From the McGill Cancer Centre and the
§§ Departments of Biochemistry, Medicine, and
Oncology, McGill University, Montreal, Quebec H3G 1Y6, Canada and the
** Samuel Lunenfeld Institute, Toronto, Ontario M5G 1X5, Canada
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ABSTRACT |
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Biliary glycoprotein (Bgp, C-CAM, or CD66a) is an
immunoglobulin-like cell adhesion molecule and functions as a tumor
suppressor protein. We have previously shown that the Bgp1 isoform
responsible for inhibition of colonic, liver, prostate, and breast
tumor cell growth contains within its cytoplasmic domain two tyrosine
residues positioned in immunoreceptor
tyrosine-based inhibition motif
(ITIM) consensus sequences. Moreover, we determined that these
residues, upon phosphorylation, associate with the protein-tyrosine
phosphatase SHP-1. In this report, we have further evaluated the
structural bases of the association of Bgp1 with Tyr phosphatases.
First, we demonstrate that Bgp1 also associates with the SHP-2 Tyr
phosphatase, but not with an unrelated Tyr phosphatase, PTP-PEST.
Association of Bgp1 and SHP-2 involves the Tyr residues within the Bgp1
ITIM sequences, Val at position +3 relative to the second Tyr
(Tyr-515), and the SHP-2 N-terminal SH2 domain. In addition, our
results indicate that residues +4, +5, and +6 relative to Tyr-515 in
the Bgp1 cytoplasmic domain play a significant role in these
interactions, as their deletion reduced Bgp1 Tyr phosphorylation and
association with SHP-1 and SHP-2 by as much as 80%. Together, these
results indicate that both SHP-1 and SHP-2 interact with the Bgp1
cytoplasmic domain via ITIM-like sequences. Furthermore, they reveal
that the C-terminal amino acids of Bgp1 are critical for these interactions.
Biliary glycoprotein
(Bgp),1 also known as C-CAM
or CD66a, is a cell-surface immunoglobulin-like glycoprotein and a
member of the carcinoembryonic antigen family (1, 2). Whereas one BGP gene has been identified in human, two very similar
Bgp genes (Bgp1 and Bgp2) are
expressed in the mouse (3, 4). The BGP genes in all species
studied so far are subjected to very similar alternative splicing
events (1, 3, 5). The most commonly encountered isoforms of the mouse
Bgp1 gene exhibit four extracellular Ig domains and
cytoplasmic domains, which include either a short 10-amino acid peptide
(BgpS) or a longer version of 73 amino acids (BgpL) (3, 6). Bgp1 is
expressed in epithelial cells of the gastrointestinal tract (7). In
addition, the Bgp1 protein is expressed in epithelial cells of the
reproductive system, where its expression is hormonally controlled (7).
Bgp1 is also present at the surface of endothelial cells and in
hemopoietic cells, in particular in B cells, macrophages, and
interleukin-2-activated T cells (7-10).
Several functions have been attributed to the biliary glycoprotein.
First, it functions as an intercellular adhesion molecule (6, 11, 12).
This function requires the first Ig domain (13, 14) and is independent
of the cytoplasmic region (15, 16). Second, mouse Bgp1 and Bgp2 behave
as receptors for all strains of the mouse hepatitis viruses (4, 17),
whereas human BGP binds to bacterial proteins from Escherichia
coli, Salmonella typhimurium, or Neisseria
gonorrhoeae (18-20). Furthermore, we have recently shown that
Bgp1 acts as a negative regulator of colonic tumor cell growth and that
this role is dependent on the presence of the longer cytoplasmic domain
of Bgp1 (21). Similar findings have been reported in human prostate and
breast carcinoma models (22, 23).
Recent reports have also suggested that Bgp1 behaves as a signal
transduction molecule. Several physiological events promote the Tyr
phosphorylation of Bgp1 on one or two Tyr residues within its
cytoplasmic domain (Tyr-488 and Tyr-515). BGP becomes
Tyr-phosphorylated by Src-like Tyr kinases in activated neutrophils
(24) and in human colon carcinoma cells (25) and is a Tyr
phosphorylation substrate for the insulin receptor (26). Moreover, we
(27) and others (28) have shown that Tyr-488 also undergoes
phosphorylation when cellular phosphatases are inactivated by the
pharmacological inhibitor vanadate. Whereas the physiological
significance of Bgp1 Tyr phosphorylation remains largely to be defined,
it has been reported that stimulation of Bgp1 (CD66a) in neutrophils leads to activation of Rac1, PAK, and Jun N-terminal kinase (29).
Interestingly, the amino acid sequences surrounding Tyr-488 and Tyr-515
of Bgp1 perfectly match that of an immunoreceptor tyrosine-based inhibition motif
(ITIM). This includes a phosphorylated Tyr residue preceded by a Val,
Ile, or Leu residue at position As our studies related to Bgp1-dependent tumor inhibition
were being conducted in mouse colonic carcinoma cells (21), we questioned whether Bgp1 Tyr phosphorylation was conducive to any significant protein associations in this cellular background. We have
recently shown that Tyr phosphorylation of the mouse Bgp1 cytoplasmic
domain in CT51 mouse colonic carcinoma cells led to its binding to the
protein-Tyr phosphatase SHP-1 and that this event required the presence
of both Tyr-488 and Tyr-515 (27). In vitro binding assays
confirmed that either one of the SHP-1 SH2 domains could bind to Bgp1
(27). We now report that, in addition to its interaction with SHP-1,
Bgp1 physically associates with another ubiquitously expressed
cytosolic protein-Tyr phosphatase, SHP-2 (36, 37). The association with
SHP-2, like that with SHP-1, is dependent upon the expression of both
Bgp1 cytoplasmic Tyr residues and the phosphorylation of at least one
Tyr residue. Binding of SHP-2 requires a Val residue within the second
ITIM sequence of the Bgp1 cytoplasmic domain. In addition, the
N-terminal SH2 domain of SHP-2 interacts with Tyr-phosphorylated Bgp1.
Furthermore, in reconstituting the association of Bgp1 deletion and
point mutants with the Tyr phosphatases in CT51 mouse colonic
epithelial cells and 293 human embryonic kidney cells, we found that
the Bgp1 carboxyl-terminal region is critical for the regulation of its
association with the protein-Tyr phosphatases SHP-1 and SHP-2. Residues
+4, +5, and +6 relative to Tyr-515 within the second Bgp1 ITIM are
necessary for maximal phosphatase association with Bgp1. These results
represent the first report that residues located outside the classical
ITIM consensus sequence influence the in vivo binding of the
Tyr phosphatases SHP-1 and SHP-2. These three residues may consequently
imprint a particular conformation on the Bgp1 cytoplasmic domain or may be involved in anchoring other Bgp1 cytosolic partners to the cell surface.
