From the Dana-Farber Cancer Institute, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, October 24, 2000, and in revised form, January 9, 2001
The DF3/MUC1 mucin-like glycoprotein is
aberrantly overexpressed in most human carcinomas. The cytoplasmic
domain of MUC1 interacts with glycogen synthase kinase 3
(GSK3
)
and thereby decreases binding of MUC1 and
-catenin. The present
studies demonstrate that MUC1 associates with the c-Src tyrosine
kinase. c-Src phosphorylates the MUC1 cytoplasmic domain at a YEKV
motif located between sites involved in interactions with GSK3
and
-catenin. The results demonstrate that the c-Src SH2 domain binds
directly to pYEKV and inhibits the interaction between MUC1 and
GSK3
. Moreover and in contrast to GSK3
, in vitro and
in vivo studies demonstrate that c-Src-mediated
phosphorylation of MUC1 increases binding of MUC1 and
-catenin.
The findings support a novel role for c-Src in regulating
interactions of MUC1 with GSK3
and
-catenin.
 |
INTRODUCTION |
-catenin, a component of the adherens junctions of mammalian
epithelial cells, binds directly to the cytoplasmic domain of the
transmembrane E-cadherin protein that functions in
Ca2+-dependent epithelial cell-cell
interactions (1). In turn,
-catenin binds to
-catenin and thereby
links the complex to the actin cytoskeleton (2). Formation of the
cadherin-catenin complex is essential for adherens junction function
(3). In the cytosol,
-catenin binds directly to the adenomatous
polyposis coli (APC)1 tumor
suppressor (4-6). Phosphorylation of APC and
-catenin by GSK3
increases the formation of APC-
-catenin complexes (7) and targets
-catenin for ubiquitination and degradation by the 26 S proteosome
(8-10). Cells that express certain APC mutants or are APC deficient
thus exhibit increased levels of cytosolic
-catenin (11). Other
studies have shown that
-catenin forms complexes with members of the
T-cell factor/leukocyte-enhancing factor (Tcf/LEF-1) family of
transcription factors (12-14) and functions in the activation of gene
expression (13-15).
The finding that
-catenin and GSK3
interact with the cytoplasmic
domain of the DF3/MUC1 mucin-like glycoprotein has supported the
involvement of an additional pathway in
-catenin signaling (16, 17).
MUC1 is highly overexpressed by human carcinomas (18). In addition,
whereas MUC1 expression is restricted to the apical borders of normal
secretory epithelial cells, MUC1 is aberrantly expressed by carcinoma
cells at high levels throughout the cytoplasm and over the entire cell
surface (18-20). The MUC1 protein consists of an N-terminal ectodomain
with variable numbers of 20-amino acid tandem repeats that are subject
to extensive O-glycosylation (21, 22). The C-terminal region
includes a transmembrane domain and a 72-amino acid cytoplasmic tail.
MUC1 is subject to proteolytic cleavage and the large ectodomain
containing the tandem repeats can remain complexed to the 25-kDa
C-terminal subunit or undergo release from the cell surface (23).
-catenin binds directly to MUC1 at a SAGNGGSSL motif in the
cytoplasmic domain (16). Similar SXXXXXSSL sites in
E-cadherin and APC are responsible for
-catenin interactions (4-6).
GSK3
also binds directly to MUC1 and phosphorylates serine in a
DRSPY site adjacent to that for the
-catenin interaction (17).
GSK3
-mediated phosphorylation of MUC1 decreases the association of
MUC1 and
-catenin (17).
The present studies demonstrate that the c-Src tyrosine kinase
interacts directly with MUC1. A YEKV motif in the MUC1 cytoplasmic domain (CD) has been identified as a site for c-Src phosphorylation. The results demonstrate that c-Src regulates the interactions of MUC1
with GSK3
and
-catenin.
 |
MATERIALS AND METHODS |
Cell Culture--
Human ZR-75-1 breast carcinoma cells were
grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine
serum, 100 µg/ml streptomycin, 100 units/ml penicillin, and 2 mM L-glutamine. 293 cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated
fetal bovine serum, 100 µg/ml streptomycin, and 100 units/ml penicillin.
Lysate Preparation--
Subconfluent cells were disrupted on ice
in lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM
NaCl, 0.1% Nonidet P-40, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM
dithiothreitol) for 30 min. Lysates were cleared by centrifugation at
14,000 × g for 20 min.
