From the Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received for publication, September 21, 2000, and in revised form, October 16, 2000
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ABSTRACT |
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Ligation of the
Basement membranes regulate the survival, proliferation, and
differentiation of cells through ligation of integrin receptors (1-4).
The Shc is an SH2/PTB1 domain
adaptor protein that couples a variety of receptor and nonreceptor
tyrosine kinases, cytokine receptors, immune receptors, and adhesion
receptors to Ras signaling (7, 8). In most cases, Shc binds to upstream
tyrosine phosphorylated molecules through its SH2 domain, PTB domain,
or both. It is then phosphorylated on tyrosine and recruits the
Grb2/SOS complex, which can subsequently activate Ras. By this
mechanism, Shc participates in mediating the proliferative functions of
many receptors. In addition, it regulates cell migration (9-11).
Although many receptor tyrosine kinases must interact directly with Shc
to induce its tyrosine phosphorylation and activation, the EGF receptor
is capable of signaling through Shc even when prevented from binding to
it (12, 13). In addition, whereas Dominant negative studies suggest that Shc is required to couple the
In addition to its signaling function, the cytoplasmic domain of
Recent studies have indicated that ligation of
Immunochemical Reagents--
The rabbit polyclonal antibody and
the mAb against the extracellular domain of Constructs--
cDNAs encoding human Cell Culture and Transfections--
293T cells were provided by
Dr. David Levy (New York University School of Medicine) and cultured in
Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine
serum (FBS) on dishes coated with gelatin and transfected by the
calcium phosphate method with equimolar amounts of pRK5 Fusion Proteins--
GST fusion proteins were expressed in
BL21-RES cells (Stratagene) treated with 0.1 mM
isopropyl-1-thio- Far Western Analysis--
After overnight serum starvation, 293T
cells were left untreated or treated for 5 min at 37 °C with 100 µM orthovanadate and 3 mM hydrogen peroxide,
washed with ice-cold PBS, and lysed in lysis buffer (50 mM
Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 5 mM EDTA) supplemented with
phosphatase inhibitors (1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 25 mM sodium fluoride)
and protease inhibitors (10 µg/ml pepstatin A, aprotinin, and
leupeptin and 0.4 mM 4-(2-aminoethyl)-benzene
sulfonylfluoride). Aliquots containing 1 mg of total proteins
were immunoprecipitated for 3 h with 5 µg of 3E1 plus 30 µl of
packed protein-G agarose. Immunocomplexes were separated by SDS-PAGE
and transferred to nitrocellulose. Membranes were blocked with TBS,
0.1% Tween (TBST) containing 5% milk for 1 h at room temperature
and incubated with 1 µg/ml GST Shc SH2 or PTB domain fusion proteins
in TBST-2.5% milk, 1 mM dithiothreitol, 10 µg/ml
aprotinin, leupeptin, and pepstatin A. After three washes with TBST,
the membranes were incubated with 1 µg/ml anti-GST rabbit polyclonal
antibody in TBST-2.5% milk, washed again, and then incubated with
protein A-HRP in TBST-5% milk. After five washes with TBST and two
washes with TBST-0.2% Triton X-100, the membranes were rinsed with TBS and incubated with ECL for 1 min before exposure to film. To assess phosphorylation of Peptide Competition Assay--
HaCat cells were serum starved
and stimulated as described above. Immunoprecipitation and Immmunoblotting--
To examine tyrosine
phosphorylation of Shc, the cells were serum starved, detached with 2 mM EDTA, washed with DMEM, 0.2% BSA, resuspended at
107/ml in DMEM, 0.2% BSA, and subdivided in 300-µl
aliquots. Cells were incubated on ice for 40 min with 10 µg of
anti- Mitogen-activated Protein Kinase Assay--
Dishes were coated
with 10 µg/ml rabbit anti-mouse IgGs for 2 h at room
temperature, saturated with 0.5% heat-denatured BSA (fatty acid,
globulin-free), and then incubated with 20 µg/ml 3E1 mAb overnight at
4 °C. Serum starved cells were detached with 2 mM EDTA,
washed with DMEM, 0.2% BSA, and kept in suspension in DMEM, 0.2% BSA
for 1 h at 37 °C. They were then plated onto 3E1-coated dishes
for indicated times at 37 °C and lysed in RIPA buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% SDS, 10% glycerol, 1 mM EGTA, 4 mM EDTA, and phosphatase and
protease inhibitors). Aliquots containing 20 µg of total proteins
were separated by SDS-PAGE and probed by immunoblotting with an
antibodies to phospho-ERK.
Immunofluorescence--
804G cells were cultured on glass
coverslips for 24 h in DMEM, 10% FBS and then overnight in
DMEM, 1% FBS in the presence of the indicated concentrations of sodium
orthovanadate. The cells were fixed with methanol at Identification of the
After immunoprecipitation from cells treated with pervanadate,
The connecting segment contains two potential tyrosine phosphorylation
sites that conform to the consensus for binding to the SH2 domain of
Shc (31). We thus mutated these two tyrosines (Tyr1422 and
Tyr1440) to phenylalanine either separately or in
combination. The resulting versions of
The PTB domain of Shc binds to phosphorylated tyrosines in the context
of NXXY motifs (24, 29). The cytoplasmic domain of
Phosphopeptide competition assays were performed to compare the
relative affinities of the potential binding sites for the SH2 or PTB
domain of Shc in
As shown in Fig. 3A, the
peptides
Fig. 3B shows that the phosphorylated peptide
Signaling by
We examined the ability of Signaling by
To verify that
We next analyzed the relative efficiency of phosphorylation of the
To examine whether the interaction of Shc with
Finally, we examined the relative importance of the two potential PTB
binding sites in Mutation of the Major Tyrosine Phosphorylation Sites Does Not
Prevent Incorporation of the Canonical Form A of
The 804G cells are a rat bladder carcinoma cell line that expresses
endogenous Phosphorylation of the
We have previously shown that treatment with EGF induces tyrosine
phosphorylation of Ligation of the The primary The SH2 domain of Shc interacts in a
phosphorylation-dependent manner primarily with
Tyr1440 and secondarily with Tyr1422 in
Our results indicate that tyrosine phosphorylation of The finding that the region of the 6
4 integrin induces tyrosine
phosphorylation of the
4 cytoplasmic domain, followed by
recruitment of the adaptor protein Shc and activation of
mitogen-activated protein kinase cascades. We have used Far
Western analysis and phosphopeptide competition assays to map the sites
in the cytoplasmic domain of
4 that are required for
interaction with Shc. Our results indicate that, upon phosphorylation,
Tyr1440, or secondarily Tyr1422,
interacts with the SH2 domain of Shc, whereas Tyr1526, or
secondarily Tyr1642, interacts with its phosphotyrosine
binding (PTB) domain. An inactivating mutation in the PTB domain of
Shc, but not one in its SH2 domain, suppresses the activation of Shc by
6
4. In addition, mutation of
4 Tyr1526, which binds to the PTB domain of
Shc, but not of Tyr1422 and Tyr1440, which
interact with its SH2 domain, abolishes the activation of ERK by
6
4. Phenylalanine substitution of the
4 tyrosines able to interact with the SH2 or PTB domain
of Shc does not affect incorporation of
6
4 in the hemidesmosomes of 804G cells.
Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of
4 and disrupts the hemidesmosomes of
804G cells expressing recombinant wild type
4. This
treatment, however, exerts a decreasing degree of inhibition on the
hemidesmosomes of cells expressing versions of
4
containing phenylalanine substitutions at Tyr1422 and
Tyr1440, at Tyr1526 and Tyr1642, or
at all four tyrosine phosphorylation sites. These results suggest that
4 Tyr1526 interacts in a
phosphorylation-dependent manner with the PTB domain of
Shc. This event is required for subsequent tyrosine phosphorylation of
Shc and signaling to ERK but not formation of hemidesmosomes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6
4 integrin is a major receptor for
the basement membrane component laminin-5 and is expressed in a variety
of epithelial cells, in Schwann cells, in certain endothelial cells, and in CD4
CD8
T cells (5, 6). The
cytoplasmic domain of
4, which is unusually long and
dissimilar in amino acid sequence from the corresponding portions of
other integrin
subunits, enables
6
4 to recruit the adaptor protein Shc as well as to promote the assembly of hemidesmosomes (5, 6).
6
4 can
interact directly with Shc (14), a subset of
1 and
v integrins recruit Shc indirectly through Src family
kinases (15, 16).
6
4 integrin to Ras and thereby both the
Raf-ERK and Rac-JNK signaling cascades. Through these pathways,
6
4 cooperates with growth factor
receptors to promote immediate-early gene expression and progression
through the G1 phase of the cell cycle (17). Mice carrying
a targeted deletion of the cytoplasmic domain of
4
display proliferative defects in the skin and gastro-intestinal tract,
suggesting that signaling pathways activated by the cytoplasmic domain
of
4, probably through Shc, are required for optimal
epithelial cell proliferation in vivo (18).
4 plays a crucial role in the assembly of hemidesmosomes (18, 19). The hemidesmosomes are adhesive junctions that mediate stable
attachment of stratified and transitional epithelia to the basement
membrane. They differ from focal adhesions because they are linked to
the keratin instead of the actin cytoskeleton. In accordance with the
role of hemidesmosomes in mediating strong adhesion to the basement
membrane, defects in hemidesmosome integrity cause epidermal fragility
and skin blistering (6, 20). The assembly of hemidesmosomes is likely
to require interaction of the cytoplasmic domain of
4 with
HD1/plectin and BPAG2 (21-23).
6
4 promotes phosphorylation of the
4
cytoplasmic domain through activation of an integrin-associated Src
family kinase.2,3 Because Src
kinases are known to activate Shc (24),
it is possible that
6
4 activates Shc
through the integrin-associated Src kinase independently of direct
binding of Shc to the cytoplasmic domain of
4. We have
thus examined whether
6
4-mediated Shc
signaling requires direct binding of Shc to the
4
cytoplasmic domain. Our results indicate that the SH2 and PTB domain of
Shc bind to separate phosphotyrosines in the cytoplasmic domain of
4. The interaction mediated by the PTB domain is
essential for Shc signaling to ERK in vivo, whereas that
mediated by the SH2 domain is dispensable. In addition, we provide
evidence that phosphorylation of the Shc binding sites in
4 antagonizes formation of hemidesmosomes.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
4 (3E1) were
characterized previously (17, 25). The rabbit polyclonal antibodies to
GST were affinity purified on a GST-Sepharose column from sera of
rabbits immunized with GST fusion proteins. The biotinylated
anti-phosphotyrosine mAb 4G10 and protein A-agarose were purchased from
Upstate Biotechnologies. The recombinant anti-phosphotyrosine antibody
RC20 was from Transduction Laboratories. The anti-Myc tag mAb 9E10 was
obtained from the Sloan-Kettering Hybridoma Facility and horseradish
peroxidase (HRP)-conjugated mAb 9E10 from Roche Molecular Biochemicals.
The mouse monoclonal antibody M2 to FLAG tag was from Kodak. The rabbit polyclonal antibodies to phospho-ERK were purchased from New England Biolabs. The rabbit polyclonal antibodies to ERK2 and the Myc tag were
purchased from Santa Cruz. Unconjugated rabbit and horse anti-mouse
secondary antibodies for cross-linking and protein G-agarose were
from Pierce. FITC-conjugated anti-mouse IgGs were purchased from
Molecular Probes. HRP-conjugated protein A and streptavidin were
purchased from Amersham Pharmacia Biotech.
6 and
wild type and mutant versions of
4 were subcloned into
the EcoRI site of the pRK5 expression vector. The generation
of cDNAs encoding the canonical form of wild-type
4,
A, and the mutant versions C, E (17, 19), G, Y1422F, Y1440F, and
Y1422F/Y1440F (14) were described previously.
