(Received for publication, February 7, 1997, and in revised form, April 3, 1997)
From the Engagement of Integrins are Cas was originally identified as one of the major
tyrosine-phosphorylated proteins in v-crk- or
v-src-transformed cells (26, 27). Cas belongs to a new
family of structurally related proteins that are thought to act as
"docking molecules," i.e. regulating the assembly of
several SH2 and SH3 domain-containing proteins into signaling
complexes. This family includes three members so far: Cas, HEF1/Cas-L
(25, 28), and Efs/Sin (embryonal Fyn-associated substrate/Src-interacting or
signal-integrating protein) (29, 30). They all
contain an SH3 domain in the N-terminal region; a cluster of SH2
domain-binding motifs that have been named the "substrate domain"
(27); and, with the exception of HEF1/Cas-L, several potential binding
motifs for SH3 domains.
Following Proteins interacting with Cas include FAK and PTP-1B, which bind to the
Cas SH3 domain (31-33); Crk family members, which interact with
tyrosine-phosphorylated Cas through SH2 domain-binding motifs (23,
34-36); c-Src, which interacts with tyrosine-phosphorylated Cas
through SH2 and SH3 domain-binding motifs in the Cas C-terminal region
(37); and the protein-tyrosine phosphatase PTP-PEST (38). Cas also
associates with the focal adhesion proteins paxillin and tensin (39).
Binding of Crk family members to tyrosine-phosphorylated Cas
illustrates the assembly of signaling complexes since the SH3 domain of
Crk proteins can bind in turn to a number of proteins, including two
guanine nucleotide exchange factors, Sos and C3G, which regulate Ras
and Rap1 activation, respectively (40-44). The Cas-Crk-Sos or
Cas-Crk-C3G signaling complexes are potentially involved in the
propagation of downstream signals.
Studies performed in fibroblasts have shown that c-Src is primarily
responsible for integrin-mediated Cas phosphorylation and that FAK
might recruit Src family kinases to phosphorylate Cas (34, 35). To
date, the identification of Cas-associated Src kinases in
nontransfected cells is not well documented. In this study, we present
evidence that in the human multiple myeloma cell line ARH-77,
p59Fyn tyrosine kinase (Fyn) and SHP2 tyrosine phosphatase
are recruited to the Cas complex following integrin ligation. This
study provides insight into the control of integrin-mediated tyrosine
phosphorylation of Cas in B cells.
ARH-77 cells were maintained in
RPMI 1640 medium containing 10% heat-inactivated fetal calf serum.
Antibodies used in this study were directed against the following:
CD29/ ARH-77 cells were stimulated with
anti-integrin antibodies plus rabbit anti-mouse Ig as described
previously (14). Cells were then lysed in 0.5% Nonidet P-40 buffer
containing 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
10 µg/ml aprotinin, 10 mM NaF, and 1 mM
sodium vanadate.
Fyn cDNA inserted into the pSR For
immunoprecipitation studies, cell lysates were precleared with protein
G-Sepharose beads (Pharmacia, Uppsala) and then preincubated with
specific antibody for 1 h at 4 °C, followed by the addition of
protein G-Sepharose beads for 1 h at 4 °C. For precipitations
with GST fusion proteins, lysates were incubated for 2 h at
4 °C with 25 µg of fusion proteins bound to glutathione beads
(Pharmacia). Precipitated proteins were washed four times with lysis
buffer and subjected to kinase assay or eluted by boiling in sample
buffer (2% SDS, 10% glycerol, 0.1 M Tris, pH 6.8, 0.02% bromphenol blue). For sequential immunoprecipitation, washed beads were
boiled for 5 min in the presence of 2% SDS, and the supernatants were
reprecipitated with antibodies in lysis buffer containing a 0.1% final
SDS concentration. In vitro kinase assays were performed by
washing Cas immunoprecipitates once in kinase buffer (10 mM Hepes, pH 7.3, containing 50 mM NaCl, 5 mM
MnCl2, 5 mM MgCl2, and 100 µM sodium vanadate) and incubating the pellet in kinase buffer containing 0.1 mM ATP (Sigma) for 10 min at room
temperature. Proteins were separated by SDS-polyacrylamide gel
electrophoresis under reducing conditions and transferred to
Immobilon-PTM membranes (Millipore Corp., Bedford, MA). Membranes were
blocked using 5% nonfat dried milk in TBS-T (20 mM Tris,
pH 7.6, 130 mM NaCl, 0.1% Tween 20) and incubated for
1 h with specific antibodies in TBS-T. Immunoreactive bands were
visualized using secondary horseradish peroxidase-conjugated antibodies
(Promega, Madison, WI) and chemiluminescence (ECL, Amersham
International, Buckinghamshire, United Kingdom).