Immunoprecipitation and Immunoblotting--
Equal amounts of
protein from cell lysates were incubated with normal mouse IgG, MAb DF3
(anti-MUC1) (18), anti-c-Src (Upstate Biotechnology, Lake Placid, NY),
or the rabbit anti-DF3-E antibody prepared against a peptide derived
from the MUC1 extracellular domain (HDVETQFNQYKTEAAS). After incubation
for 2 h at 4 °C, the immune complexes were precipitated with
protein G-agarose. The immunoprecipitates were washed with lysis
buffer, separated by SDS-PAGE, and transferred to nitrocellulose
membranes. The immunoblots were probed with 500 ng/ml anti-MUC1 or 1 µg/ml anti-c-Src. Reactivity was detected with horseradish
peroxidase-conjugated second antibodies and chemiluminescence (ECL,
Amersham Pharmacia Biotech).
Preparation of MUC1 and c-Src Mutants--
The MUC1/CD(Y46F) and
MUC1(Y46F) mutants were generated using site-directed mutagenesis
(QuikChange; Stratagene, La Jolla, CA) to change Tyr-46 to Phe.
Kinase-inactive c-Src was similarly generated by mutation of Lys-295 to
Arg (K295R) (24).
In Vitro Phosphorylation--
Purified wild-type and mutant
MUC1/CD proteins were incubated with 1.5 units of purified c-Src
(Oncogene Research Products, Cambridge, MA) in 20 µl of kinase buffer
(20 mM Tris-HCl, pH 7.6, 10 mM
MgCl2, 5 mM dithiothreitol). The reaction was
initiated by addition of 10 µCi [
-32P]ATP. After
incubation for 15 min at 30 °C, the reaction was stopped by addition
of sample buffer and boiling for 5 min. Phosphorylated proteins were
separated by SDS-PAGE and analyzed by autoradiography.
Binding Studies--
Purified wild-type and mutant MUC1/CD
proteins were incubated with 1.5 units of c-Src in the presence or
absence of 200 µM ATP for 30 min at 30 °C. GST,
GST-Src-SH3, GST-Src-SH3De90/92 (Ref. 25, provided by Dr. J. Brugge,
Harvard Medical School), GST-Src-SH2, or GST-
-catenin bound to
glutathione beads was then added, and the reaction was incubated for
1 h at 4 °C. After washing, the proteins were subjected to
SDS-PAGE and immunoblot analysis with the anti-MUC1/CD antibody that
was generated against the cytoplasmic domain (17). In other studies,
GST-MUC1/CD bound to glutathione beads was incubated with 1.5 units of
c-Src in the presence and absence of 200 µM ATP for 30 min at 30 °C before adding 0.1 mg of purified GSK3
(New England
BioLabs) for an additional 1 h. Precipitated proteins were
analyzed by immunoblotting with anti-GSK3
.
Transient Transfection Studies--
ZR-75-1 or 293 cells were
transiently transfected with pCMV, pCMV-MUC1, pCMV-c-Src (provided by
Dr. R. Rickles, ARIAD Pharmaceuticals, Inc., Cambridge, MA) or
pCMV-c-Src(K295R) using electroporation methods. Efficiency of
transient transfections ranged from 40-50% of ZR-75-1 cells and
70-80% of 293 cells. Cell lysates were prepared at 48 h after transfection.
 |
RESULTS AND DISCUSSION |
To determine whether DF3/MUC1 forms a complex with c-Src,
anti-MUC1 immunoprecipitates from lysates of human ZR-75-1 cells were
analyzed by immunoblotting with anti-c-Src. The results demonstrate that c-Src coprecipitates with MUC1 (Fig.
1A, left). In the
reciprocal experiment, analysis of anti-c-Src immunoprecipitates by
immunoblotting with anti-MUC1 confirmed the association of MUC1 and
c-Src (Fig. 1A, right). Similar results have been
obtained in human HeLa cells (data not shown). To assess whether the
binding is direct, we incubated purified His-tagged MUC1 cytoplasmic
domain (His-MUC1/CD) with a GST fusion protein that contains the c-Src
SH3 domain. Analysis of the adsorbate to glutathione beads by
immunoblotting with anti-MUC1/CD demonstrated binding of MUC1/CD to
GST-Src SH3, and not GST or a GST-Src SH2 fusion protein (Fig.