4
Y1422F/Y1440F without the Gap was generated by replacing the BssHII fragment containing the Gap with the
BssHII fragment from wild type
4. The Gap
construct was generated by replacing the BssHII fragment
from wild type
4 with the BssHII fragment
containing the Gap. The
4 construct I was generated by
digesting pRC/CMV
4 A with NotI and
SacI followed by blunt end ligation. The
4 construct J was generated by digesting pRC/CMV
4 G with
NotI and XbaI followed by blunt end ligation,
which created an in-frame stop codon. The
4 construct M
was generated by digesting pRC/CMV
4 E with
NotI and XbaI followed by blunt end ligation,
resulting in an in-frame stop codon. pRK5
4 Y1526F and
Y1642F were generated by two-step polymerase chain reaction of
fragments comprised between the NotI and EcoRV or
EcoRV and XbaI sites of pRK5
4 A. Double and quadruple tyrosine to phenylalanine mutants of
4 were then generated by shuffling fragments of
NotI/EcoRV, EcoRV/XbaI, or NotI/XbaI. For stable transfection in 804G cells,
4 cDNAs were subcloned into the EcoRI
site of pcDNA3. Expression vectors encoding Myc-tagged wild type
and mutant murine p52 Shc proteins containing mutations in either the
PTB domain (F198V) or SH2 domain (R397K) or both domains (R397K and
F198V), were provided by Dr. Ben Margolis and were described previously
(13). Expression vectors encoding FLAG-tagged dominant negative Shc (Y
239/317 F) were described previously (16). Plasmids encoding GST-Shc
SH2 or PTB (residues 1-209) domains were described previously
(26). Mutant versions of the GST-Shc SH2 (R397K) and PTB (F198V)
domains were kindly provided by Dr. Ben Margolis. Correctness of all
newly generated vectors was verified by sequencing.
6 and
4. Immortalized human keratinocytes (HaCat) were
cultured in DMEM with 10% FBS. Human umbilical vein endothelial cells
(HUVECs) were purchased from VEC Technologies and cultured on
gelatin-coated dishes in endothelial cell SFM (Life Technologies, Inc.)
supplemented with 20% FBS (Life Technologies, Inc.), 10 ng/ml EGF, 20 ng/ml basic fibroblast growth factor, and 1 µg/ml Heparin
(Intergen). HUVECs were electroporated at 300 V and 450 microfarads with equimolar amounts of pRK5
6 and
4 supplemented with pBS to a total of 30 µg of DNA.
HeLa cells were purchased from American Type Culture Collection and
cultured in DMEM with 10% FBS. For transient transfection, 4.5 × 106 cells were plated on 15-cm-diameter plates for 24 h and then transfected with 20 µg of DNA and 100 µl of Lipofectin
(Life Technologies, Inc.) in a total of 10 ml of DMEM for 5 h,
according to manufacturer's recommendations. Cells were allowed to
recover for 24 h before serum starvation. Rat bladder carcinoma
804G cells (27) were cultured in DMEM with 10% FBS and transfected by
electroporation at 260 V and 975 microfarads in 300 µl of PBS, 1.25%
Me2SO with 10 µg of pcDNA3
4 and 10 µg of pBS. They were then replated onto gelatin-coated dishes in DMEM
10% FBS containing 1.25% Me2SO and cultured for
additional 24 h. Stably transfected cells were selected with 400 µg/ml G418 (Life Technologies, Inc.) and maintained in 200 µg/ml
G418. Clones were pooled and
4 expressing cells were selected by detaching cells with PBS/2 mM EDTA until cells
rounded followed by brief treatment with 0.2% trypsin/EDTA and
collection in DMEM, 10% FBS. Cells were then panned for 5 min at
37 °C over dishes coated overnight at 4 °C with 20 µg/ml 3E1
mAb followed by blocking with 1% heat-inactivated BSA. Adherent cells
were allowed to grow until confluency. Cells expressing equivalent levels of human recombinant
4 were obtained by
fluorescence-activated cell sorting with the 3E1 mAb.
-D-galactopyranoside for 3 h
at 37 °C and purified on glutathione-agarose as described previously
(28). Proteins were eluted by incubating glutathione-agarose beads
three times for 5 min at room temperature with an equal volume of 100 mM Tris, pH 8.5, 120 mM sodium chloride, 0.1%
Triton X-100, and freshly added 20 mM glutathione. Aliquots
were stored frozen at
20 °C until use.
4, the membranes were stripped for 30 min at 50 °C in 62.5 mM Tris, pH 6.7, 2% SDS, and 100 mM
-mercaptoethanol, rinsed extensively with TBS, and
then probed by immunoblotting with RC20 according to the
manufacturer's recommendations. To control for equal levels of
4, membranes were stripped again, blocked with TBST, 5%
milk and probed by immunoblotting with 1 µg/ml anti-
4
exo in TBS, 3% BSA followed by protein A-HRP.
4 was
immunoprecipitated with the 3E1 mAb from lysates containing 2 mg of
total proteins and transferred to nitrocellulose. Membranes were cut,
and Far Western blotting was performed as above in the absence or
presence of various concentrations of peptides. Purified phosphorylated
(pY) or nonphosphorylated peptides were produced by the Microchemistry
Core Facility of Sloan-Kettering Institute. They included
4 pY1422 (LTRDpYNSLTRSE),
4 pY1440 (LPRDpYSTLTSVS),
4 pY1526 (DLLPNHSpYVFRV),
4 Y1526 (DLLPNHSYVFRV), and
4 pY1642
(GLSENVPpYKFKV). Phosphopeptides modeled after the high affinity
binding site for the SH2 domain of Shc at Tyr579 in the
platelet-derived growth factor receptor (DGHEpYIYVDPMQ) and after the
high affinity binding site for the PTB domain of Shc in the middle T
antigen (SLLSNPTpYSVMR) (29, 30) were used as controls.
4 mAb 3E1, washed with 700 µl of cold DMEM,
resuspended in 200 µl of cold DMEM containing 5 µg of horse
anti-mouse IgG, and incubated at 37 °C for 5 min. After one wash
with cold PBS, the cells were pelleted and lysed in 800 µl of lysis
buffer with phosphatase and protease inhibitors. Recombinant Shc
proteins were immunoprecipitated with a rabbit anti-Myc polyclonal
antibody, separated by SDS-PAGE, and transferred to nitrocellulose. The
membranes were probed by immunoblotting with 1 µg/ml of
biotin-conjugated 4G10 followed by streptavidin-HRP. To examine
tyrosine phosphorylation of
4, HUVECs were transfected
with mutant versions of
4 as described above, allowed to
recover for 24 h, serum starved overnight, and treated with 1 µM sodium orthovanadate and 3 mM hydrogen
peroxide. After extraction in lysis buffer,
4 was
immunoprecipitated with 5 µg of 3E1 mAb and 30 µl of protein
G-agarose, and probed by immunoblotting with RC20.
