(Received for publication, September 9, 1996, and in revised form, February 14, 1997)
From the Division of Hematologic Malignancies,
Dana-Farber Cancer Institute, Boston, Massachusetts 02115 and the
§ Institute for Cancer Research, Fox Chase Cancer Center,
Philadelphia, Pennsylvania 19111
CRKL is an SH2-SH3-SH3 adapter protein that is a
major substrate of the BCR/ABL oncogene. The function of CRKL in normal
cells is unknown. In cells transformed by BCR/ABL we have previously shown that CRKL is associated with two focal adhesion proteins, tensin
and paxillin, suggesting that CRKL could be involved in integrin
signaling. In two hematopoietic cell lines, MO7e and H9, we found that
CRKL rapidly associates with tyrosine-phosphorylated proteins after
cross-linking of 1 integrins with fibronectin or anti-
1 integrin
monoclonal antibodies. The major tyrosine-phosphorylated CRKL-binding
protein in the megakaryocytic MO7e cells was identified as
p120CBL, the cellular homolog of the v-Cbl oncoprotein.
However, in the lymphoid H9 cell line, the major
tyrosine-phosphorylated CRKL-binding protein was p110HEF1. In
both cases, this binding was mediated by the CRKL SH2 domain. Interestingly, although both MO7e and H9 cells express p120CBL
and p110HEF1,
1 integrin cross-linking induces tyrosine
phosphorylation of p120CBL (but not p110HEF1) in MO7e
cells and of p110HEF1 (but not p120CBL) in H9 cells. In
both cell types, CRKL is constitutively complexed to C3G, SOS, and
c-ABL through its SH3 domains, and the stoichiometry of these complexes
does not change upon integrin ligation. Thus, in different cell types
CRKL and its SH3-associated proteins may form different multimeric
complexes depending on whether p120CBL or p110HEF1 is
tyrosine-phosphorylated after integrin ligation. The shift in
association of CRKL and its SH3-associated proteins from
p120CBL to p110HEF1 could contribute to different
functional outcomes of "outside-in" integrin signaling in different
cells.
Integrins play a role in cell movement and apoptosis and also act
as costimulatory molecules. The integrin receptors are /
heterodimeric transmembrane proteins that mediate cell-cell or cell-extracellular matrix interactions. Activation of integrin receptors leads to the formation of focal adhesions where integrin cytoplasmic domains are connected with actin-containing cytoskeleton components, thereby providing a link between the extracellular environment and intracellular elements. Tyrosine phosphorylation of
cellular proteins is an early event after integrin receptor stimulation
and is believed to initiate a series of signaling events involving
protein-protein interactions leading to changes in viability,
proliferation, or other functions in various cells (1, 2). One tyrosine
kinase that is localized to the focal adhesion and is activated after
integrin ligation has been identified as p125FAK (3). This
kinase may have a negative regulatory role in the formation of focal
adhesions (4). Also, another non-receptor tyrosine kinase (related
adhesion focal tyrosine kinase) has been found to be partially
associated with the actin cytoskeleton and is activated by integrins
(5, 6). Recently, investigators have begun to identify the major
cellular proteins that are tyrosine-phosphorylated after cross-linking
of integrins by ligands. For example, p120CBL is
tyrosine-phosphorylated after
1 integrin ligation in the human B
cell line Nalm-6 and after
1 and
2 integrin ligation in the
megakaryoblastic cell line MO7e (7, 8).
In many signal transduction pathways activated by tyrosine kinases, adapter molecules have been shown to play a key role in mediating transient protein-protein interactions. We have previously shown that the adapter protein CRKL is associated with the focal adhesion protein paxillin in cells transformed by the oncogenic tyrosine kinase BCR/ABL (9). CRKL is a 39-kDa protein with one SH2 and two SH3 domains (10). CRKL has a high homology to c-CRK-II and belongs to the CRK family of adapter proteins, which includes v-CRK, c-CRK-II, and c-CRK-I (11-13). The CRK and CRKL SH3 domains have been shown to specifically bind to c-ABL, SOS, or C3G (14-19). The SH2 domain of CRKL has been shown to bind to p120CBL in cells transformed by oncogenic tyrosine kinases (19, 20), and CRKL binds p120CBL inducibly after epidermal growth factor receptor stimulation (21) or after T cell receptor stimulation (22).