To examine
Cas-associated tyrosine kinase(s), the B cell line ARH-77 was
stimulated with the anti-
The C terminus of Cas contains a proline-rich sequence
(RPLPSPP) and a YDYV motif, which have been shown to bind to Src SH3 and SH2 domains, respectively (37). Since we have previously reported
that a GST fusion protein containing the RPLPSPP sequence bound to Fyn
(24), we further examined the requirement of this region of Cas for
Fyn/Cas interaction. cDNAs encoding HA-tagged wild-type Cas or
deletion mutants of Cas were transiently expressed in COS-7 cells in
the presence or absence of cDNA encoding Fyn. The mutants of Cas
included the following: Cas
To
further study the interaction between Cas and Fyn, we performed binding
experiments with truncated GST-Fyn fusion proteins corresponding to the
SH2 or SH3 domain of Fyn. Fusion proteins were incubated with lysates
of unstimulated (Fig. 4A, 0 lanes) or
To investigate the presence of a Cas-associated
tyrosine phosphatase, Cas immunoprecipitates from
Integrins are involved in the regulation of proliferation,
differentiation, and cell survival in a variety of cell types, events
that are dependent upon tyrosine phosphorylation (49). Cas has been
identified to be a major tyrosine-phosphorylated substrate following
integrin ligation (20-24). In this study, we found that precipitations
of Cas immune complexes from integrin-stimulated cells contained both
tyrosine kinase and tyrosine phosphatase activities, which
modulated the in vitro phosphorylation of Cas. Furthermore,
Fyn tyrosine kinase and SHP2 tyrosine phosphatase were recruited
in Cas complexes, suggesting that they participate in modulating Cas
phosphorylation.
The C-terminal proline-rich region of Cas can associate in
vitro with several Src kinases, including p59Fyn,
p59/62Hck, and p53/56Lyn (24). In this study,
we found that in ARH-77 cells, Fyn was the most obvious kinase
detectable. The focal adhesion kinases FAK and RAFTK can associate with
Cas (18, 31, 32, 36). In B cells, RAFTK and, to a lesser extent, FAK
are both tyrosine-phosphorylated following integrin ligation (14, 18).
Similar to Src kinases, the kinase activity of RAFTK and FAK correlates
with an increase in autophosphorylation activity in certain cell types
(15, 50). However, we previously reported that Cas associated with
RAFTK is mainly nonphosphorylated on tyrosine residues (18). In
addition, we did not observe tyrosine-phosphorylated bands
corresponding in size to RAFTK and FAK, 120 and 125 kDa, respectively.
This results suggest that in nontransfected cells and under the
conditions of these assays, these two kinases may not be sufficient for
Cas phosphorylation. Further support of this is the observation that Cas phosphorylation is reduced in fibroblasts lacking Src kinases, but
remains unaffected in fibroblasts lacking FAK (34, 35).