20 °C for 20 min and stained with 10 µg/ml 3E1 mAb followed by 2 µg/ml
FITC-conjugated anti-mouse IgGs. Samples were examined with a Zeiss
fluorescent microscope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
4 Tyrosine Phosphorylation
Sites Required for Interaction with the SH2 and the PTB Domain of
Shc--
GST pull-down assays with SDS-denatured extracts have shown
that the isolated SH2 and PTB domain of Shc can interact directly with
the tyrosine phosphorylated
4 subunit in
vitro (17). To identify the sequences of
4
cytoplasmic domain mediating the interaction with the SH2 and the PTB
domain of Shc, we initially transfected 293T cells with the constructs
encoding the wild type or deleted
4 subunits illustrated
in Fig. 1. Antibody or laminin-5-mediated ligation of
6
4 promotes tyrosine
phosphorylation of
4 in vivo, but this event
is rapidly reversed, presumably by tyrosine phosphatases. By contrast,
treatment of the cells with the tyrosine phosphatase inhibitor
pervanadate causes high level and persistent tyrosine phosphorylation of
4. We thus used this protocol to
increase tyrosine phosphorylation of
4 in mapping
experiments.
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Fig. 1.
Structure of integrin
4 constructs. The
features of wild type (A) and mutant forms of human
4
used in this study are shown. TM, transmembrane domain;
black box, type III Fn-like repeat; gray box,
connecting segment; F, phenylalanine substitution. For
brevity, some of the mutants are referred to in the text as indicated
on the right.
6
4 was separated by SDS-PAGE and probed
by Far Western blotting with GST fusion proteins containing the SH2 or
PTB domain of Shc or by immunoblotting with anti-phosphotyrosine
antibodies. As shown in Fig.
2A, the SH2 domain of Shc
bound to wild type
4 and to
4 mutants
containing the connecting segment (E and M) in a tyrosine
phosphorylation-dependent manner. It did not, however, bind
to mutants lacking the connecting segment (G and I), despite the fact
that they were still phosphorylated on tyrosine. We consistently observed that the mutants E and M, which lack the membrane proximal region of the cytoplasmic domain, were phosphorylated to a higher stoichiometry than wild type
4. It is possible that the
segment deleted in E and M is necessary for efficient association with a tyrosine phosphatase able to reverse
4
phosphorylation. Alternatively, the deletion may bring some of the
4 tyrosine phosphorylation sites in closer proximity to
the tyrosine kinase that phosphorylates them. The mutants truncated
after the second fibronectin (Fn) type III repeat (J and C) did not
become phosphorylated on tyrosine. Because these mutants can still
associate with Src kinases,2 it is likely that the major
tyrosine phosphorylation sites in
4 reside downstream of
the second Fn type III module. Taken together, these observations
suggest that the SH2 domain of Shc binds to phosphorylated tyrosines
located within the connecting segment of
4.
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Fig. 2.
Far Western analysis indicates that
4 Tyr1440 is the major
binding site for the SH2 domain of Shc and Tyr1526 is the
major binding site for its PTB domain. 293T cells were transiently
transfected with
6 and either wild type (A) or the indicated mutant
versions of
4. Cells were either left untreated (
) or
treated with pervanadate (+). After immunoprecipitation,
6
4 was separated by SDS-PAGE and
transferred to nitrocellulose. Membranes were incubated with GST fusion
proteins comprising the SH2 (A) or PTB domain of Shc
(B), followed by immunoblotting with anti-GST antibodies
(top panels). Membranes were stripped and reprobed with
antibodies against phosphotyrosine (middle panels) and then
stripped again and reprobed with antibodies against the extracellular
domain of
4 (bottom panels).
4 were tested for
their ability to bind the SH2 domain of Shc. Fig. 2A shows
that the SH2 domain of Shc does not bind to
4
Y1422F/Y1440F and binds to a modest extent to
4 Y1440F. By contrast, the SH2 domain of Shc interacts effectively with
4 Y1422F. These results suggest that the SH2 domain of
Shc binds primarily to phosphorylated Tyr1440 and
secondarily to phosphorylated Tyr1422 in the connecting
segment of
4.
4 contains three NXXY motifs: one in the
region between the transmembrane domain and the first Fn type III
module and the other two downstream of the connecting segment, one in
the third and the other in the fourth Fn type III repeat. As shown in
Fig. 2B, the PTB domain of Shc bound to phosphorylated
4 mutant E as effectively as to phosphorylated wild type
4, but it did not interact with phosphorylated mutant M. This result suggests that the major binding site for the PTB domain of
Shc in
4 resides downstream of the connecting segment
and possibly corresponds to the NXXY motif in the third or
that in the fourth Fn type III repeat. Therefore, versions of
4 containing phenylalanine substitutions of the
tyrosines within each of these two NXXY sites
(Tyr1526 and Tyr1642) were analyzed by Far
Western blotting with the Shc PTB domain (Fig. 2B).
Phenylalanine substitution of either Tyr1526 or
Tyr1642 decreased only partially binding of the PTB domain
to
4, with substitution of Tyr1526 resulting
in a significantly greater reduction. Mutation of both sites in
combination (Y1526F/Y1642F) completely prevented the binding of PTB
domain to
4. These results suggest that the PTB domain
of Shc binds primarily to Tyr1526 and secondarily to
Tyr1642 in
4.
4 (see Table
I for peptide sequences). For these
experiments, we used HaCat keratinocytes, which express endogenous
6
4. Cells were either left untreated or
treated with pervanadate. After immunoprecipitation,
4
was transferred to nitrocellulose and probed with GST fusion proteins
containing the SH2 or PTB domain of Shc in the absence or presence of
tyrosine phosphorylated synthetic peptides modeled after the sequences surrounding Tyr1422, Tyr1440,
Tyr1526, or Tyr1642 in
4 (Table
I). As positive controls, we used tyrosine phosphorylated peptides
reproducing the high affinity binding sites for the SH2 and PTB domain
of Shc in the platelet-derived growth factor receptor and middle T
antigen, respectively (29, 30).
Sequences of peptides used in competition studies
4 pY1422 and
4 pY1440 inhibited
the binding of the SH2 domain of Shc to wild type
4 to a
similar extent, suggesting that Tyr1422 and
Tyr1440 are both potential binding sites for the SH2 domain
of Shc. It is likely that the SH2 domain of Shc binds preferentially to
Tyr1440 (Fig. 2B) because this tyrosine is more
efficiently phosphorylated than Tyr1422 in vivo
(14, 17). Tyr1440 may thus be the main physiologic binding
site for the SH2 domain of Shc in
4. Neither
4 Tyr(P)1526 nor a nonphosphorylated peptide
(
4 Tyr1526) were able to inhibit the binding
of the SH2 domain of Shc to
4.