In this study, we examined the involvement of CRKL in signal
transduction pathways activated after cross-linking of 1 integrins in two hematopoietic cell lines, the megakaryoblastic cell line MO7e
and a T cell line, H9. In both cell lines,
1 integrin stimulation resulted in the rapid association of CRKL with a single major tyrosine-phosphorylated cellular protein. Surprisingly, however, this
protein was of a different apparent molecular mass in the two cell
lines. We found that p120CBL was the major
tyrosine-phosphorylated CRKL-binding protein in MO7e cells, and
p110HEF1 was the major tyrosine-phosphorylated CRKL-binding
protein in H9 cells. In both cases the binding was mediated through the
CRKL SH2 domain, while proteins constitutively associated with the CRKL
SH3 domain, including C3G, SOS, and c-ABL, did not appear to be
affected by
1 integrin stimulation. These results indicate that CRKL
and its associated signaling proteins can interact with more than one
signaling pathway activated by
1 integrin ligation.
The human megakaryoblastic cell line MO7e (obtained from Dr. Steve Clark, Genetics Institute, Cambridge, MA) was maintained in Dulbecco's modified Eagle's medium (Mediatech, Washington, D. C.), 10 ng/ml granulocyte-macrophage colony-stimulating factor (Genetics Institute), and 20% (v/v) fetal calf serum (PAA Laboratories Inc., Newport Beach, CA) at 37 °C with 10% CO2. The BCR/ABL-expressing MO7e cell line MO7/p210 was generated by transfection with the pGD vector containing the sequence for the p210BCR/ABL cDNA (obtained from Dr. George Daley, MIT, Cambridge, MA). For stimulation studies, MO7e cells were washed with Dulbecco's phosphate-buffered saline (DPBS)1 and deprived of growth factors for 20 h at 37 °C in serum-free medium with 1% (w/v) bovine serum albumin (Sigma). The human T cell line H9 (obtained from Dr. Jerome Ritz, Dana-Farber Cancer Institute) was maintained in RPMI 1640 (Mediatech) and 10% (v/v) fetal calf serum (PAA Laboratories Inc.) at 37 °C with 5% CO2. Starved H9 cells were prepared by washing with DPBS and were deprived of serum for 2 h at 37 °C in serum-free medium.
Stimulation of Cells and Preparation of Cellular LysatesStarved MO7e or H9 cells were first incubated for 15 min
on ice with antibodies against CD29/1 integrin (4B4, obtained from Dr. C. Morimoto, Dana-Farber Cancer Institute), CD3 (OKT3, Coulter Corp., Miami, FL), or an irrelevant antibody (3C11C8, an
anti-interferon-
murine monoclonal antibody) and then stimulated by
cross-linking using affinity-purified rabbit anti-mouse Ig (Dako Corp.,
Carpinteria, CA) at 37 °C for 10 min. For
integrin subunit
cross-linking, starved MO7e and H9 cells were incubated for 30 min on
ice with antibodies against
4 integrins (8F2 from Dr. C. Morimoto
and B5G10 from Dr. M. Hemler, Dana-Farber Cancer Institute) or against
5 integrins (2H6 from Dr. C. Morimoto and A5-PUJ2 from Dr. M. Hemler) and then stimulated by cross-linking for 20 min as described above. Either starved H9 cells or MO7e cells (washed three times in
DPBS after starvation and resuspended in Dulbecco's modified Eagle's
medium) were used for stimulation with fibronectin (Life Technologies,
Inc.) in the same fashion. Cell lysates were prepared as described
(23).