In a cotransfection assay in COS-7 cells, we found that the Fyn/Cas
physical interaction and subsequent Fyn-mediated Cas phosphorylation required amino acids 638-889 in the C-terminal region of Cas. This
sequence contains a polyproline stretch (RPLPSPP) and a YDYV motif to
which both Fyn SH3 and SH2 domains could bind in vivo. In vitro studies using GST fusion proteins derived from the
SH2 and SH3 domains of Fyn indicated that both domains could
participate in this Fyn/Cas interaction. However, the in
vivo association of Fyn and Cas was transient and correlated with
Cas tyrosine phosphorylation, implying a regulation of this
interaction. Whether the Fyn SH3 domain is primarily involved in Cas
binding or is subsequent to Fyn SH2 binding to reinforce the
interaction between the two proteins is not known. A precedent for the
predominant binding of the SH2 domain over the SH3 domain has been
described for the interaction of p120Cbl with phosphoinositol
3 The Cas-like protein Sin leads to activation of Src kinase activity on
binding of Sin to the c-Src SH3 domain (30). Similarly, it is possible
that Fyn binding to Cas will stimulate the kinase activity of Fyn,
which in turn will lead to the effective phosphorylation of Cas and
allow the recruitment of SH2 domain-containing molecules such as the
adapter proteins of the Crk family (23,
24).2 In support of this is the finding
that Fyn-phosphorylated Cas in COS cells interacts with Crk (data not
shown). The assembly of this complex of proteins might further enable
recruited proteins to interact with and potentially be phosphorylated
by Fyn.
Since the phosphorylation of Cas is likely to be an important event in
the propagation and amplification of downstream signals of integrin
ligation, a mechanism of down-regulating that process is critical. A
role for SHP2 tyrosine phosphatase in modulating the phosphorylation of
Cas in B cells came from the finding that SHP2 was recruited in Cas
complexes following integrin ligation. The Cas-SHP2 association
correlated with the extent of Cas tyrosine phosphorylation. In view of
the recent finding that SHP2 associates with HEF1/Cas-L through its SH2
domains (25), it is possible that a similar mechanism occurs for
Cas-SHP2 assembly. This might result in activation of SHP2 activity
since engagement of its SH2 domains following binding to
platelet-derived growth factor receptor The precise mechanism by which integrin cross-linking leads to the
activation of the kinase(s) and phosphatase(s) is presently unclear.
Integrin-mediated phosphorylated Cas localizes to focal adhesions,
whereas nonphosphorylated Cas remains in the cytosol (22, 27). Such Cas
localization may be driven by its constitutive association through its
SH3 domain with FAK, due to the FAK C-terminal focal adhesion targeting
domain (50). Therefore, redistributed Cas during integrin stimulation
might localize Cas to a region of the cell where Cas will be accessible
to tyrosine kinases. Support for this hypothesis is that optimal Cas
phosphorylation requires an intact cytoskeleton since inhibitors of
cytoskeletal assembly also inhibit integrin-mediated Cas tyrosine
phosphorylations (20, 24). A potential sequence of events leading to
Cas phosphorylation in ARH-77 cells could be that integrin-mediated
cytoskeletal reorganization allows the co-compartmentalization of Cas
and Fyn. Activated Fyn, possibly by its binding to Cas, phosphorylates
Cas, leading to the formation of a signaling complex including Crk
family members and SHP2. SHP2, which becomes activated following
SH2-mediated binding to phosphorylated Cas, could then participate in
downstream signaling events and/or attenuate the activity of the
complex by dephosphorylating Cas.
Cas and the Cas-like protein HEF1 are both expressed in normal B cells
and most B cell lines (24). However, Cas is the predominant substrate
that is phosphorylated in terminally differentiated B cell lines
following We thank Prof. Alain Bernard for K20
antibody, Drs. Joan Brugge and Martyn Botfield for Fyn constructs, and
Dr. Antonio Da Silva for providing Fyn cDNA. We greatly appreciate
Dr. Andreas Beck for help in preparing Cas constructs. We also thank
Drs. Andreas Beck, Antonio Da Silva, Bernard Mari, Susan Law, and Erica Golemis for helpful discussions. We thank Janet Walsh for assistance in
preparing the manuscript.
Department of Medicine,
Third
Department of Internal Medicine, University of Tokyo, Hongo,
Tokyo 113, Japan
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 integrins in terminally
differentiated human B cell lines, such as ARH-77, leads to prominent
tyrosine phosphorylation of the p130 Crk-associated substrate (Cas).