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Fig. 3.
Phosphopeptides modeled after
4 sequences comprising
Tyr1422 and Tyr1440 inhibit binding of
4 to the SH2 domain of Shc,
whereas a phosphopeptide including Tyr1526 prevents
interaction with the PTB domain. HaCat cells were either left
untreated (
) or treated with pervanadate (+). After
immunoprecipitation,
6
4 was transferred
to nitrocellulose. Pieces of membrane containing individual bands of
phosphorylated
4 were incubated with GST-Shc-SH2
(A) or GST-Shc-PTB (B) in the absence or presence
of 100 µM of the indicated phosphorylated and
nonphosphorylated peptides (top panels). Membranes were
stripped and reprobed with anti-phosphotyrosine antibodies
(middle panels) and then stripped again and reprobed with
antibodies against the extracellular domain of
4
(bottom panels).
4 Tyr(P)1526, but not
4
Tyr(P)1642, inhibited the binding of the PTB domain of Shc
to wild type
4. Unphosphorylated
4
Tyr1526 as well as phosphorylated
4
Tyr(P)1440 had no effect. Together with those of mutational
analysis (Fig. 2B), these results indicate that
Tyr1526 is the primary binding site for the PTB domain of
Shc in
4.
6
4 Requires
the PTB, but Not SH2, Domain of Shc--
To examine whether Shc
signaling by
6
4 required direct
interaction of the SH2 and/or PTB domain of Shc with the
4 cytoplasmic domain, we used versions of Shc carrying
inactivating mutations in either the SH2 domain (R397K), PTB domain
(F198V), or both domains. The SH2 domain mutation resides within the
conserved FLVRES motif and prevents the interaction with
phosphotyrosine, whereas the PTB domain mutation prevents interaction
with the hydrophobic residue at position
5 and with the asparagine at position
3 in the
XNXXY motif (32). As shown
in Fig. 4A, each of these
mutations prevented the binding of a GST fusion protein containing the
corresponding domain of Shc to tyrosine phosphorylated
4
in vitro (Fig. 4A).
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Fig. 4.
6
4-mediated
phosphorylation of Shc requires an intact PTB, but not SH2,
domain. A, mutations that inactivate the SH2 or PTB
domain of Shc abolish binding of the corresponding domain to
4 in vitro. HaCat cells were either left
untreated (
) or treated with pervanadate (+). After
immunoprecipitation,
6
4 was transferred
to nitrocellulose. Pieces of membrane containing bands of
phosphorylated
4 were probed with wild type
(WT) or mutant (R397K) GST-Shc-SH2 domain or with wild type
or mutant (F198V) GST-Shc-PTB domain (top panels). Membranes
were then stripped and reprobed with anti-phosphotyrosine antibodies
(middle panels) and then stripped again and reprobed with
antibodies against the extracellular domain of
4
(bottom panels). B, an inactivating mutation in
the PTB domain of Shc, but not one in its SH2 domain, prevents
phosphorylation of Shc upon ligation of
6
4. HeLa cells were transiently
transfected with Myc-tagged versions of wild type Shc or versions of
Shc carrying mutations in the SH2 domain (R397K), the PTB domain
(F198V), or both (F198V/R397K). Cells were incubated in suspension with
the anti-
4 mAb 3E1 followed by anti-mouse IgGs. Control
cells (lanes C) were stimulated with secondary anti-mouse
IgGs alone. After immunoprecipitation, the Myc-tagged recombinant
proteins were probed with anti-phosphotyrosine antibodies (top
panel). The membranes were then stripped and reprobed with
anti-Myc antibodies (bottom panel). C, as a
control, adherent cells were either left untreated (
) or were treated
with 250 ng/ml EGF for 5 min (+). After immunoprecipitation, the
Myc-tagged recombinant proteins were probed with anti-phosphotyrosine
antibodies (top panel). The membranes were then stripped and
reprobed with anti-Myc antibodies (bottom panel). EGF
receptor-mediated phosphorylation of Shc is not prevented by mutation
of the SH2 domain of Shc, its PTB domain, or both, as reported
previously.
6
4 to activate
in vivo versions of Shc containing inactivating mutations in
either or both the SH2 and PTB domains. HeLa cells were transfected
with Myc-tagged wild type or mutant versions of Shc. HeLa cells were
chosen because they are easily transfectable, express endogenous
6
4, and were used previously to study
6
4 signaling (17) as well as EGF receptor
activation of the same Shc mutants (13). To specifically ligate
6
4, cells were incubated in suspension
with the anti-
4 mAb 3E1, followed by an anti-mouse
secondary antibody. The recombinant Shc proteins were
immunoprecipitated with an anti-Myc antibody and analyzed by
immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig.
4B, both wild type and SH2 mutant Shc (R397K) were
efficiently phosphorylated on tyrosine in response to ligation of
6
4. By contrast, the PTB mutant (F198V)
and double mutant (F198V/R397K) versions of Shc were not phosphorylated
on tyrosine upon
6
4 stimulation. All four
versions of Shc were efficiently phosphorylated upon treatment of the
cells with EGF (Fig. 4C), as shown previously (13). These
results indicate that a functional PTB domain is necessary for
6
4-mediated Shc signaling and imply that
Shc has to bind to
4 through this domain to become
phosphorylated on tyrosine by the
6
4-associated kinase.
6
4 Requires Interaction
with the PTB, but Not SH2, Domain of Shc--
In HeLa cells,
6
4 signaling to ERK proceeds through Shc
(17). We thus examined whether the binding of Shc to
4
was required also for activation of ERK. To test this hypothesis, we
chose to use primary HUVECs because they do not express
6
4 and can therefore be transfected with
various mutant versions of
4. These cells offer a better
model for examining
6
4 signaling than
fibroblastic cells because
6
4 is
expressed in certain endothelial cells in vivo (5, 6). In
addition, because ERK is often constitutively activated in established
cell lines, nonimmortalized cells such as the HUVECs enable a
more accurate assessment of the activation of ERK.
6
4 activated ERK in a
Shc-dependent manner in HUVECs, these cells were
transfected with constructs encoding
6
4
alone or in combination with increasing doses of a FLAG-tagged version
of dominant negative Shc carrying phenylalanine permutations at both
potential Grb2 binding sites. After serum starvation, the cells were
detached and replated onto dishes coated with the anti-
4
mAb 3E1. As shown in Fig. 5A,
dominant negative Shc inhibited
6
4-induced activation of ERK in a
dose-dependent manner, suggesting that
6
4 signaling to ERK proceeds through Shc
also in HUVECs.