1B). As an additional control, His-MUC1/CD was incubated
with a GST fusion protein containing a mutated c-Src SH3 domain
(GST-Src SH3De90/92) (26). The finding that MUC1/CD binds to wild-type
c-Src SH3 but not the mutant supported a direct interaction between
MUC1 and c-Src (Fig. 1C).

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Fig. 1.
Interaction of MUC1 with c-Src.
A, lysates from ZR-75-1 cells were subjected to
immunoprecipitation with anti-MUC1 (MAb DF3; left panel) or
anti-c-Src (right panel). Mouse IgG was used as a control.
The immunoprecipitates and lysates not subjected to immunoprecipitation
were analyzed by immunoblotting with anti-c-Src (left panel)
and anti-MUC1 (right panel). B, purified MUC1/CD
was incubated with GST, GST-Src-SH2, or GST-Src-SH3 for 1 h at
4 °C. Proteins precipitated with glutathione-Sepharose 4B beads were
subjected to SDS-PAGE and immunoblot analysis with anti-MUC1/CD.
C, purified MUC1/CD was incubated with GST, GST-Src-SH3, or
GST-Src-SH3De90/92 (deletion of amino acids 90/92). Adsorbates to
glutathione beads were subjected to immunoblot analysis with
anti-MUC1/CD (left panel). The gel was stained with
Coomassie Blue to assess loading of the wild-type and mutant SH3
domains (right panel).
|
|
To determine whether MUC1/CD is a substrate for c-Src, we incubated
MUC1/CD with purified c-Src and [
-32P]ATP. Analysis of
the reaction products by SDS-PAGE and autoradiography demonstrated
c-Src-mediated phosphorylation of MUC1/CD (Fig.
2A). Previous
studies have demonstrated that GSK3
phosphorylates MUC1/CD on Ser at
a DRSPYEKV site (17). As the adjacent YEKV sequence represents a
consensus for c-Src phosphorylation, MUC1/CD was generated with a FEKV
mutation (Fig. 2B). Incubation of MUC1/CD(Y46F) with c-Src
demonstrated a decrease in phosphorylation as compared with that found
with wild-type MUC1/CD (Fig. 2C). These findings indicate
that c-Src phosphorylates MUC1/CD predominantly but not exclusively at
the YEKV site. As the c-Src SH2 domain interacts with a preferred pYEEI
sequence (27), c-Src-mediated phosphorylation of YEKV in MUC1/CD
provides a potential site for c-Src SH2 binding. To determine whether
the c-Src SH2 domain binds to phosphorylated MUC1/CD, we incubated
MUC1/CD with c-Src and ATP and then assessed binding to GST-Src SH2.
The results demonstrate that GST-Src SH2 associates with phosphorylated
but not unphosphorylated MUC1/CD (Fig. 2D). Moreover,
compared with MUC1/CD, there was substantially less binding of GST-Src
SH2 to the MUC1/CD(Y46F) mutant that had been incubated with c-Src and
ATP (Fig. 2D). These results support c-Src-mediated
phosphorylation of MUC1/CD and thereby a direct interaction of
phosphorylated MUC1/CD with the c-Src SH2 domain.
As the c-Src phosphorylation site on MUC1/CD resides next to the
binding and phosphorylation site for GSK3
(17), we asked if the
interaction of MUC1/CD with c-Src affects that with GSK3
. GST-MUC1/CD was incubated with c-Src and ATP before addition of GSK3
. Analysis of proteins precipitated with glutathione beads demonstrated that c-Src-mediated phosphorylation of MUC1/CD is associated with a decrease in binding of MUC1/CD and GSK3
(Fig. 3A). To assess the effects of
c-Src on the interaction of MUC1/CD and GSK3
in vivo,
ZR-75-1 cells were transfected to express the empty vector or c-Src.
Anti-MUC1 immunoprecipitates were analyzed by immunoblotting with
anti-GSK3
. The results demonstrate that c-Src also decreases the
interaction of MUC1 and GSK3
in vivo (Fig.
3B). These findings indicate that GSK3
interacts with
MUC1/CD by a c-Src-dependent mechanism.
We thank Joan Brugge for GST-Src-SH3 and
GST-Src-SH3De90/92, Ricky Rickles for pCMV-c-Src, and John Hilkens for
pCMV-MUC1 constructs.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.C000754200
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