-
most surely involve the regulation of specific protein tyrosine
phosphatases (PTPs). It is likely that adhesion promotes the
activation of a PTP or the interaction of a PTP with Trask,
thereby shifting the phosphorylation state equilibrium toward
the dephosphorylation of Trask.
Interest in Trask (also known as CDCP1) has grown because
of a growing body of evidence suggesting that it may have
cellular functions that are particularly important in tumor pro-
gression. A number of studies have documented increased
expression of Trask/CDCP1 in some cancers of the lung, kid-
ney, and pancreas, with a poorer prognosis conferred by its
elevated expression (3, 24, 31). Some studies have suggested
that Trask/CDCP1 promotes anoikis resistance in cancer cells
(45). This may be a cell-specific finding, since anoikis resis-
tance was not affected when Trask was knocked down in
MDA-468 breast cancer cells (data not shown). Other studies
suggest that Trask/CDCP1 may be more important for tumor
metastasis. Consistent with this, a number of experimental
studies using overexpression, knockdown, or monoclonal anti-
body targeting approaches show that the functions of Trask/
CDCP1 are important in tumor invasion and metastasis (12,
19, 41, 46).
The link between Trask/CDCP1 and cell adhesion has been
reported by others as well. Brown et al. reported that Trask/
CDCP1 undergoes tyrosine phosphorylation upon loss of ad-
hesion, but they reported that the phosphorylation event is
specifically linked with the proteolytic cleavage of the Trask/
CDCP1 ECD and does not occur with EDTA-induced detach-
ment (8). However, this study was done without the benefit of
anti-Trask/CDCP1 antibodies, and the data were only indi-
rectly inferred from the cross-reactive activities of an anti-p-
FAK antibody. Our current data obtained using specific anti-
Trask/CDCP1 antibody reagents as well as myc-tagged Trask/
CDCP1 constructs now clearly show that the phosphorylation
of Trask/CDCP1 is tightly linked with the state of anchorage
and is not a consequence of proteolytic cleavage (Fig. 1A, C,
D, and F, 4B, 5A, and 6A). In panels of the cell types studied,
we found that the relative expression levels of the 85- and
140-kDa forms of Trask vary considerably among different cell
types but that both forms undergo phosphorylation when an-
chorage is lost. Both examples were seen in this study. MDA-
468 cells express predominantly the 85-kDa form of Trask,
which undergoes phosphorylation upon detachment (Fig. 4B),
mouse keratinocytes express predominantly the 140-kDa form
of Trask, which undergoes phosphorylation upon scraping
(Fig. 1F), and MCF10A cells express a mixture of the 85- and
140-kDa forms of Trask, both of which undergo phosphoryla-
tion upon detachment (Fig. 1A). The evidence is now clear that
the phosphorylation of Trask/CDCP1 is not a consequence of
the proteolytic cleavage of its ECD.
It should be noted that overexpressed Trask does not follow
the same cleavage ratio as endogenous Trask. Although MDA-
468 cells express predominantly the 85-kDa form of Trask, its
overexpression by transfection produces both forms. This is
likely due to the fact that the overexpression exceeds the cel-
lular capacity for full cleavage. However, both forms are phos-
phorylated and both forms are found in integrin immune com-
plexes when expressed (Fig. 8A). Therefore, Trask cleavage
does not appear to play a role in the regulation of integrins.
The adhesion functions of Trask appear to be entirely medi-
ated through the tyrosine phosphorylation of its intracellular
This work was funded by the National Institutes of Health (grant
CA113952 to M.M.M.). D.S.S. is funded by a Susan G. Komen for the
Cure Postdoctoral Fellowship. C.H.W. was funded by a California
Breast Cancer Research Program Postdoctoral Fellowship.
We thank Michael McManus and the UCSF Sandler Lentiviral
RNAi core facility. Data for the TIRF microscopy analysis were ac-
quired at the Nikon Imaging Center at UCSF/QB3.
We have no conflicts of interest to declare.
1. Arias-Salgado, E. G., et al. 2003. Src kinase activation by direct interaction
with the integrin beta cytoplasmic domain. Proc. Natl. Acad. Sci. U. S. A.
2. Arnaout, M. A., B. Mahalingam, and J. P. Xiong. 2005. Integrin structure,
allostery, and bidirectional signaling. Annu. Rev. Cell Dev. Biol. 21:381–410.
3. Awakura, Y., et al. 2008. Microarray-based identification of CUB-domain
containing protein 1 as a potential prognostic marker in conventional renal
cell carcinoma. J. Cancer Res. Clin. Oncol. 134:1363–1369.
4. Bellis, S. L., J. T. Miller, and C. E. Turner. 1995. Characterization of
VOL. 31, 2011 p-Trask INHIBITS INTEGRIN CLUSTERING781
tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase. J. Biol.