View larger version (56K):
[in a new window]
Fig. 5.
6
4-mediated
activation of ERK requires phosphorylation of
4 Tyr1526, which mediates
interaction with the PTB domain of Shc. A, dominant
negative Shc inhibits activation of ERK by
6
4 in HUVECs. HUVECs were transiently
transfected with wild type
6 and
4 in
combination with increasing amounts of vector encoding FLAG-tagged
dominant negative (Dn) Shc (7.5, 15, and 30 µg). Cells
were detached and replated for 60 min on dishes coated with the
anti-
4 mAb 3E1. Because dishes were post-coated with
BSA, only the cells expressing
6
4
(approximately 30%) attached. Total proteins from cells in suspension
(lane S) or adhering to the 3E1 mAb (3E1) were probed with
antibodies against phosphorylated ERK (top panel) or the
FLAG epitope (middle panel). Blots were stripped and
reprobed with antibodies against total ERK (bottom panel) as
control. B, phenylalanine substitution of
Tyr1440 and Tyr1526 inhibits vanadate-induced
phosphorylation of
4. HUVECs were transiently
transfected with
6 and either wild type (A) or the
indicated mutant versions of
4 (Y1422F, Y1440F, Y1526F,
Y1642F, and 4F). Cells were either left untreated (
) or treated with
pervanadate (+) and lysed. After immunoprecipitation,
6
4 was separated by SDS-PAGE and probed
by immunoblotting with antibodies against phosphotyrosine (top
panel) and then stripped and reprobed with antibodies against the
extracellular domain of
4 (bottom panel).
C, a version of
4 containing phenylalanine
substitutions at the PTB domain binding sites (Y1526F/Y1642F) does not
activate ERK as efficiently as wild type
4 or a version
with phenylalanine substitutions at the SH2 domain binding sites
(Y1422F/Y1440F). HUVECs were transfected with
6 together with wild
type (A) or the indicated mutant forms of of
4
(Y1526F/Y1642F and Y1422F/Y1440F). Total proteins from cells in
suspension or plated for the indicated times on dishes coated with the
3E1 mAb were probed by immunoblotting with antibodies against
4 (top panel) or phosphorylated ERK
(middle panel). The blot was stripped and re-probed with an
antibody to total Erk2 (bottom panel) as control.
D, phenylalanine substitution of the primary PTB domain
binding site in
4 prevents Erk activation. HUVECs
transfected with
6 together with wild type (A) or the indicated
mutant versions of
4 (Y1526F, Y1642F, Y1526F/Y1642F,
L) were plated on 3E1-coated dishes for 60 min. Total proteins
were probed by immunoblotting with antibodies against
4
(top panel) or phosphorylated ERK (middle panel).
The blot was then stripped and reprobed with an antibody recognizing
total Erk2 (bottom panel).
4 tyrosines involved in binding to the SH2 and PTB
domain of Shc. Previous studies using phosphopeptide mapping had
identified Tyr1440 as a major tyrosine phosphorylation site
in
4 and revealed that
4 is
phosphorylated at several additional tyrosines in vivo (14, 17). HUVECs were transfected with mutant forms of
4
carrying phenylalanine substitutions at each one or all four tyrosines (Tyr1422, Tyr1440, Tyr1526, and
Tyr1642) capable of binding to Shc and stimulated with
pervanadate (Fig. 5B). The phosphorylation of
4 was analyzed by immunoblotting with antibodies against
phosphotyrosine. Phenylalanine substitution of either
Tyr1440 or Tyr1526 resulted in a significant
decrease in total phosphorylation of
4, whereas
phenylalanine substitutions at Tyr1422 or
Tyr1642 had a negligible effect. Mutation of all four
tyrosines to phenylalanine (4F) resulted in an almost complete block in
tyrosine phosphorylation. With the caveat that the anti-phosphotyrosine
antibodies may not interact with equal affinity with all phosphorylated
tyrosines in
4, these results suggest that
Tyr1440 and Tyr1526 are the major
phosphorylation sites in
4.
4 was
required for signaling to ERK, the HUVECs were transfected with
constructs encoding
6 in combination with versions of
4 unable to bind to either the SH2 domain
(Y1422F/Y1440F) or the PTB domain (Y1526F/Y1642F) of Shc. Cells were
then replated onto 3E1-coated dishes and analyzed by immunoblotting
with anti-phospho-ERK antibodies. As shown in Fig. 5C,
ligation of
4 Y1422F/Y1440F caused activation of ERK, although slightly less efficiently than ligation of wild type
4, whereas ligation of
4 Y1526F/Y1642F
did not induce activation of ERK. Immunoblotting with a polyclonal
antibody against the extracellular domain of
4 (Fig.
5C, top panel) and fluorescence-activated cell
sorting analysis (data not shown) confirmed that the different versions
of
4 were expressed at comparable levels. These results provide evidence that
6
4 signaling to ERK
requires interaction of the PTB domain of Shc with
4.
This finding is consistent with the observation that
6
4 signaling to ERK proceeds through Shc (Fig. 5B) and the activation of Shc by
6
4 requires a functional PTB domain (Fig.
4B).
4 for activation of ERK. As shown in
Fig. 5D, ligation of
4 Y1526F did not cause
activation of ERK, whereas ligation of
4 Y1642F induced
activation of ERK, although slightly less efficiently than wild type
4. These results indicate that
4
Tyr1526, the primary binding site for the PTB domain of
Shc, is required for activation of ERK.
4 into
Hemidesmosome-like Adhesions--
The cytoplasmic domain of
4, and in particular the segment comprising the first
two Fn type III modules and the connecting segment, is required for
formation of hemidesmosomes (18, 19). Our initial studies had suggested
that Tyr1422 and Tyr1440 (which resemble a
tyrosine-based activation motif or TAM) were necessary for
incorporation of recombinant
4 into the
hemidesmosome-like adhesions formed by 804G cells in culture (14).