Western blotting
using a chemiluminescence technique was performed as described (23).
Immunochemical detection of tyrosine-phosphorylated proteins in Western
blots utilized monoclonal antibody 4G10 (kindly provided by Dr. B. Druker, Oregon Health Science University, Portland, OR). Polyclonal
rabbit antisera against p120CBL (Santa Cruz Biotechnology,
Santa Cruz, CA), CRKL (Santa Cruz Biotechnology), p110HEF1
(HEF1 SB) (24), and mouse monoclonal antibodies against c-ABL (AB-3
from Oncogene Science, Manhasset, NY) and CRKL (the mouse monoclonal
was generated as described elsewhere (25) and only used for Western
blotting) were used for this study. The pGEX vector containing the SH2
and SH3-SH3 domains of CRKL was obtained from Dr. J. Groffen,
Children's Hospital, UCLA, Los Angeles, CA. The GST-fusion proteins
were expressed in Escherichia coli (DH-5) by
isopropyl-1-thio-
-D-galactopyranoside induction and
isolated from sonicated bacterial lysates using glutathione-Sepharose
beads (Pharmacia Biotech Inc.) according to the manufacturer's
directions.
MO7e and H9 cells (0.5 × 106 cells/sample) were incubated with murine monoclonal
antibodies against integrin receptors including 1 (TS2/7),
2
(A2-2E10),
3 (A3-2F5),
4 (B5G10),
5 (A5-PUJ2),
6 (A6-ELE)
(all anti-
integrin receptor antibodies were obtained from Dr. M. Hemler, Dana-Farber Cancer Institute),
1 (4B4), or an irrelevant
monoclonal antibody (3C11C8) for 20 min on ice and then washed once
with DPBS. Cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse serum (Southern Biotechnology Assoc., Birmingham, AL) for an additional 20 min and subsequently washed twice in DPBS before analysis using a Coulter Epics XL flow
cytometer (Coulter Corp.) for analysis.
Using previously established techniques (26), far-Western blotting was performed as described previously (19). In brief, immunoprecipitated proteins were transferred after SDS-PAGE to Immobilon-P (polyvinylidene difluoride) membrane (Millipore) and blocked with 5% nonfat dry milk in 0.1% Tween 20 in phosphate-buffered saline, pH 7.4. The specific direct in vitro binding was evaluated by probing the membrane with GST-fusion proteins and visualized with a combination of anti-GST monoclonal antibody (Santa Cruz Biotechnology) and horseradish peroxidase-coupled anti-mouse IgG antibody by chemiluminescence.
To investigate
the potential role of CRKL in integrin signaling we looked for
tyrosine-phosphorylated proteins that coprecipitate with CRKL, since
tyrosine phosphorylation of cellular proteins is an early event
following integrin receptor ligation. To determine if CRKL associates
with tyrosine-phosphorylated proteins, we investigated two different
hematopoietic cell lines with known differences in tyrosine
phosphorylation of cellular proteins after 1 integrin stimulation.
For initial experiments we stimulated MO7e and H9 cells with
fibronectin, a natural ligand for some integrin receptors including VLA-4 (4
1) and VLA-5 (
5
1), which are the major
1
integrin receptors in MO7e cells as well as in H9 cells. In MO7e cells, fibronectin induced association of CRKL with a prominent 120-kDa tyrosine phosphoprotein (Fig. 1A, left
panel). We have previously shown that
1 integrin ligation
induces tyrosine phosphorylation of p120CBL in MO7e cells. We
therefore asked if the 120-kDa protein coprecipitating with CRKL in
MO7e cells was p120CBL. This blot was stripped, and the
phosphoprotein was identified as p120CBL by immunoblotting
(Fig. 1A, upper right panel). The same results were obtained when the immunoprecipitation and blotting antibodies were
reversed (data not shown). The lower right panel in Fig. 1A demonstrates that equal amounts of CRKL were loaded in
each lane.