Cell Culture--
Growth of CT51 mouse colon carcinoma cells,
generously provided by Dr. Michael Brattain (Medical College of Ohio,
Toledo, OH), and insertion of the Bgp1 cDNA constructs
by retrovirus-mediated infection have been described previously (21).
Cell clones were selected from G418-resistant populations (G418: 1.5 mg/ml), and Bgp1-positive clones were identified by
fluorescence-activated cell sorter analyses and immunoblotting as
reported previously (21). Experiments were performed with either cell
populations or a minimum of two clones for each transfectant cell line.
Pervanadate treatment of cells was performed by incubating transfected
CT51 cells in Cloning of the SHP-2 cDNA from a Mouse Thymocyte cDNA
Library--
The mouse Syp cDNA (GenBankTM
accession number L08663) was kindly provided by Dr. Gen-Sheng Feng
(Indiana University School of Medicine, Indianapolis, IN) (37). The
C-terminal regions of mouse BALB/c 3T3 Syp and brain SHP-2 are
different; to obtain a complete SHP-2 cDNA, the
Syp cDNA was therefore used as a probe on a C57Bl6 mouse
thymocyte cDNA library, kindly provided by Dr. L. A. Matis
(Yale University School of Medicine, New Haven, CT). Several cDNA
clones were isolated and subjected to nucleotide sequencing (40). Clone
9 encoded a 4-kilobase pair cDNA encompassing the complete coding
region of 1778 base pairs identical to that found in a mouse brain
SHP-2 cDNA (GenBankTM accession number
D84372). Cloning of the mouse PTP-PEST cDNA has
previously been reported (41).
Site-directed Mutagenesis--
Mutations were introduced in the
cytoplasmic domain of the BgpL cDNA by single-strand
excision with M13K07 helper virus of a uracil-containing BlueScript
SK
A SHP-1 phosphatase-inactive mutant was generated by replacing Cys-453
with Ser (27). Phosphatase-inactive SHP-2 mutants were generated by
converting Cys-459, present in the active site of the enzyme, to Ser.
These mutations were created by the overlap PCR technique (42) using
the following combination of primers: nucleotides 1353-1369
(5'-GCGTGTTAGGAACGTCA-3') with nucleotides 1583-1603
(5'-TCCCAGCGCTCGAGTGAACCA-3') and nucleotides 1583-1603 (5'-TGGTTCACTCGAGCGCTGGGA-3') with nucleotides 2187-2200
(5'-CGCAGAGCAGTCTC-3'). The overlap fragments were cleaved with
PflMI-HincII and substituted with that present
within the full-length SHP-2 cDNA. All mutants were
subjected to DNA sequencing to confirm their integrity.
Antibodies--
An anti-mouse Bgp1 polyclonal antibody, specific
to the extracytoplasmic Ig domains of Bgp1, has previously been
described (6). Antibodies specific to the C-terminal regions of either SHP-1 (27) or SHP-2 were raised in rabbits using TrpE fusion proteins.
A fragment from the mouse SHP-2 cDNA corresponding to amino acids 553-597 was amplified by PCR and cloned into the pATH11 vector, and antibodies were raised against the induced purified fusion
protein. Neither the SHP-1 nor the SHP-2 antibodies exhibit cross-reactivity. A polyclonal antibody raised against the
non-catalytic portion of mouse PTP-PEST has previously been described
(41). This antibody does not cross-react with the human PTP-PEST
phosphatase. A mouse monoclonal antibody specific to v-Src was
purchased from Oncogene Research Products (Cambridge, MA), whereas an
anti-phosphotyrosine (Tyr(P)) antibody (4G10) was obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY). The polyclonal antibody to the
GST protein was a kind gift from Drs. Philip Branton and Joseph Lee
(Department of Biochemistry, McGill University).
Immunoprecipitation and Immunoblotting--
CT51 or HEK293 cells
were scraped from the dishes; collected by centrifugation; and
resuspended in lysis buffer containing 50 mM Tris-HCl (pH
8.0), 20 mM EDTA, 1% Brij 96, 10 µg/ml protease inhibitors (leupeptin, aprotinin, phenylmethylsulfonyl fluoride, N Generation of GST-SHP-2 Fusion Proteins and in Vitro Binding
Assays--
GST fusion proteins containing various domains of the
SHP-2 protein-Tyr phosphatase were generated by polymerase chain
reaction amplification using the indicated primers. The N-terminal SH2 domain fragment (amino acids 6-102; referred to as N-SH2 on
Fig. 5) was amplified using primers 5'-GTGGTTTCACCCCAAC-3' and
5'-TTCAGCGGGTACTTGAG-3', whereas the C-terminal SH2 domain fragment
(amino acids 112-208; referred to as C-SH2 on Fig. 5) was
produced with primers 5'-GTGGTTCCATGGTCAC-3' and
5'-ACTGTGCCCAGCGTCTC-3'. A fragment encoding the two SH2 domains (amino
acids 6-208; referred to as SH2 ×2 on Fig. 5) was obtained using the first and last of these primers. Finally, the SHP-2 fragment
(amino acids 6-594) was produced using primers 5'-GTGGTTTCACCCCAAC-3' and 5'-ACGGGTCCTGGGAACGCTCGT-3'. The oligonucleotides were tagged with
restriction cleavage sites, and the resulting PCR fragments were
submitted to digestion with restriction enzymes and cloned into the
pGEX2T vector (Amersham Pharmacia Biotech). The cloned PCR fragments
were subjected to DNA sequencing (40). The constructs were transformed
into E. coli BL21, and GST fusion proteins were induced with
isopropyl- Quantification--
Quantification of the radioactive bands was
performed on a Fuji BioAnalyzing System 2000. Values were estimated
from a minimum of three independent experiments. Ratios of proteins
present in immunoprecipitated complexes were derived by dividing the
value of the band by the corresponding values of bands in the total cell lysate proteins. Differences were then expressed as percentages of
values obtained with wild-type Bgp1.
Association of Bgp1 and SHP-2 in CT51 Mouse Colonic Carcinoma Cells
and HEK293 cells--
We have previously demonstrated that SHP-1
associates with Bgp1 in mouse colonic carcinoma cells (27). Another
SH2-containing protein-Tyr phosphatase, SHP-2, is also recruited to the
cell surface following activation of Tyr-phosphorylated receptors (36, 37). We therefore verified whether SHP-2 was expressed in CT51 cells
and whether it also associated with Bgp1. None of the available antibodies directed to the mouse Bgp1 protein elicited Bgp1 Tyr phosphorylation. As no specific ligands for Bgp1 capable of provoking its Tyr phosphorylation in colonic cells have yet been identified, we
therefore made use of the pharmacological inhibitor pervanadate, which
is known to inactivate endogenous protein phosphatases, thereby
mimicking activation of protein-tyrosine kinases (35, 38). A
Bgp1-expressing CT51 cell clone was subjected to pervanadate treatment;
the cells were lysed with non-ionic detergent-containing buffers; and
the Bgp1 protein was immunoprecipitated with anti-Bgp1 antibodies.