Subsequent studies have, however, indicated that residues C-terminal to
residue 1355 in the
4 cytoplasmic domain are not
required for the formation of hemidesmosome-like adhesions by cultured
cells (23, 33). To resolve this discrepancy, we have performed a number
of additional studies. Among them, an analysis of the entire coding
sequence of the constructs used in our previous study revealed that the
4 mutant Y1422F/Y1440F (called the TAM mutant or YZ in
(14)) originated from a version of
4 that contained an
in-frame deletion of the sequences encoding amino acids 880-886
(DHTIVDT). Because this sequence is located in the membrane proximal
portion of the cytoplasmic domain of
4, which previous
studies had indicated to be dispensable for incorporation in
hemidesmosomes-like adhesion structures (19, 23), we had not covered it
during our initial sequence reanalysis. The nature and origin of the
variant cDNA lacking amino acids 880-886 remains to be examined.
As shown below, this deletion (termed the Gap) in combination with the
Y1422F/Y1440F mutation prevents incorporation of
4 into
hemidesmosomes in 804G cells, in agreement with our original result.
6
4 and forms
hemidesmosome-like adhesions in culture. Immunofluorescence staining of
hemidesmosomal components reveal that the hemidesmosome-like adhesions
of these cells are arranged in a "Swiss cheese" pattern (34). We
transfected 804G cells with constructs encoding human versions of
either the canonical (A) or various variant forms of
4
(Y1422F/Y1440F, Y1526F/Y1642F, 4F, Gap or Gap-Y1422F/Y1440F). Pools of
cells stably expressing these recombinant forms of
4
(see "Materials and Methods") were stained with the 3E1 mAb that
recognizes human but not rat
4. As shown in Fig.
6A, the
4
variants Y1422F/Y1440F, Y1526F/Y1642F, 4F, and Gap localized to
hemidesmosomes as efficiently as wild type
4 (A),
whereas the
4 mutant Gap- Y1422F/Y1440F displayed a
significantly decreased ability to localize to hemidesmosomes. Thus,
mutation of the potential
4 TAM impairs localization of
4 to hemidesmosomes only in the context of the Gap
version of
4. These results indicate that the integrity
of Tyr1422 and Tyr1440 is not required for
incorporation of
4 in hemidesmosome-like adhesions, as
shown previously by others (23). However, the synergy between the TAM
mutation and the Gap suggests that Tyr1422 and
Tyr1440 may play a role in assembly of hemidesmosomes. It
is possible that a full assessment of this role requires examination of
the assembly of bona fide hemidesmosomes in skin organ
culture systems or in vivo.
View larger version (44K):
[in a new window]
Fig. 6.
Tyrosine phosphorylation of the
4 cytoplasmic domain
antagonizes assembly of hemidesmosomes. A, 804G cells
expressing either wild type (A) or the indicated mutant versions of
4 (Y1422/1440F, Y1526/1642F, 4F, Gap, Gap-Y1422/1440F)
were cultured on coverslips for 2 days, fixed, and stained with the
anti-
4 mAb 3E1 followed by FITC-conjugated anti-mouse
IgGs. B, phenylalanine substitution of the major tyrosine
phosphorylation sites in
4 antagonizes the disruption of
hemidesmosomes caused by orthovanadate. 804G cells expressing either
wild type (A) or the indicated mutant versions of
4
(Y1422/1440F, Y1526/1642F, and 4F) were cultured for 24 h and then
serum starved overnight in the presence of 20 or 100 µM
orthovanadate. Cells were fixed and stained with 3E1 mAb followed by
FITC-conjugated anti-mouse IgGs.
4 Tyrosines Involved in the
Recruitment of Shc Antagonizes Assembly of
Hemidesmosomes--
Hemidesmosomes are structures that provide stable
adhesion of epithelial cells to the underlying basement membranes and
are therefore disassembled during cell migration (34, 35). Because phenylalanine substitution of the tyrosines that bind to Shc does not
prevent incorporation of the canonical form of
4 in
hemidesmosomes, it is very unlikely that the phosphorylation of these
sites participates in the assembly of hemidesmosomes (Ref. 23 and Fig.
6A).
4 and disassembly of hemidesmosomes in keratinocytes, although we did not establish a cause-effect relationship (36). To examine whether tyrosine phosphorylation of
4 causes disassembly of hemidesmosomes, we used 804G
cell lines expressing wild type
4 (A) or the
4 mutants Y1422F/Y1440F, Y1526F/Y1642F, or 4F. Because
these cells express very low levels of EGF receptor, they were treated
with either 20 or 100 µM orthovanadate for 15 h to
increase tyrosine phosphorylation of
4. When
orthovanadate is added in the absence of hydrogen peroxide, it behaves
as a competitive inhibitor of tyrosine phosphatases, whereas in the presence of hydrogen peroxide it is converted to the irreversible inhibitor pervanadate (37). We used orthovanadate for these experiments
because prolonged exposure to pervanadate causes cell toxicity.
Treatment with 20 µM orthovanadate induced tyrosine phosphorylation of wild type
4 but not of the mutant 4F
in 804G cells (data not shown). As shown in Fig. 6B, 20 µM orthovanadate caused disruption of hemidesmosomes in
804G cells expressing wild type
4 (A) but not in those
expressing any of the mutant versions of
4
(Y1422F/Y1440F, Y1526F/Y1642F, or 4F). In the presence of 100 µM orthovanadate, most 804G cells expressing wild type
4 (A) rounded up and almost completely detached (>90%
detached), whereas cells expressing the mutant versions of
4 were protected from disruption of hemidesmosomes to
varying degrees. Those expressing the
4 mutant 4F were
the most protected (~20% detached, some remaining hemidesmosomes),
whereas those expressing the
4 mutant Y1526F/Y1642F were
protected to a lesser degree (~30% detached), and those expressing
the
4 mutant Y1422F/Y1440F were the least protected
(~60% detached). Therefore, phosphorylation of these tyrosines can
lead to a reduction of hemidesmosomes and may be a physiologic
mechanism for regulating hemidesmosome turnover. Treatment with 100 ng/ml EGF for 15 h resulted in a modest and equivalent disruption
of hemidesmosomes in all four transfectants (data not shown),
suggesting that the partial disassembly of hemidesmosomes caused by EGF
in 804G cells may also involve an additional mechanism, as suggested
previously (38).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6
4 integrin induces
tyrosine phosphorylation of the cytoplasmic domain of
4,
recruitment of Shc, and activation of Ras-dependent
mitogen-activated protein kinase cascades (14, 17). Biochemical and
genetic evidence implies that
6
4-dependent signaling
promotes proliferation of both keratinocytes and intestinal epithelial
cells (17, 18). To assess the specific role of Shc in these processes,
it is necessary to identify the mechanism by which Shc binds to
4 and determine whether this binding is required for
activation of downstream signaling pathways. The results of this study
provide evidence that the PTB domain of Shc interacts in a
phosphorylation-dependent manner with Tyr1526 in
4, whereas the SH2 domain binds to Tyr1440.