In H9 cells, CRKL was also found to associate with tyrosine-phosphorylated proteins. However, a 110-kDa tyrosine-phosphorylated protein coprecipitated with CRKL after fibronectin stimulation (Fig. 1B, left panel). This protein did not react with a p120CBL antibody (data not shown). Based on its molecular mass and the presence of multiple potential CRKL SH2 binding motifs (Tyr-X-X-Pro), we examined p110HEF1 for possible coprecipitation with CRKL. The blot was stripped, and the phosphoprotein was identified as p110HEF1 by immunoblotting (Fig. 1B, upper right panel). The lower right panel in Fig. 1B demonstrates that comparable amounts of CRKL were loaded. These results demonstrate that integrin receptor activation with fibronectin can induce the formation of a CRKL-p120CBL complex in MO7e cells and a CRKL-p110HEF1 complex in H9 cells. However, we did not detect significant association of p120CBL with CRKL in H9 cells or with p110HEF1 in MO7e cells at any time points tested between 0 and 60 min (data not shown).
p120CBL and p110HEF1 Are Differentially Tyrosine-phosphorylated afterSince we observed differential association of
tyrosine-phosphorylated p120CBL and p110HEF1 with CRKL
in MO7e cells or H9 cells, respectively, we asked if these proteins
were differentially tyrosine-phosphorylated after 1 integrin
ligation in these cells. Stimulation of the megakaryocytic MO7e cells
or the T cell line H9 with a monoclonal antibody to cross-link
1
integrins induced rapid tyrosine phosphorylation of cellular proteins
compared with unstimulated cells (Fig. 2A, left panel). Mock stimulation with an irrelevant antibody
(3C11C8, an anti-interferon-
murine monoclonal antibody) did not
induce tyrosine phosphorylation (data not shown). The major
tyrosine-phosphorylated proteins in MO7e cells include proteins with
apparent molecular masses of 145, 120, 95, 70, and 40 kDa, whereas in
H9 cells two prominent proteins of 110 and 95 kDa were
tyrosine-phosphorylated. H9 cells treated with an irrelevant antibody
also did not induce tyrosine phosphorylation of cellular proteins. The
tyrosine phosphorylation pattern induced by fibronectin was virtually
identical to the
1 integrin-induced pattern.
We identified the 120-kDa protein as p120CBL in phosphotyrosine immunoprecipitations of stimulated MO7e cells but not H9 cells (Fig. 2A, middle panel). In contrast, the 110-kDa protein in the phosphotyrosine immunoprecipitation of H9 cells was found to be the recently cloned p130CAS-related protein p110HEF1 (Fig. 2A, right panel). In addition, p120CBL and p110HEF1 were also inducibly (but again selectively) tyrosine-phosphorylated with fibronectin stimulation in MO7e and H9 cells, respectively (data not shown). Interestingly, p110HEF1 is not tyrosine-phosphorylated in MO7e cells. The increased tyrosine phosphorylation of p120CBL and p110HEF1 is likely to mediate the specific interaction with CRKL after integrin cross-linking (Fig. 1). The differences in phosphorylation of p120CBL or p110HEF1 could not be attributed to differential expression of p120CBL and p110HEF1 as expression of these proteins in MO7e and H9 cells by Western blotting was comparable (Fig. 2B). In addition to p110HEF1, the antiserum to p110HEF1 recognized a 95-kDa protein in Western blot experiments (Fig. 2B, right panel). The identity of the 95-kDa protein is not known at this time. However, our preliminary data suggest that it may be the SH3 domain-containing p130CAS-related protein p95EFS/SIN (27, 28) (data not shown).