Cas regulates the assembly of several SH2 and SH3 domain-containing
proteins into signaling complexes, which are potentially involved in
the propagation of downstream signals. We demonstrate here that
immunoprecipitated Cas from
1 integrin-stimulated ARH-77 cells was
associated with tyrosine kinase and phosphatase activities and that
integrin ligation led to the recruitment of at least
p59Fyn tyrosine kinase and SHP2 tyrosine phosphatase
in Cas immune complexes. Cotransfection studies in COS-7 cells further
indicated that Fyn/Cas physical interaction and Fyn-mediated Cas
phosphorylation required amino acids 638-889 in the C-terminal region
of Cas. This sequence contains both c-Src SH2 and SH3 domain-binding
motifs. In vitro binding studies using glutathione
S-transferase fusion proteins derived from the SH2 or SH3
domains of Fyn suggested that both Fyn domains can participate in
Fyn/Cas interaction. These data implicate Fyn and SHP2 as potential
modulators of Cas signaling complexes in B cells.
/
heterodimeric adhesion receptors that are
involved in cell/cell and cell/matrix interactions (1, 2). With regard
to B lymphocytes, integrins are involved in cell localization within
specific microenvironments (3, 4) and in regulating cell survival
(5-8). One of the intracellular signaling events initiated by
integrins is the activation of a cascade of tyrosine phosphorylation
events (9). In many cell types, including B lymphocytes, there is
prominent tyrosine phosphorylation of proteins of 105-130 kDa. Several
of these substrates have been identified, including p125FAK
(FAK (focal adhesion kinase))
(10-14); RAFTK (related adhesion focal tyrosine kinase; also known
as PYK2 and CAK
) (15-18); p120c-cbl, the
cellular homologue of the oncogene v-cbl (19);
p130Cas (Cas (Crk-associated
substrate)) (20-24); and the Cas-like molecule p105HEF1 (human enhancer of
filamentation 1), also known as Cas-L for lymphocyte-type
Cas protein (24, 25).
1 integrin cross-linking, Cas phosphorylation was most
prominent in B cell lines representative of a more differentiated state, such as the multiple myeloma cell lines ARH-77, IM-9, and RPMI
8226, and was minimally detectable in normal mature B cells (24). In
contrast, HEF1 was consistently tyrosine-phosphorylated in all
immature, mature, and terminally differentiated B cell lines as well as
in normal B cells following both
1 integrin and B cell antigen
receptor ligation. Therefore, the phosphorylation of these two related
molecules appears to be differentially regulated in B cells.
Cell Lines and Materials
1 integrin (mAb1 K20, provided by
Prof. Alain Bernard, U146 INSERM, Nice, France), phosphotyrosine (mAb
4G10), p59Fyn or p130Cas (Cas) (rabbit
polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA), GST (GST
mAb, Santa Cruz Biotechnology, Santa Cruz, CA), SHP2 (PTP-1D mAb,
Transduction Laboratories, Lexington, KY), hemagglutinin (HA mAb,
Boehringer Mannheim), and affinity-purified rabbit anti-mouse Ig
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). GST-Fyn
fusion proteins were provided by Drs. Joan Brugge and Martyn Botfield
(Ariad Pharmaceuticals, Cambridge, MA).
2
expression vector was provided by Dr. Antonio Da Silva (Dana-Farber
Cancer Institute) (45). Rat Cas cDNA and deletion mutants of Cas
cDNA were cloned into a modified version of the pcDL-SR
296
expression plasmid, termed pSP65-SR
.2-HAtag-Hygro, containing a
hygromycin B phosphotransferase gene and an HA epitope tag sequence in
frame with the Cas cDNAs (46, 47). Briefly, 5
-fragments of the
cDNAs with an in-frame XbaI site were generated using
polymerase chain reaction and cloned together with the corresponding
3
-fragment into the restriction sites XbaI and
EcoRI of pSP65-SR
.2-HAtag-Hygro. The expression plasmids
were used to transiently transfect COS-7 cells by the DEAE-dextran/Me2SO method as described (48).