SHP-2 was revealed by blotting with its specific antibody (Fig.
1A). SHP-2 was absent from
immune complexes prepared from untreated cells (Fig. 1A,
lane 1), but was found associated with Bgp1 immune complexes
prepared from cells treated with pervanadate (lane 2).
Expression of Bgp1 and SHP-2 in the CT51 cells was monitored by
immunoblotting cell lysate proteins with their respective antibodies (Fig. 1A, lower panels). Therefore, upon Bgp1 Tyr
phosphorylation, the SHP-2 Tyr phosphatase associates with Bgp1,
presumably via its cytoplasmic domain.
To evaluate the potential association of Bgp1 with SHP-2 in a different
cellular system, we also performed transfection experiments in HEK293
cells. HEK293 cells were therefore transiently transfected with the
wild-type Bgp1 cDNA in combination with the
SHP-1 and SHP-2 Tyr phosphatase constructs (Fig.
1B). As it has previously been shown that Src induces Tyr
phosphorylation of Bgp1 (25), we included the constitutively activated
mouse c-src Y529F cDNA in these transfection assays.
Levels of Bgp1 Tyr phosphorylation and Bgp1 association with the
various phosphatases were determined after lysis of the transfected
cells, immunoprecipitation of Bgp1, and detection with either
anti-Tyr(P) antibodies or the phosphatase-specific antibodies (Fig.
1B). HEK293 cells do not express the endogenous BGP proteins
as revealed by immunoprecipitation/immunoblotting experiments using
BGP-specific antibodies (Fig. 1B, third panel, lane
1). As revealed on the anti-Tyr(P) immunoblots (Fig.
1B, first panels, lanes 2 and
5), transfection of the Bgp1 and
c-src Y529F cDNAs in these cells led to Bgp1 Tyr
phosphorylation. HEK293 cells do not endogenously express the SHP-1 Tyr
phosphatase, but SHP-2 was present in the lysates (Fig. 1B,
fourth panels, lanes 1 and 2 versus lane 5).
We then examined whether adding the wild-type SHP-1 or
SHP-2 cDNA or their catalytically inactive versions
(C453S or C459S, respectively) altered the Bgp1 Tyr phosphorylation
status and consequent Tyr phosphatase association. The Cys-to-Ser
mutations in the SHP-1 and SHP-2 Tyr phosphatases inactivate their
phosphatase activity, but do not compromise their binding
characteristics (35). The levels of Bgp1 expressed in the transfected
cells were uneven, with this protein being particularly abundant in Fig. 1B (lane 3). However, this reinforced the
fact that, despite the large amount of Bgp1 in this sample, its Tyr
phosphorylation level was characteristically lower when the wild-type
SHP-1 phosphatase was present compared with samples in which the
catalytically inactive SHP-1 phosphatase was expressed (17-fold higher
in this experiment) (Fig. 1B, lane 4 versus lane
3). The -fold values of Bgp1 Tyr phosphorylation were normalized
to the expression of the Bgp1 protein present in each lane.
Furthermore, when equal levels of Bgp1 were expressed as in Fig.
1B (lanes 6 and 7), the levels of Bgp1
Tyr phosphorylation were again lower when the wild-type SHP-2
phosphatase was included in the assay relative to those when the
Cys-to-Ser SHP-2 mutant was used (7-fold higher) (Fig. 1B, lane 7 versus lane 6). This result suggested that the
wild-type SHP-1 and SHP-2 phosphatases may be using Bgp1 as a
substrate. Correspondingly, in both cases, the association of the SHP-1
or SHP-2 phosphatase with Bgp1 was concomitant with the Tyr
phosphorylation levels of the latter protein. This was quantified as a
15-fold increase in Cys-to-Ser SHP-1 or SHP-2 binding relative to
wild-type SHP-1 or SHP-2 phosphatase binding.
To investigate the specificity of the Bgp1 interaction with the SHP Tyr
phosphatases, we considered whether other protein-Tyr phosphatases
would also bind to this protein. PTP-PEST is a ubiquitous enzyme that
contains an N-terminal phosphatase domain followed by a long
non-catalytic carboxyl-terminal region with several PEST-like sequences
(41). When similar transfections were performed with the
Bgp1, src, and PTP-PEST phosphatase
constructs, no PEST phosphatase was found in Bgp1 immune complexes
(Fig. 1C, first panel, lanes 1 and 2),
although the PEST phosphatase and Bgp1 were adequately expressed
(second and third panels) and phosphorylated in
these cells (data not shown). Due to the specificity of the antibody used, the mouse PTP-PEST phosphatase, was not detected in
untransfected HEK293 cells (Fig. 1C, third panel, lane 1). Hence, there is specificity in the association of Tyr-phosphorylated Bgp1 with protein-Tyr phosphatases containing SH2 domains, such as
SHP-1 and SHP-2.
Association of Bgp1 with SHP-2 Requires Both Bgp1 Tyr
Residues--
We have previously demonstrated that the presence of
both Bgp1 Tyr residues and the phosphorylation of Tyr-488 were
necessary for maximal association of the SHP-1 phosphatase with Bgp1
(27). As SHP-2 also binds to Bgp1, we therefore questioned whether
association of Bgp1 and SHP-2 also necessitated both Bgp1 Tyr residues.
The mutants used in this study are graphically depicted in Fig.