The failure to tyrosine-phosphorylate p110HEF1 in MO7e cells
and p120CBL in H9 cells could be due to defects in signaling
pathways leading to tyrosine phosphorylation of these proteins. To
address this issue, other pathways known to induce phosphorylation of
p120CBL and p110HEF1 were examined. We found
p110HEF1 in phosphotyrosine immunoprecipitates of MO7e cells
expressing the oncogenic tyrosine kinase BCR/ABL but not in
untransfected cells, demonstrating apparent phosphorylation of
p110HEF1 in response to BCR/ABL (Fig. 2C). Also,
p120CBL was found to be inducibly tyrosine-phosphorylated
after CD3 cross-linking in H9 (Fig. 2D). These data suggest
that p120CBL and p110HEF1 can be
tyrosine-phosphorylated in both cell lines by stimuli other than
1 integrin receptor cross-linking. Tyrosine-phosphorylated p120CBL and p110HEF1 were also found to be inducibly
and selectively associated with CRKL after cross-linking with
4
integrin in MO7e and H9 cells, respectively (Fig. 2E).
Cross-linking of
5 integrin in MO7e cells produced similar results;
however, the increased association of CRKL with p110HEF1 was
very small in H9 cells (data not shown). We further tested if different
signaling was due to differences in
integrin or
1 integrin
receptor expression. We found that both cell lines had comparable
expression of the
1 integrin as well as
4,
5, and
6
integrins. Expression of
1,
2, and
3 integrins was lower or
negligible (Fig. 2F). Overall these results demonstrate that similar integrin receptors can activate distinct signaling proteins in
different cell lines.
The above results
suggest the potential induction of one or more multimeric protein
complexes containing CRKL, p120CBL, or p110HEF1. The
binding of CRKL to p120CBL and p110HEF1 appears to
require tyrosine phosphorylation of these proteins. Since CRKL has one
SH2 and two adjacent SH3 domains, we sought to determine the mechanism
of CRKL binding to p120CBL and p110HEF1 using
GST-fusion proteins containing various segments of each protein. The
SH2 domain of CRKL precipitated p120CBL from lysates of
stimulated (but not unstimulated) MO7e cells (Fig.
3A). The blots were stripped and reprobed
with antibodies against c-ABL demonstrating that GST-CRKL-SH3 but not
GST-CRKL-SH2 constitutively precipitated c-ABL (Fig. 3A). We
also found constitutive coprecipitation of C3G and SOS with the ABL-SH3
domain (data not shown). Using lysates from H9 cells, the SH2 domain of
CRKL precipitated p110HEF1 after fibronectin stimulation, while
the GST-CRKL SH3 domain did not (Fig. 3B).
The in vitro GST-fusion protein precipitations with
p120CBL, c-ABL, and CRKL do not indicate if binding of the SH2
or SH3 domains is direct or indirect. We therefore used a far-Western
technique to examine possible direct in vitro interactions.
Cellular lysates from unstimulated and integrin-stimulated MO7e
cells were used for immunoprecipitations with anti-p120CBL or
anti-CRKL antibody. Fig. 3C shows that GST protein alone
does not bind to proteins in p120CBL or CRKL
immunoprecipitations. Direct binding of a single 120-kDa protein band
in CRKL immunoprecipitations using the GST-CRKL SH2 protein as a probe
was found only after
integrin ligation. This protein was identified
as p120CBL in the p120CBL immunoprecipitation and the
CRKL SH2 far-Western blot. We also observed binding of the CRKL SH3
domain to a set of proteins between 140 and 160 kDa; however, this
interaction was not changed upon
integrin ligation (data not
shown). The binding of SH2 domains to p120CBL is likely to
require phosphotyrosine, since no binding was observed to
p120CBL in lysates from unstimulated cells where tyrosine
phosphorylation of p120CBL is not induced. These results
indicate that in MO7e cells, CRKL is linked through its SH2 domain to a
pathway involving p120CBL, whereas in H9 cells CRKL is linked
to a pathway involving p110HEF1.