In Vivo Association between p59Fyn and Cas following
1 Integrin Ligation in the ARH-77 B Cell Line
1 integrin mAb K20 followed by rabbit
anti-mouse Ig. Cell lysates were then immunoprecipitated with anti-Cas
antibody, and subjected (+) or not (
) to an in vitro
kinase assay for 10 min (Fig. 1A, upper
panels). Anti-phosphotyrosine Western blot analysis showed that
Cas immune complexes contained a transient tyrosine kinase(s) activity
(maximally detected at 5 and 15 min), which resulted in increased
in vitro tyrosine phosphorylation of Cas plus an additional
main band ranging from 55 to 60 kDa. The same membrane was reprobed
with anti-Cas antibody to show that equivalent amounts of
immunoprecipitated Cas were loaded in each lane (Fig. 1A,
middle panels). The in vitro phosphorylated 55-60-kDa band contained a sharp 59-kDa protein, which was suggestive of the presence of p59Fyn kinase. To investigate this
possibility, the membrane was reprobed with anti-p59Fyn
antibody. As shown in Fig. 1A, (lower panels),
anti-Fyn antibody reacted with a faint band detectable only in the
5 and 15 min lanes of stimulation. To further
confirm the identity of this 59-kDa protein, anti-Cas or control
(Ct) immunoprecipitates from 15-min
1 integrin-stimulated
ARH-77 cells were subjected to in vitro kinase assays
(Ip 1), and half of the samples were then reimmunoprecipitated with anti-Fyn antibody (Ip 2). As shown
in Fig. 1B (lower panel), Fyn could be
reimmunoprecipitated from Cas immune complexes, but not from control
immunoprecipitates. Fyn was tyrosine-phosphorylated (Fig.
1B, upper panel) and comigrated with the sharp
pp59. These results indicate that Fyn kinase associates in an
integrin-regulated manner with Cas and strongly suggest that it
participates in Cas phosphorylation.
Fig. 1.
In vivo association between
p59Fyn and Cas following 1 integrin ligation in the
ARH-77 B cell line. A, the human B cell line ARH-77 was
stimulated with anti-
1 integrin antibodies plus rabbit anti-mouse Ig
and then lysed as a function of time. Cell lysates were
immunoprecipitated (IP) with anti-Cas antibody and subjected
(+) or not (
) to an in vitro kinase assay
(IVKA). Samples were then immunoblotted with
anti-phosphotyrosine antibody (P-Tyr; upper
panels), stripped, and reblotted with anti-Cas antibody (middle panels) or antiserum to Fyn (lower
panels) as indicated. B, lysates of 15-min anti-
1
integrin-stimulated ARH-77 cells were first immunoprecipitated
(Ip 1) with anti-Cas antibody (Cas) or an
irrelevant control antibody (Ct) and subjected to an
in vitro kinase assay. A second immunoprecipitation
(Ip 2) with anti-Fyn antibodies (Fyn) was then
performed on eluates of the first immunoprecipitations. All eluates
were then analyzed by Western blotting with anti-Tyr(P) antibody
(upper panels) and after stripping, with anti-Cas
(middle panel) or anti-Fyn (lower panel)
antibodies as indicated. The positions of Cas and Fyn proteins and
immunoglobulin (bracket) as well as molecular mass markers
(in kDa) are shown. Blots were imaged by chemiluminescence.
[View Larger Version of this Image (74K GIF file)]
SD, in which the sequence from amino
acids 213 to 514, which contains the substrate domain with 15 out of 27 potential sites of tyrosine
phosphorylation within Cas, was deleted; and Cas
SB, in which the
sequence from amino acids 638 to 889, which contains the Src SH2 and
SH3 domain-binding motifs, was deleted (37) (Fig. 2). To
discriminate transfected Cas proteins from endogenous Cas proteins,
lysates of transfected COS-7 cells were immunoprecipitated with anti-HA
tag antibody. Anti-phosphotyrosine immunoblotting of the anti-HA tag
immunoprecipitations indicated that the presence of cotransfected Fyn
led to Cas phosphorylation (Fig. 3A). The Cas
SD mutant, although lacking some of the potential sites of tyrosine
phosphorylation, showed increased tyrosine phosphorylation in the
presence of Fyn. In contrast, the Cas
SB mutant cotransfected with
Fyn demonstrated reduced tyrosine phosphorylation when compared with
Cas or Cas
SD. Therefore, the SB domain of Cas was necessary for
Fyn-mediated Cas phosphorylation. Comparable levels of expression of
Cas, Cas mutants (Fig. 3B), and Fyn (Fig. 3D)
were detected regardless of the cotransfection conditions. More
important, Fyn kinase was co-immunoprecipitated with Cas and Cas
SD,
but not with Cas
SB (Fig. 3C), indicating that the SB
region was required for a physical interaction between Fyn and Cas.