2. We investigated this by subjecting
CT51 cells expressing Bgp1 Tyr mutants to pervanadate treatment. As
demonstrated in Fig. 3 (third
panel), the wild-type CT51 cells or the control transfected cells
(neo) do not endogenously express Bgp1, whereas the SHP-2 protein is reasonably abundant in this cell line (fourth panel, lanes 1-4). However, when the wild-type Bgp1 construct
was transfected into this cell line and cell clones were isolated, the
Bgp1 protein was expressed at significant levels (Fig. 3, third
panel, lanes 5-8). Furthermore, the Bgp1 protein was endogenously
Tyr-phosphorylated, albeit at low levels (Fig. 3, first
panel, lane 5). More important, however, inactivating
cellular phosphatases with the pervanadate inhibitor led to enhancement
of this Tyr phosphorylation by 34 ± 3-fold (Fig. 3, first
panel, lanes 6 and 8) as well as recruitment of the SHP-2 Tyr phosphatase (second panel, lanes
6 and 8). Mutation of either Tyr-488 or Tyr-515 within
the Bgp1 cytoplasmic domain still gave rise to a Tyr-phosphorylated
Bgp1 protein (Fig. 3, first panel, lanes 10 and
12). This result is at variance with our own previously
reported data (27), where we had been unable to detect phosphorylation
of Tyr-515 when Tyr-488 was mutated. In this report, we used a
different anti-Tyr(P) antibody (4G10), which detected a
Tyr-phosphorylated Bgp1 protein in the Y488F mutant clone. Note that
the Bgp1 expression in this clone was lower than in the Y515F clone
(Fig. 3, third panel, compare lanes 9 and
10 with lanes 11 and 12), yet Bgp1 Tyr
phosphorylation was still detectable (Fig. 3, first panel, lane
12). Mutation of either one of the Bgp1 Tyr residues led to
abrogation of SHP-2 Tyr phosphatase association (Fig. 3, second
panel, lanes 10 and 12). This result was
further confirmed using Bgp1 deletion mutants in which Tyr-488 or
Tyr-515 was removed from the cytoplasmic domain (see below) or in
transient transfection assays in HEK293 cells (data not shown); in all
these cases, loss of SHP-2 association was noticed when either one of
the Tyr residues was mutated. As expected, mutation of both Tyr
residues together revealed neither Bgp1 Tyr phosphorylation nor SHP-2
association (Fig. 3, first and second panels,
lane 14). Therefore, both Bgp1 Tyr residues are necessary for maximal association of the SHP-1 (27) and SHP-2 Tyr
phosphatases.
Molecular Requirements for Maximal Bgp1 Tyr Phosphorylation and
Association with the SHP-1 and SHP-2 Tyr Phosphatases--
To
investigate whether other subregions or residues of the Bgp1
cytoplasmic domain were involved in binding to SHP-1 or SHP-2, a series
of deletion mutants depicted in Fig. 2 was prepared. The
As the three C-terminal Lys residues appeared to be crucial in these
interactions and since this represented the first observation that
residues outside the ITIM consensus sequence affected the in
vivo binding of the phosphatases to a cell-surface protein, we
further investigated the possible mechanisms responsible for this
reduction. We first focused on two issues: the charge of these residues
and their role within a potential protein kinase C consensus sequence,
S516XXK1-3 (45). Accordingly,
additional Bgp1 mutants were generated in which the three Lys residues
were converted to either Arg (3K In Vitro Binding of SHP-2 to Bgp1--
We have previously
described that the two SH2 domains of SHP-1 bind equally well to
Tyr-phosphorylated Bgp1 (27). To determine whether similar binding
properties could be demonstrated for SHP-2, various bacterially
expressed GST fusion domains of SHP-2 bound to glutathione-Sepharose
beads were incubated with proteins from Bgp1-expressing CT51 cell
lines. After several washes, bound proteins were eluted, resolved by
electrophoresis, and detected by immunoblotting with anti-Bgp1 or
anti-GST antibodies (Fig. 5). Equal molar
amounts of the GST fusion proteins were adsorbed onto the
glutathione-Sepharose beads, as revealed on an anti-GST immunoblot
(Fig. 5B). In both unstimulated and pervanadate-treated
cells, no Tyr-phosphorylated Bgp1 bound to the GST protein alone (Fig.
5A, lanes 9 and 10) or to the
carboxyl-terminal SH2 domain (C-SH2) of SHP-2 (lanes 5 and 6), whereas a low but consistent amount of Bgp1
associated with the SHP-2 N-terminal SH2 domain (N-SH2) in
each of four repeated experiments (lanes 7 and
8). Using fusion proteins containing the two SHP-2 SH2
domains increased Bgp1 binding by a factor of 11 relative to binding
with the N-terminal SH2 domain alone (Fig. 5A, compare
lanes 3 and 7). Interestingly, under
pervanadate-treated conditions, the binding of Bgp1 to full-length
SHP-2 increased by 14-15-fold (Fig. 5A, lane 1)
relative to the binding under untreated conditions (lane 2).
Therefore, in contrast to the results obtained with SHP-1 in similar
experiments (27), the SHP-2 N-terminal SH2 domain binds to the Bgp1
cytoplasmic domain. Moreover, full-length SHP-2 demonstrated a
40-45-fold increased binding relative to the N-terminal SH2 domain
alone, suggesting that the presence of other domains or the overall
conformation of the SHP-2 Tyr phosphatase may influence its binding to
Bgp1. A low amount of Bgp1 from the untreated transfected CT51 cells
was found to be associated with full-length SHP-2 (Fig. 5A,
lane 2), demonstrating that a fraction of Bgp1 is
endogenously Tyr-phosphorylated in these cells, as previously shown in
Fig. 3.
In this work, we have demonstrated that the protein-Tyr
phosphatase SHP-2 associates with the Tyr-phosphorylated form of Bgp1 in CT51 mouse colon carcinoma cells and in 293 human embryonic kidney
cells. The association of Bgp1 with SHP-2 seems to parallel that
observed previously with SHP-1 (27), except that only the N-terminal
SH2 domain of SHP-2 appears capable of this interaction, whereas either
SH2 domain of SHP-1 was competent in binding to the Bgp1 cytoplasmic
domain. There may be, however, large differences in the relative
affinities of the SH2 domains of SHP-1 or SHP-2 for binding to the
cytoplasmic domain of Bgp1; this is currently being measured using
surface plasmon resonance technology.
We have also investigated the molecular requirements leading to the
association of Bgp1 with the Tyr phosphatases. Mutational analyses had
previously shown that Tyr-488 was subject to Tyr phosphorylation events
(27); we have now confirmed using point mutations that Tyr-515 is also
capable of sustaining Tyr phospho-transfer in vivo, albeit
with less apparent efficiency than Tyr-488. Yet, association of both
Tyr phosphatases requires the presence of both Bgp1 Tyr residues as
well as their Tyr phosphorylation. Interestingly, deletion of the Lys
residues located at the C-terminal end of Bgp1 significantly diminished
SHP-2 binding, suggesting some direct or indirect interactions between
the C-terminal residues and the Tyr residues for efficient
phosphorylation of the cytoplasmic domain. Further deletions of a few
residues within the Bgp1 cytoplasmic domain ( The terminal Lys residues of Bgp1 are located at positions +4, +5, and
+6 relative to the second ITIM consensus sequence. Although they are
positioned outside the classical ITIM consensus sequence, they appear
to play a determining role in the association of either of the Tyr
phosphatases. Several reports have suggested that residues +3, +4, and
+5 relative to the ITIM sequences can dramatically affect either
maximal SHP-1 or SHP-2 binding or levels of activity (31, 46, 47).