5. Bhatt, A. S., H. Erdjument-Bromage, P. Tempst, C. S. Craik, and M. M.
Moasser. 2005. Adhesion signaling by a novel mitotic substrate of src ki-
nases. Oncogene 24:5333–5343.
6. Blaschke, F., et al. 2002. Hypoxia activates beta(1)-integrin via ERK 1/2 and
p38 MAP kinase in human vascular smooth muscle cells. Biochem. Biophys.
Res. Commun. 296:890–896.
7. Bockholt, S. M., and K. Burridge. 1995. An examination of focal adhesion
formation and tyrosine phosphorylation in fibroblasts isolated from src-, fyn-,
and yes- mice. Cell Adhes. Commun. 3:91–100.
8. Brown, T. A., et al. 2004. Adhesion or plasmin regulates tyrosine phosphor-
ylation of a novel membrane glycoprotein p80/gp140/CUB domain-contain-
ing protein 1 in epithelia. J. Biol. Chem. 279:14772–14783.
9. Calderwood, D. A., et al. 2002. The phosphotyrosine binding-like domain of
talin activates integrins. J. Biol. Chem. 277:21749–21758.
10. Chodniewicz, D., and R. L. Klemke. 2004. Regulation of integrin-mediated
cellular responses through assembly of a CAS/Crk scaffold. Biochim. Bio-
phys. Acta 1692:63–76.
11. David-Pfeuty, T., and S. J. Singer. 1980. Altered distributions of the cytoskel-
etal proteins vinculin and alpha-actinin in cultured fibroblasts transformed
by Rous sarcoma virus. Proc. Natl. Acad. Sci. U. S. A. 77:6687–6691.
12. Deryugina, E. I., et al. 2009. Functional role of cell surface CUB domain-
containing protein 1 in tumor cell dissemination. Mol. Cancer Res. 7:1197–
13. Dittel, B. N., J. B. McCarthy, E. A. Wayner, and T. W. LeBien. 1993.
Regulation of human B-cell precursor adhesion to bone marrow stromal
cells by cytokines that exert opposing effects on the expression of vascular
cell adhesion molecule-1 (VCAM-1). Blood 81:2272–2282.
14. Engen, J. R., et al. 2008. Structure and dynamic regulation of Src-family
kinases. Cell. Mol. Life Sci. 65:3058–3073.
15. Fincham, V. J., and M. C. Frame. 1998. The catalytic activity of Src is
dispensable for translocation to focal adhesions but controls the turnover of
these structures during cell motility. EMBO J. 17:81–92.
16. Fincham, V. J., et al. 1996. Translocation of Src kinase to the cell periphery
is mediated by the actin cytoskeleton under the control of the Rho family of
small G proteins. J. Cell Biol. 135:1551–1564.
17. Frame, M. C. 2004. Newest findings on the oldest oncogene; how activated
src does it. J. Cell Sci. 117:989–998.
18. Frame, M. C., V. J. Fincham, N. O. Carragher, and J. A. Wyke. 2002. v-Src’s
hold over actin and cell adhesions. Nat. Rev. Mol. Cell Biol. 3:233–245.
19. Fukuchi, K., et al. 2010. Inhibition of tumor metastasis: functional immune
modulation of the CUB domain containing protein 1. Mol. Pharm. 7:245–
20. Glenney, J. R., Jr., and L. Zokas. 1989. Novel tyrosine kinase substrates from
Rous sarcoma virus-transformed cells are present in the membrane skeleton.
J. Cell Biol. 108:2401–2408.
21. Harte, M. T., J. D. Hildebrand, M. R. Burnham, A. H. Bouton, and J. T.
Parsons. 1996. p130Cas, a substrate associated with v-Src and v-Crk, local-
izes to focal adhesions and binds to focal adhesion kinase. J. Biol. Chem.
22. Hooper, J. D., et al. 2003. Subtractive immunization using highly metastatic
human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phos-
phorylated glycoprotein antigen. Oncogene 22:1783–1794.
23. Huveneers, S., and E. H. Danen. 2009. Adhesion signaling—crosstalk be-
tween integrins, Src and Rho. J. Cell Sci. 122:1059–1069.
24. Ikeda, J., et al. 2009. Expression of CUB domain containing protein
(CDCP1) is correlated with prognosis and survival of patients with adeno-
carcinoma of lung. Cancer Sci. 100:429–433.
25. Kaplan, K. B., et al. 1994. Association of the amino-terminal half of c-Src
with focal adhesions alters their properties and is regulated by phosphory-
lation of tyrosine 527. EMBO J. 13:4745–4756.
26. Kaplan, K. B., J. R. Swedlow, D. O. Morgan, and H. E. Varmus. 1995. c-Src
enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-inde-
pendent mechanism. Genes Dev. 9:1505–1517.
27. Klinghoffer, R. A., C. Sachsenmaier, J. A. Cooper, and P. Soriano. 1999. Src
family kinases are required for integrin but not PDGFR signal transduction.
EMBO J. 18:2459–2471.