Both phenylalanine substitution of Tyr1526 in
4 and inactivation of the PTB domain in Shc suppress
6
4-mediated phosphorylation of Shc and
signaling to ERK. By contrast, mutation of both the primary and
secondary binding site for the Shc SH2 domain in
4 or
inactivation of the SH2 domain itself exert only a minor effect on Shc
signaling to ERK. These observations suggest that the binding of the
PTB domain of Shc to Tyr1526 in
4 is crucial
for subsequent phosphorylation of Shc by the
6
4-associated kinase and thus activation
of Ras-dependent pathways. It remains, however, possible
and indeed likely that the interaction mediated by the SH2 domain of
Shc contributes to some extent to the stability of the association of
Shc with
4 in vivo.
4 binding site for the PTB domain of Shc,
Tyr1526, is located in the third Fn type III module.
Interestingly, all known type III Fn repeats contain a tyrosine at this
position. Fn type III repeats are found not only in extracellular
matrix proteins (fibronectin and tenascin) and cell surface receptors (for growth hormone, prolactin, and insulin) but also in cytoplasmic components (twitchin, titin, and the
4 cytoplasmic
domain) (39). To our knowledge, this study is the first to identify a
physiologically relevant phosphorylation site in a Fn type III module.
Because several of these repeats have been crystallized, including the N-terminal pair in
4, it is possible to predict the
conformation of
4 Tyr1526 and surrounding
amino acids. The residues N-terminal to tyrosine 1526 that are also
important for interaction with the PTB domain of Shc, namely the
asparagine and leucine at the
3 and
5 positions, are predicted to
be in a loop between the E and F strands of the
-sandwich. Based on
its location in a loop between
strands, this sequence motif is
likely to be exposed to solvent in the intact molecule. This position
would facilitate its phosphorylation and subsequent interaction with
the PTB domain of Shc. In addition, there is evidence suggesting that
the C-terminal portion of the
4 tail binds to a more
proximal segment comprising the second Fn type III repeat and the
connecting segment in vitro (23). If this intramolecular
interaction occurs also in vivo, it may bring the third Fn
type III repeat closer to the plasma membrane and thus facilitate the
interaction of the Src family kinase with Tyr1526 in
4 and thereby the recruitment of Shc. This mechanism
would also enable Shc to position Grb2/SOS in closer proximity to its target Ras.
4. In agreement with the observation that these two
sites are homologous to one another and both conform well to the
consensus for binding to the SH2 domain of Shc (YXXL), our
results show that phosphopeptides encompassing both sites inhibit to a
similar extent the interaction of the SH2 domain of Shc with
4. It is likely that Tyr1440 is more
important in both in vitro and in vivo
experiments because it is more efficiently phosphorylated (see also
Refs. 14 and 17). Based on the observation that phenylalanine
substitutions at Tyr1422 and Tyr1440 prevent
incorporation of
4 in the hemidesmosome-like adhesions of 804G cells, we had made the hypothesis that phosphorylation of both
tyrosines, which encompass a potential TAM, be required for assembly of
hemidesmosomes (14). This hypothesis now has to be reevaluated.
Together with the results of studies by others (21, 23), our current
observations indicate that the phosphorylation of Tyr1422
and Tyr1444, as well as that of Tyr1526 and
Tyr1642, disrupts hemidesmosomes or inhibits their
assembly. The simplest hypothesis is that these tyrosines of
4 interact with a component, such as BPAG2 (21, 23) that
confers stability to hemidesmosomes. Phosphorylation of these tyrosines
would interfere with the association of
4 with this
component and thus destabilize hemidesmosomes. This model is attractive
also because it potentially explains why phenylalanine substitution
may exert an effect similar to, although much smaller than,
phosphorylation of Tyr1440 and Tyr1422. In
particular, a previous study has shown that phenylalanine substitution
of these tyrosines reduces association of BPAG2 with hemidesmosomes
(21, 23), and we have shown here that it prevents incorporation of a
form of
4 lacking amino acids 880-886 in the hemidesmosome-like adhesions of 804G cells. Further experiments will be
required to test this model.
4
induces Shc signaling but antagonizes formation of hemidesmosomes, suggesting that these two processes may be mutually exclusive. It has
been reported that processing of the
6
4
ligand laminin-5 inhibits assembly of hemidesmosomes and promotes cell
migration (40-42). It will be interesting to determine whether this
process also affects
6
4 signaling to Shc.
If so, differential processing of laminin-5 may help direct whether
6
4 ligation leads to tyrosine phosphorylation and Shc signaling or hemidesmosome formation.
4 cytoplasmic domain
required for Shc signaling is distinct from that required for
hemidesmosome formation may enable further studies on the role of
6
4 signaling. For example, analysis of
knock-in mice expressing a version of
4 unable to signal
through Shc but still able to promote assembly of hemidesmosomes will
enable a more comprehensive understanding of the physiologic role of
6
4 signaling.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Ben Margolis and David Levy for
reagents and members of our laboratory for discussion. We are grateful
to Paul Tempst for assistance in our attempts to map the major tyrosine
phosphorylation sites of 4 by mass spectrometry.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01-CA58976 and P30-CA08748.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.
Student in the M.D.-Ph.D. Program of New York University School of Medicine.
§ Recipient of a fellowship from INSERM (France).
¶ Supported by National Institutes of Health Postdoctoral Fellowship F32-CA79516.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Cellular Biochemistry and
Biophysics Program, Memorial Sloan-Kettering Cancer Center, Box 216, 1275 York Ave., New York, NY 10021. Tel.: 212-639-6998; Fax:
212-794-6236; E-mail: F-Giancotti@ski.mskcc.org.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M008663200
2 L. Gagnoux-Palacios, M. Dans, W. van t'Hoff, M. Resh, and F. G. Giancotti, manuscript in preparation.
3 A. Mariotti, P. Kedeshian, M. Dans, A.M. Curatola, L. Gagnoux-Palacios, and F. G. Giancotti, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SH2, Src homology 2; PTB, phosphotyrosine binding; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; mAb, monoclonal antibody; GST, glutathione S-transferase; HRP, horseradish peroxidase; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; Fn, fibronectin; TAM, tyrosine-based activation motif.
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