Substitutions at positions +1, +2, +4, and +5 relative to the first
ITIM of the natural killer cell receptor decreased phosphatase
activation and presumably binding by approximately 50% (31). These
results were obtained using synthetic peptides encompassing residues
adjacent to the first Tyr residue within the cytoplasmic domain of the
natural killer cell inhibitory receptor (31). Similarly, the crystal structure of the SHP-2 phosphatase complexed with peptides indicated that the side chains of phosphatase residues +3 and +5 relative to the
phosphotyrosine interacted with the peptides (46). Furthermore, high
affinity binding of the SHP-2 N-terminal SH2 domain requires contact
with residues beyond position +3 of the ITIM (47). The results
presented in this report thereby further reinforce these findings and
provide additional evidence that residues +4, +5, and +6 downstream of
an ITIM (TXY515XXV) can dramatically
influence the in vivo association and presumably the
activity of the protein-Tyr phosphatases.
Several mechanisms have been explored to explain the decreased Bgp1 Tyr
phosphorylation following the deletion of the three Lys residues
located at the C-terminal end of Bgp1. These Bgp1 residues are likely
to represent important hallmarks as they are conserved across species
(KK(K/Q)). First and most obvious, we considered the effect of loss of
net charge of the cytoplasmic domain. This could possibly lead to
altered conformation of this domain, which would render Tyr-488 more or
less accessible to Tyr kinases and phosphatases. However, replacement
of the Lys residues with either uncharged Ala residues or charged Arg
residues restored the binding of both SHP-1 and SHP-2 to Bgp1,
indicating that the presence of three residues, irrespective of their
identity, constitutes the most determinant factor. Furthermore, we had
speculated that the three Lys residues might be part of a protein
kinase C consensus sequence leading to the phosphorylation of Ser-516 and potentially to variations in the levels of Bgp1 Tyr
phosphorylation. However, replacement of the three Lys residues with
Ala residues or mutation of Ser-516 to Ala did not alter Bgp1 Tyr
phosphorylation levels and consequent association of the Tyr
phosphatases. Alternative mechanisms remain to be investigated; for
instance, the proximity of the last Bgp1 ITIM sequence to the
carboxyl-terminal end of the protein could potentially result in
greater accessibility of the phosphatases to the Bgp1 phosphotyrosine
residues, and this should be more closely examined. This is not a
unique occurrence as SHPs or SIRP proteins also exhibit a similar ITIM
sequence near their carboxyl-terminal ends (48, 49). In addition, other Ig-like proteins such as ILT1 and ILT2 also contain an ITIM sequence in
this same position (50). Moreover, the CD22 adhesion molecule, shown to
act as an accessory receptor to the B cell receptor complex and to
associate with SHP-1 (51), contains six potential ITIM consensus
sequences, the last one of which is at the extreme carboxyl-terminal end of the protein (52). In this particular case, it has been shown
that competition with a peptide corresponding to the sixth ITIM
sequence of CD22 can inhibit binding of SHP-1 to this glycoprotein (52).
Another mechanism to consider is that this Bgp1-conserved Lys sequence
motif may also be involved in its own protein-protein interactions, and
disruption of such associations through deletion of the Lys residues
might induce dramatic changes in the local environment. We have
recently identified Bgp1 cytosolic partners in a yeast two-hybrid
screen, and the potential role of the Lys residues in regulating the
association of these proteins with Bgp1 is currently being
examined.2 Another mechanism
involved might be the disruption of Bgp1 dimers. Hunter et
al. (53) have reported that Bgp1 forms homodimers in equilibrium
with monomers either in intact epithelial cells or as purified
proteins. Conformation of the Bgp1 cytoplasmic domain may be governed
as a complex dynamic process, as suggested by recent models proposed by
Öbrink (54). One of these models proposes that Ser and/or Tyr
phosphorylation of the Bgp1 cytoplasmic domain could lead to
dissociation of dimerized cytoplasmic domains, thereby rendering them
capable of sustaining novel interactions such as those observed with
the SHP-1 or SHP-2 Tyr phosphatase (54). In this context, modulation of
the Tyr phosphorylation levels of Bgp1 dependent upon the presence
and/or accessibility of its three C-terminal Lys residues would
constitute a regulatory event.
It should be noted that our study represents the first report (to our
knowledge) of either Tyr phosphatase associating with a cell-surface
adhesion molecule in intestinal cells. A few reports have examined the
roles played by SHP-1 or SHP-2 in association with cell-surface
receptors in epithelial cells. For instance, both SHP-1 and SHP-2 are
known to associate with the epidermal growth factor receptor in A431
human epidermoid carcinoma cells, where SHP-1 mediates the
dephosphorylation of this receptor (55). In MCF-7 human breast
carcinoma cells or in TRMP canine kidney epithelial cells, SHP-1
associates with the platelet-derived growth factor receptor and the p85
subunit of phosphatidylinositol 3-kinase and negatively regulates
platelet-derived growth factor receptor-mediated activation of the
c-fos promoter (56). This same receptor as well as the
epidermal growth factor receptor were also shown to bind to SHP-2 in
epithelial cells (57), where, in the latter case, SHP-2 acts as a
positive mediator of epidermal growth factor signaling (58). Similarly,
in primary mammary epithelial cells, the protein-Tyr phosphatases may
act as regulatory targets via the extracellular matrix proteins in
integrin-mediated milk production (59). Finally, in normal mouse liver
and kidney, in vivo peroxyvanadate treatment induces the
association of various proteins with SHP-1 and SHP-2 (60).
What could be the functional consequences of SHP-1 or SHP-2 association
with Bgp1 in intestinal cells? In normal colon, the Bgp1 protein is
found both in the crypts and in the lining of the villi (61).
Interestingly, the Bgp1 protein expressing the longer cytoplasmic
domain is located primarily in epithelial cells confined to the crypts
(61), a region involved in active proliferation. As Bgp1 is also
involved in negative regulation of tumor cell growth (21-23), it will
be important to determine whether SHP-1 and SHP-2 may in some way
modulate the signals leading to intestinal growth arrest. This negative
proliferation signal could potentially trigger the start of the
differentiation program as intestinal cells begin their migration up
the villus. Results of in vivo tumor inhibition assays
clearly suggest, however, that the Bgp1-dependent mechanisms leading to inhibition of colon tumor development are complex
and involve several motifs and/or cytosolic partners associating with
the Bgp1 cytoplasmic
domain.2,3 The identification
of the SHPs or SIRP proteins as substrates of SHP-1 or SHP-2 is also of
interest in considering functional consequences of association of the
Tyr phosphatases with Bgp1 (48, 49). These proteins contain, within
their cytoplasmic domain, four ITIM consensus sequences, one or several
of which are involved in their association with the SHP-1 and SHP-2 Tyr phosphatases. These proteins also convey negative proliferation signals
in response to growth factor stimulation or upon cell adhesion-induced signaling.