Fig. 2.
Schematic diagram representing the various
p130Cas constructs used. Shown are intact Cas; Cas
SD, in which the substrate domain containing 15 YXXP
motifs was deleted; and
SB, in which the C-terminal domain
containing the Src SH3-binding motif (RPLPSPP) and the Src SH2-binding
motif (YDYV) was deleted.
[View Larger Version of this Image (16K GIF file)]
Fig. 3.
Tyrosine phosphorylation of Cas by
coexpression with Fyn kinase in COS-7 cells requires the Src-binding
site of Cas. COS-7 cells were transiently transfected with control
plasmid (Mock) or with plasmid encoding HA-tagged wild-type
Cas (Cas WT), HA-tagged Cas with the Src-binding site
deleted (SB), or HA-tagged Cas with the substrate domain
deleted (
SD). A second set of cells were also
cotransfected with a plasmid encoding Fyn (+ Fyn).
Transfected Cas was immunoprecipitated (Ip) using anti-HA
antibodies and analyzed by Western blotting with anti-Tyr(P) antibody
(P-Tyr) (A). The positions of Cas, Cas
SB, Cas
SD, and Fyn are indicated. The membrane was then stripped and
reblotted with anti-Cas (Cas; B) or anti-Fyn
(Fyn; C) antibodies as indicated. Cotransfected
cell lysates were also immunoprecipitated with anti-Fyn antibodies to
control for the amount of Fyn expression within the cells
(D).
[View Larger Version of this Image (36K GIF file)]
1 integrin-stimulated (
1 lanes) ARH-77 cells, and
the presence of Cas was analyzed by immunoblotting. To control for the
integrin-mediated Cas tyrosine phosphorylation, anti-phosphotyrosine
immunoprecipitations from the same samples were also analyzed. Fig.
4A shows that Cas did not bind to GST protein alone. In
contrast, Cas that was derived from stimulated cells was able to bind
to the GST-Fyn SH2 domain, whereas Cas from both unstimulated and
stimulated cells bound to the GST-Fyn SH3 domain. In this experiment,
Cas was seen to migrate as 105- and 130-kDa bands as described
previously (24), with the main increased phosphorylation in the 130-kDa
form. Hyperphosphorylated Cas precipitated with anti-phosphotyrosine
antibody or GST-Fyn SH2 protein resolved with a slower migration (27).
The membrane was reprobed with anti-GST antibody to show that
comparable amounts of GST fusion proteins were used to precipitate Cas
(Fig. 4B). These results indicate that the Fyn SH3 domain
binds to Cas in vitro and that integrin-stimulated
phosphorylation of Cas creates a binding site for the Fyn SH2
domain.
Fig. 4.
Interaction of Cas in vitro with
Fyn SH2 and SH3 domains. ARH-77 cells were unstimulated (0 lanes) or stimulated for 15 min with cross-linked anti-1
integrin antibody (
1 lanes), and cell lysates were either
immunoprecipitated with anti-Tyr(P) antibody (P-Tyr) or
precipitated with the following GST fusion proteins as indicated: GST
alone, the GST-Fyn SH2 domain, and the GST-Fyn SH3 domain. Eluates were
immunoblotted with anti-Cas antibody (Cas; A),
stripped, and reblotted with anti-GST antibodies (GST;
B).