28. Mamat, S., et al. 2010. Prognostic significance of CUB domain containing
protein expression in endometrioid adenocarcinoma. Oncol. Rep. 23:1221–
29. McCleverty, C. J., D. C. Lin, and R. C. Liddington. 2007. Structure of the
PTB domain of tensin1 and a model for its recruitment to fibrillar adhesions.
Protein Sci. 16:1223–1229.
30. Mitra, S. K., and D. D. Schlaepfer. 2006. Integrin-regulated FAK-Src sig-
naling in normal and cancer cells. Curr. Opin. Cell Biol. 18:516–523.
31. Miyazawa, Y., et al. 2010. CUB domain-containing protein 1, a prognostic
factor for human pancreatic cancers, promotes cell migration and extracel-
lular matrix degradation. Cancer Res. 70:5136–5146.
32. Moser, M., K. R. Legate, R. Zent, and R. Fassler. 2009. The tail of integrins,
talin, and kindlins. Science 324:895–899.
33. Nigg, E. A., B. M. Sefton, T. Hunter, G. Walter, and S. J. Singer. 1982.
Immunofluorescent localization of the transforming protein of Rous sar-
coma virus with antibodies against a synthetic src peptide. Proc. Natl. Acad.
Sci. U. S. A. 79:5322–5326.
34. Parsons, S. J., and J. T. Parsons. 2004. Src family kinases, key regulators of
signal transduction. Oncogene 23:7906–7909.
35. Playford, M. P., and M. D. Schaller. 2004. The interplay between Src and
integrins in normal and tumor biology. Oncogene 23:7928–7946.
36. Sakai, T., R. Jove, R. Fassler, and D. F. Mosher. 2001. Role of the cytoplas-
mic tyrosines of beta 1A integrins in transformation by v-src. Proc. Natl.
Acad. Sci. U. S. A. 98:3808–3813.
37. Schaller, M. D., et al. 1994. Autophosphorylation of the focal adhesion
kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol. Cell.
38. Schaller, M. D., and J. T. Parsons. 1995. pp125FAK-dependent tyrosine
phosphorylation of paxillin creates a high-affinity binding site for Crk. Mol.
Cell. Biol. 15:2635–2645.
39. Scherl-Mostageer, M., et al. 2001. Identification of a novel gene, CDCP1,
overexpressed in human colorectal cancer. Oncogene 20:4402–4408.
40. Shattil, S. J. 2005. Integrins and Src: dynamic duo of adhesion signaling.
Trends Cell Biol. 15:399–403.
41. Siva, A. C., et al. 2008. Targeting CUB domain-containing protein 1 with a
monoclonal antibody inhibits metastasis in a prostate cancer model. Cancer
42. Spassov, D. S., F. L. Baehner, C. H. Wong, S. McDonough, and M. M.
Moasser. 2009. The transmembrane src substrate Trask is an epithelial
protein that signals during anchorage deprivation. Am. J. Pathol. 174:1756–
43. Timpson, P., G. E. Jones, M. C. Frame, and V. G. Brunton. 2001. Coordi-
nation of cell polarization and migration by the Rho family GTPases requires
Src tyrosine kinase activity. Curr. Biol. 11:1836–1846.
44. Turner, C. E. 2000. Paxillin and focal adhesion signalling. Nat. Cell Biol.
45. Uekita, T., et al. 2007. CUB domain-containing protein 1 is a novel regulator
of anoikis resistance in lung adenocarcinoma. Mol. Cell. Biol. 27:7649–7660.
46. Uekita, T., et al. 2008. CUB-domain-containing protein 1 regulates perito-
neal dissemination of gastric scirrhous carcinoma. Am. J. Pathol. 172:1729–
47. Volberg, T., L. Romer, E. Zamir, and B. Geiger. 2001. pp60(c-src) and
related tyrosine kinases: a role in the assembly and reorganization of matrix
adhesions. J. Cell Sci. 114:2279–2289.
48. Vuori, K. 1998. Integrin signaling: tyrosine phosphorylation events in focal
adhesions. J. Membr. Biol. 165:191–199.
49. Wegener, K. L., et al. 2007. Structural basis of integrin activation by talin.
50. Wong, C. H., et al. 2009. Phosphorylation of the SRC epithelial substrate
Trask is tightly regulated in normal epithelia but widespread in many human
epithelial cancers. Clin. Cancer Res. 15:2311–2322.
51. Yamana, N., et al. 2006. The Rho-mDia1 pathway regulates cell polarity and
focal adhesion turnover in migrating cells through mobilizing Apc and c-Src.
Mol. Cell. Biol. 26:6844–6858.
52. Yokosaki, Y., et al. 1994. The integrin alpha 9 beta 1 mediates cell attach-
ment to a non-RGD site in the third fibronectin type III repeat of tenascin.
J. Biol. Chem. 269:26691–26696.
782SPASSOV ET AL.MOL. CELL. BIOL.