In addition, Bgp1 is an intercellular adhesion molecule whose
homophilic abilities are likely to be controlled by intracytoplasmic associating partners. Recent studies on PECAM-1 (platelet
endothelial cell adhesion
molecule-1) give credence to this suggestion
(62-64): PECAM-1 is a member of the Ig superfamily and is responsible
for contacts between leukocytes or platelets with the surface of
endothelial cells. Indeed, the last 10 amino acids of Bgp1, surrounding
Tyr-515, demonstrate striking homology to a region of 18 amino acids
within PECAM-1, particularly those centered around Tyr-686 (Bgp1,
SSPRATETVYSEVKKK; PECAM-1,
LGTRATETVYSEIRKVDP). Recently, Jackson et
al. (65, 66) have demonstrated that PECAM-1 becomes
Tyr-phosphorylated on two Tyr residues during platelet aggregation
(Tyr-663 and Tyr-686) and that, consequently, SHP-2 binds to activated
PECAM-1. The maximal SHP-2-binding site in PECAM-1 is dependent upon
the presence and phosphorylation of Tyr-686, which corresponds to the
conserved sequence in Bgp1 (67). Furthermore, Famiglietti et
al. (68) have convincingly demonstrated that a point mutation of
Tyr-686 is sufficient to convert heterophilic adhesion to homophilic
binding. It is therefore tempting to speculate that SHP-1 and/or SHP-2 binding to Bgp1 may cause switches in its intercellular adhesion characteristics; this hypothesis is currently being studied.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
2 and followed by a critical Leu or
Val residue at position +3 (30, 31). In addition, as defined using
mutated phosphopeptides, other residues such as those positioned at
4
as well as +1, +2, +4, and +5 relative to the Tyr residue within the
ITIM might also contribute to the effective binding of SHP-1 with the
natural killer cell inhibitory receptors (31). The ITIM consensus
sequence is found in a number of hemopoietic cell-surface receptors
such as Fc
receptor IIB, the B cell accessory receptor CD22, and the natural killer cell inhibitory receptors. These receptors associate with the cytosolic protein-Tyr phosphatase SHP-1, which possesses two
N-terminal SH2 domains, leading to attenuation or inhibition of Tyr
phosphorylation-elicited signaling in various cellular systems (30, 32,
33). Optimal SHP-1 association with Tyr-phosphorylated proteins is
mediated by both of two tandem N-terminal SH2 domains (34). In most
cases, once SHP-1 is bound to receptors, it catalyzes their
dephosphorylation (35).
EXPERIMENTAL PROCEDURES
-minimal essential medium for 10 min at 37 °C with
a solution of 10 mM H2O2 and 100 µM sodium vanadate (38). Cells were collected from the
dishes by scraping, followed by centrifugation and subsequent washes in
the same vanadate-containing medium. Human embryonic kidney cells
(HEK293) were obtained from the American Tissue Culture Collection
(ATCC CRL1573) and grown in
-minimal essential medium supplemented
with 2 mM glutamine and 10% heat-inactivated fetal bovine
serum (Life Technologies, Inc.) at 37 °C in a humidified atmosphere
of 5% CO2. 5 µg of each individual construct,
cloned into the pXM139 plasmid, were transfected either individually or
as a mixture (total of 20 µg) into HEK293 cells (5 × 105 cells/100-mm dish) by calcium phosphate-DNA
coprecipitation (39). Calcium precipitates were removed after 18-h
incubations, and the cells were cultured for an additional 24 h
and then lysed.
plasmid containing a 1.1-kilobase pair
SacI-HindIII fragment of the BgpL
cDNA (the HindIII site is from a BlueScript
SK+ vector containing the full-length BgpL
cDNA). The mutations Y488F, Y515F, and Y488F,Y515F have been
described previously (27). The S503A mutant was produced in the same
way using the following primer, creating a new EagI site:
5'-ACCCAACCGGCCGACTGCAGCCCCTTCTT-3' (nucleotides
in boldface represent the mutations). The
483 deletion mutant
introduces a new stop codon at amino acid 484 and removes an
AatII site
(5'-TAACAAGGTGTAGGACGTGGCATACACTG-3'), whereas the
495 mutant includes a stop codon at amino acid 496 and a
new BfaI site
(5'-CTTCAATTCCTAGTAACCCAACCG-3'). The
510
deletion mutant has a stop codon at amino acid 511 and an added
StyI site
(5'-TTCTTCTCCAAGGGCCTGAGAAACAGTTTA-3'). The
518 deletion mutant has a stop codon inserted at amino acid 519 and
a new MseI site
(5'-TTATTCAGAAGTTTAATAGAAGTGAGCAT-3'). Point
mutations were also introduced in the C-terminal region of the Bgp1
cytoplasmic domain using an overlap PCR technique (42). Two PCR
fragments were generated for each mutant using a combination of common
and specific oligonucleotides. The common oligonucleotide KM4
(5'-ACACAAGGAGGCCTCTCAGATGGC-3') is located at nucleotides 1352-1375
within the BgpL cDNA and contains an endogenous
StuI site, and a common T7 oligonucleotide
(5'-TAATACGACTCACTATAGGG-3') of the Bluescript SK+ vector
was also used. Mutation of the three terminal Lys residues to three Ala
residues was accomplished using the forward primer 5'-TCAGAAGTAGCAGCGGCGTGAGCATAA-3'
and the reverse primer
5'-TTATGCTCACGCCGCTGCTACTTCTGA-3'. For
mutation of the terminal Lys residues to Arg residues, the following
primers were used:
5'-TCAGAAGTAAGAAGGAGGTGAGCATAA-3' and
5'-TTATGCTCACCTCCTTCTTACTTCTGA-3'.
Val-518 was converted to Ala using the following oligonucleotides:
5'-TATTCAGAAGCAAAAAAGAAG-3' and
5'-CTTCTTTTTTGCTTCTGAATA-3'. Mutation of Ser-516 to Ala was
performed using oligonucleotides 5'-ACAGTTTATGCAGAAGTAAAA-3' and 5'-TTTTACTTCTGCATAAACTGT-3'. All mutants were
subjected to DNA sequence analyses (40).
-p-tosyl-L-lysine
chloromethyl ketone, and N-tosyl-L-phenylalanine chloromethyl ketone), 50 mM sodium fluoride, and 1 mM sodium orthovanadate. Cells were centrifuged for 10 min
at 4 °C to remove cellular debris, and the protein content of cell
lysates was measured using the BCA protein assay kit (Pierce). Samples
of 0.5-1.5 mg of proteins were immunoprecipitated with the indicated
antibodies at 4 °C for 2 h, and immune complexes were adsorbed
onto protein A-Sepharose beads. Proteins were resolved on 8%
SDS-polyacrylamide gels and transferred to Immobilon membranes. After
blocking unspecific sites, membranes were incubated with the anti-Bgp1,
anti-SHP-1, anti-SHP-2, or anti-PTP-PEST polyclonal antibody or with
the anti-v-Src monoclonal antibody (Ab-1), respectively. Detection of
phosphotyrosine-labeled protein was performed with purified monoclonal
antibody 4G10. Proteins were visualized by incubating the membranes
with either 125I-labeled protein A (Amersham Pharmacia
Biotech) or 125I-labeled goat anti-mouse IgG antibody (ICN).