[View Larger Version of this Image (46K GIF file)]
1
integrin-stimulated ARH-77 cells were subjected to an in
vitro kinase assay with or without the tyrosine phosphatase
inhibitor sodium vanadate (Fig. 5A). The
tyrosine phosphorylation of Cas was markedly increased with the
addition of sodium vanadate in the kinase assay. These results indicate
that a tyrosine phosphatase activity is associated with Cas
immunoprecipitates. Because the tyrosine phosphatase SHP2 has been
shown recently to associate through its SH2 domains with the
Cas-related Cas-L (HEF1) protein (25), we tested whether SHP2 also
associated with Cas. Unstimulated or
1 integrin-stimulated ARH-77
cells were immunoprecipitated with anti-Cas (Cas) or control (Ct) antibodies and immunoblotted with anti-SHP2 antibody
(Fig. 5B). Compared with unstimulated cells and control
immunoprecipitates, a 72-kDa band reactive with anti-SHP2 antibody was
clearly increased in Cas complexes isolated from
1
integrin-stimulated cells. ARH-77 cells were then
1
integrin-stimulated for 0, 2, 5, and 15 min, and cell lysates were
immunoprecipitated with anti-Cas antibody (Fig. 5C). Cas
quantification was comparable at 2, 5, and 15 min and showed increased
tyrosine phosphorylation, which correlated with Cas-SHP2 complex
formation, which was increased at the 5- and 15-min time points. These
results suggest that similar to HEF1/Cas-L, SHP2 associates with Cas in
an integrin-regulated manner. Therefore, SHP2 is likely to be
associated with the phosphatase activity observed in
vitro and might play a role in the in vivo control
of Cas dephosphorylation.
Fig. 5.
In vivo association of SHP2 tyrosine
phosphatase with Cas following integrin ligation. A, ARH-77
cells were stimulated for 15 min with anti-1 integrin antibody plus
rabbit anti-mouse Ig, and cell lysates were immunoprecipitated
(Ip) with anti-Cas antibody (Cas). Cas
immunoprecipitates were then subjected to an in vitro kinase
assay in the absence (
) or presence (+) of Na3VO4 and analyzed by Western blotting with
anti-Tyr(P) antibody (P-Tyr). The same membrane were
stripped and reblotted with anti-Cas antibody as indicated.
B, 15-min
1 integrin-stimulated ARH-77 cells (
1
lanes) or unstimulated cells (0 lane) were
immunoprecipitated with anti-Cas antibody or an irrelevant control
antibody (Ct) and analyzed by Western blotting with
anti-Tyr(P) antibody (left panel). The membrane was then
stripped and reblotted with anti-Cas or anti-SHP2 antibodies
(right panels) as indicated. The position of SHP2 is
indicated by an arrow. C, ARH-77 cells were
stimulated as indicated with anti-
1 integrin antibody and lysed as a
function of time. Anti-Cas immunoprecipitates were analyzed with
anti-Tyr(P) antibody (left panel). The membrane was then
stripped and reblotted with anti-Cas or anti-SHP2 antibodies
(right panels) as indicated. The position of SHP2 is
indicated by an arrow.
[View Larger Version of this Image (46K GIF file)]
-kinase (51).
stimulates its tyrosine
phosphatase activity (52). Two other protein-tyrosine phosphatases,
namely PTP-1B and PTP-PEST, can also associate with Cas (33, 38) and
might participate in the Cas-associated phosphatase activity.
1 integrin ligation, whereas HEF1 is the major
phosphorylated substrate in normal tonsillar B cells and other B cell
lines. It is unknown why Cas or HEF1 is the favored substrate in
certain cells, although this may be related to the compartmentalization
and activation of specific kinases during integrin-induced cytoskeleton
reorganization.
*
This work was supported in part by National Institutes of
Health Grants CA55207 and CA66996, American Cancer Society Grant DHP-145, and a fellowship from the Lymphoma Foundation of America (to
S. N. M.).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.
**
To whom correspondence and reprint requests should be addressed:
Div. of Hematologic Malignancies, Dana-Farber Cancer Inst., 44 Binney
St., Boston, MA 02115. Tel.: 617-632-3441; Fax: 617-632-5167; E-mail:
arney_freedman{at}dfci.harvard.edu.
1
The abbreviations used are: mAb, monoclonal
antibody; GST, glutathione S-transferase; HA,
hemagglutinin.
2
Astier, A., Manié, S. N., Law, S. F.,
Canty, T., Hagheyeghi, N., Druker, B. J., Salgia, R., Golemis, E. A.,
and Freedman, A. S. (1997) Leukemia Lymphoma, in
press.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.