-D-thiogalactopyranoside and purified on
glutathione-Sepharose beads as described previously (27). For binding
assays, fusion proteins were bound to glutathione-Sepharose beads (~4
µg of fusion protein/binding reaction) and incubated with 1.0 mg of
CT51 BgpL-transfected pervanadate-treated or untreated cell lysate
proteins for 3 h at 4 °C in 1× lysis buffer containing 150 mM NaCl as described (27). After washing the beads four times with lysis buffer, adsorbed proteins were eluted, subjected to
electrophoresis, and analyzed by immunoblotting with an anti-Bgp1 or
anti-GST antibody.
RESULTS
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Fig. 1.
Bgp1 associates with SHP-2 in mouse colonic
carcinoma cells and HEK293 cells. A, a CT51
Bgp1-transfected cell line was treated or not with the phosphatase
inhibitor pervanadate (PV). Cells were lysed with
detergent-containing buffers, and 600 µg of cell lysate proteins were
immunoprecipitated (IP) with anti-Bgp1 antibodies. Bound
proteins were resolved by 8% SDS-polyacrylamide gel electrophoresis
and immunoblotted with an anti-SHP-2 antibody (first panel).
Expression of the Bgp1 and SHP-2 proteins was monitored by
immunoblotting 100 µg of cellular proteins with either anti-Bgp1
(second panel) or anti-SHP-2 (third panel)
antibodies. B, HEK293 cells were transiently transfected
with either wild-type Bgp1 and constitutively activated
mouse c-src Y529F together with either wild-type
(WT) SHP-1 or SHP-2 or their
catalytically inactive cDNA constructs. Cells were lysed, and 600 µg of cell
lysate proteins were immunoprecipitated with anti-Bgp1 antibodies.
Phosphorylated proteins present in the immune complexes were detected
with anti-Tyr(P) antibody 4G10 (first panels). Association
of SHP-1 and SHP-2 with the Bgp1 protein was detected with their
respective antibodies (second panels). Expression
of Bgp1 (third panels), SHP-1 or SHP-2
(fourth panels), and c-Src (fifth
panels) was monitored by immunoblotting 50 µg of cell lysate
proteins with relevant antibodies. C, similar experiments as
those described for B were performed with the
PTP-PEST phosphatase construct. Association of the PTP-PEST
phosphatase with Bgp1 was monitored using anti-PEST antibodies
(first panel), whereas expression of Bgp1 (second
panel), PTP-PEST (third panel), and
c-Src (fourth panel) was monitored with their
respective antibodies. Molecular mass standards are indicated on the
left (in kilodaltons), and the positions of Bgp1, SHP-1, SHP-2, and
PTP-PEST are shown on the right. PTP, protein-tyrosine
phosphatase.
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Fig. 2.
Graphic representation of Bgp1 mutants.
The four Ig extracellular domains of Bgp1 are represented by
ovals. A portion (amino acids 478-521) of the cytoplasmic
domain is represented as an inset. The two Tyr residues
(Tyr-488 and Tyr-515) are shown in boldface.
Asterisks correspond to the stop codon created in the
deletion mutants.
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Fig. 3.
The Bgp1 Tyr residues are necessary for SHP-2
association. CT51 mouse colonic carcinoma cells expressing various
Bgp1 mutants were treated or not with the pharmacological inhibitor
pervanadate (PV). Cells were lysed with detergent-containing
buffers, and 1 mg of cell lysate proteins was immunoprecipitated
(IP) with anti-Bgp1 antibodies. Bound proteins were resolved
by 8% SDS-polyacrylamide gel electrophoresis. The Bgp1 Tyr
phosphorylation status was monitored with anti-Tyr(P) antibody 4G10
(first panel), and association of SHP-2 was detected with an
anti-SHP-2 antibody (second panel). Expression of
the various proteins was monitored by immunoblotting 100 µg of cell
lysate proteins with either anti-Bgp1 (third
panel) or anti-SHP-2 (fourth panel)
antibodies. Molecular mass standards are indicated on the left (in
kilodaltons), as are the positions of the SHP-2 and Ig proteins.
WT cl., wild-type clone.
518 mutant
was designed to remove the three carboxyl-terminal Lys residues, well
conserved (KK(K/Q)) among the mouse, rat, and human BGP cytoplasmic
domains. Furthermore, truncations removing the residues surrounding
Tyr-515 (
510 mutant), those encompassing a conserved Ser
phosphorylation site located at Ser-503 (
495 mutant) (43, 44), and
finally those positioned close to Tyr-488 (
483 mutant) were also
created. A Ser-503 point mutant converting this residue to a
non-phosphorylatable Ala was also designed (S503A mutant). These Bgp1
mutants were stably expressed in CT51 cells, and cell populations or
clones were derived and subjected to analyses for the Bgp1 Tyr
phosphorylation status and its association with the SHP-1 and SHP-2 Tyr
phosphatases. Inactivation of the endogenous phosphatase activity was
effected by pervanadate treatment of the cells, and the Bgp1 protein
was recovered by immunoprecipitation. Proteins associated with the
immune complexes were subjected to immunoblotting with either
anti-Tyr(P) or anti-SHP-2 antibodies (Fig.
4). As shown previously, the pervanadate
treatment provoked enhanced Tyr phosphorylation of Bgp1 and its binding
to SHP-2 (Fig. 4, first and second panels, lanes
1 and 2). However, removing a fragment of the
cytoplasmic domain including both Tyr residues (
483 mutant) did not
reveal any Tyr phosphorylation or association of the Bgp1 cytoplasmic
domain with the Tyr phosphatase (Fig. 4, first and
second panels, lanes 9 and 10).
Although the expression of this Bgp1 mutant was less than that seen
with the wild-type protein (Fig. 4, third panel, compare
lanes 9 and 10 with lanes 1 and
2), longer exposures of the blots did not reveal any Bgp1 Tyr phosphorylation (data not shown). Interestingly, deleting the last
three Bgp1 Lys residues reduced Bgp1 Tyr phosphorylation by at least
50% and almost completely abrogated binding of Bgp1 to SHP-2
(inhibition of 80 ± 14%) (Fig. 4, second panel,
lanes 3 and 4). Further removal of residues at
the C terminus eliminating amino acids surrounding Tyr-515 (
510) or
Ser-503 (
495) completely abolished SHP-2 binding to Bgp1, which
exhibited barely visible levels of Bgp1 Tyr phosphorylation, detectable
only after long exposures of the blots (Fig. 4, first and
second panels, lanes 5-8). In contrast, mutating Ser-503 to
Ala did not produce significant differences when compared with results
obtained with the wild-type Bgp1 protein (Fig. 4, third
panel, compare lanes 1 and 2 with lanes 11 and 12, relative to the Bgp1
expression). SHP-1 association with these mutants was also examined and
gave essentially similar results (data not shown). It should also be
noted that comparable results were obtained by transiently transfecting
HEK293 cells with the same Bgp1 deletion or point mutants
together with c-src and catalytically inactive SHP-1
or SHP-2 phosphatase constructs (data not shown).
Therefore, the Bgp1 C-terminal region is involved in controlling the
levels of Bgp1 Tyr phosphorylation and, consequently, Bgp1 association
with the Tyr phosphatases SHP-1 and SHP-2.
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Fig. 4.
Molecular requirements for association of
Bgp1 and SHP-2. CT51 mouse colonic carcinoma cells expressing
various Bgp1 mutants were treated or not with the pharmacological
inhibitor pervanadate (PV). Cells were lysed with detergent,
and 1.5 mg of cell lysate proteins were immunoprecipitated
(IP). Bound proteins were resolved by 8% SDS-polyacrylamide
gel electrophoresis. The Bgp1 Tyr phosphorylation status was monitored
with anti-Tyr(P) antibody 4G10 (first panel), and
association of SHP-2 was detected with an anti-SHP-2 antibody
(second panel). Expression of the various
proteins was monitored by immunoblotting 100 µg of cell lysate
proteins with either anti-Bgp1 (third panel) or
anti-SHP-2 (fourth panel) antibodies. Molecular
mass standards (in kilodaltons) as well as the positions of the SHP-2
and Ig proteins are indicated on the left. WT cl., wild-type
clone; pop., population.
3R) or Ala
(3K
3A) (Fig. 2) or alternatively, Ser-516 within the
protein kinase C site was also mutated to Ala. As shown in Fig. 4,
replacing the Lys residues with Arg or Ala residues did not hinder Bgp1
Tyr phosphorylation or reduce its association with the SHP-2 Tyr
phosphatase whether tested in cell populations or clones (Fig. 4,
first and second panels, lanes
15-20). Similarly, mutation of Ser-516 to Ala had no effect on
either Bgp1 Tyr phosphorylation or the levels of SHP-2 binding (data
not shown). Similar binding results were obtained with the SHP-1
phosphatase (data not shown). Hence, it appears that the presence of
these Lys residues, more than their net charge or their role in a
potential protein kinase C-mediated Ser-516 phosphorylation, is the
determining factor in regulating association of Bgp1 with the Tyr
phosphatases. In addition, the involvement of Val-518, located within
the second ITIM consensus sequence site
(pY515XXV), was also investigated by mutating it
to Ala (V518A). This mutation completely eliminated SHP-2 and SHP-1
binding to Bgp1 (decrease of 97 ± 0.7%) (Fig. 4, second
panel, lanes 13 and 14) (data not shown),
although it did not significantly affect Bgp1 Tyr phosphorylation
(first panel, lanes 13 and 14). Thus, the second
ITIM consensus sequence is crucial for association of either SHP-1 or
SHP-2 with Bgp1.
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Fig. 5.
Domains of SHP-2 involved in association with
Bgp1. Bacterially expressed glutathione S-transferase
fusion proteins (4 µg) encompassing various domains of the SHP-2
isoforms were adsorbed onto glutathione-Sepharose beads. CT51 cells
expressing the wild-type Bgp1 protein were either treated
(odd-numbered lanes) or not
(even-numbered lanes) with the pharmacological
inhibitor pervanadate (PV). Samples of 1.0 mg of total cell
lysate proteins were incubated with the beads, and after four washes
with the cell lysis buffer described under "Experimental
Procedures," bound proteins were eluted with an SDS-containing sample
buffer. Eluted proteins were resolved by 10% SDS-polyacrylamide gel
electrophoresis. Bgp1 was detected with an anti-Bgp1 antibody
(A), and the various GST fusion proteins with an anti-GST
antibody (B). Molecular mass standards are indicated on the
left (in kilodaltons). The positions of the various proteins are
indicated on both sides of the blots.
DISCUSSION
510 mutant) almost
completely abrogated Bgp1 Tyr phosphorylation, and association with the
phosphatases was undetectable. In addition, elimination of the protein
kinase C phosphorylation site at Ser-503, previously shown to regulate
both bile transport and internalization of the insulin receptor (43,
44), produced minimal differences in Bgp1 Tyr phosphorylation compared
with that detected with wild-type Bgp1. However, conversion of Val-518
to Ala abolished SHP-2 binding and is therefore crucial for these
protein interactions.
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ACKNOWLEDGEMENTS |
---|
We express gratitude to the following researchers for providing reagents: Dr. David Shalloway (Cornell University, Ithaca, NY) for the mouse c-src cDNA, Dr. Geng-Shen Feng for the Syp cDNA and Syp antibodies, Dr. W. Muller (MOBIX, Hamilton, Ontario, Canada) for the SHP-1 cDNA, Dr. Cliff Stanners (McGill University) for the anti-human BGP monoclonal antibody, and Drs. Philip Branton and Joseph Lee for anti-GST antibodies. We thank Marielle Fournel for help in the cloning of the SHP-2 cDNA isoforms. We also thank Drs. Bjorn Öbrink and Dominique Davidson for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by Grant MT-13911 from the Medical Research Council of Canada (to N. B.) and by a grant from the National Cancer Institute of Canada (to A. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Medical Research Council of Canada fellowship.
¶ Recipient of a Fonds pour la Formation de Chercheurs et l'Aide á la Recherche Centre studentship.
Recipient of a "Fonds de la Recherche en Santé du
Québec" studentship.
Recipient of a Medical Research Council of Canada studentship.
¶¶ Recipient of a Medical Research Council of Canada Scientist award.
|| Recipient of a "Fonds de la Recherche en Santé du Québec" Senior Scientist award. To whom correspondence should be addressed: McGill Cancer Centre, McGill University, 3655 Drummond St., Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-3541; Fax: 514-398-6769; E-mail: nicoleb{at}med.mcgill.ca.
2 S. Sadekova and N. Beauchemin, manuscript in preparation.
3 L. Izzi and N. Beauchemin, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Bgp or BGP, biliary glycoprotein; ITIM, immunoreceptor tyrosine-based inhibition motif; PCR, polymerase chain reaction; GST, glutathione S-transferase..
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REFERENCES |
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