We also asked if integrin ligation changes any
complexes of CRKL with SH3-binding proteins. Fig. 4
demonstrates that 1 integrin ligation did not alter the
coprecipitation of CRKL with c-ABL, C3G, and SOS. The same results were
obtained when the immunoprecipitation and blotting antibodies were
reversed (data not shown). We did not observe detectable induction of
tyrosine phosphorylation of c-ABL after integrin ligation (data not
shown). These data demonstrate that integrin cross-linking does not
alter the constitutive complexes of CRKL with c-ABL, C3G, and SOS.
The biological effects of cross-linking integrins may vary widely from cell to cell, ranging from stimulation of proliferation to induction of apoptosis. When integrins are cross-linked through binding with a natural ligand such as fibronectin, a series of signaling events are initiated. This signaling is associated with the following changes in the actin cytoskeleton: formation of a cytoskeletal complex of proteins that includes actin, vinculin, talin, p125FAK, paxillin, and tensin, activation of tyrosine phosphorylation, and activation of other signal transduction pathways such as the p21RAS pathway. Overall, this outside-in signaling of integrins is likely to be an important part of the signals sent by the microenvironment to influence cell behavior (1, 2).
However, the mechanisms of outside-in signaling are not well understood. This is due in part to the complexity of studying a system with many related receptors (the integrin family) that are expressed heterogeneously on different cell types, coupled with the fact that different integrins may share the same ligand. Since the biological effects of outside-in signaling may vary widely in different cells, it is of interest to determine how integrin cross-linking in one cell may augment proliferation but induce apoptosis in another cell type. It would be anticipated that different integrins may activate different signaling pathways in the same cell and also that the same integrin could potentially activate different pathways in different cells. Despite this prediction, there are few examples of differential signaling by integrins and even fewer examples where specific integrin-activated signaling pathways have been directly linked to a biological event.
In this study we have investigated the specific role of CRKL (an
adapter protein that has one SH2 domain and two SH3 domains) in
integrin signaling as part of a larger effort to understand the
cellular functions of CRKL. During preliminary studies in the human
megakaryoblastic cell line MO7e, we had noted that after cross-linking
of 1 integrins by monoclonal antibody, CRKL was induced to bind
through its SH2 domain to a 120-kDa protein identified as
p120CBL. p120CBL was shown to be one of the most
prominently tyrosine-phosphophorylated proteins induced after integrin
activation in these cells (8) and virtually the only tyrosine
phosphoprotein coprecipitating with CRKL. However, in another
hematopoietic cell line, the T cell line H9, we noted that CRKL did not
coprecipitate with p120CBL after integrin cross-linking,
despite the fact that the H9 cell line was found to have the same
pattern of
1 integrin expression as MO7e as well as abundant
expression of p120CBL (29, 30). This unexpected result was made
more interesting by the finding that CRKL was induced to coprecipitate
with another tyrosine phosphoprotein in H9 cells, p110HEF1,
which is a signaling protein related to p130CAS. Again,
p110HEF1 was virtually the only tyrosine phosphoprotein
coprecipitating with CRKL after integrin stimulation, and the
interaction was mediated by the CRKL SH2 domain. Like p120CBL,
p110HEF1 has multiple copies of potential CRKL SH2 binding
motifs (phospho Tyr-X-X-Pro) (7, 24). These
combined observations suggest that CRKL is not only involved in
integrin-mediated outside-in signaling, it can also participate in
different pathways depending on which upstream molecule
(p120CBL or p110HEF1) is phosphorylated (probably at
the phospho Tyr-X-X-Pro motifs previously shown
to represent binding sites for CRK and CRKL SH2 domains). This provides
for the possibility of an intracellular signaling "switch" that
could couple integrin signaling to different biological effects.
In contrast to the effects of integrin-induced tyrosine phosphorylation on the binding of the CRKL SH2 domain to signaling molecules, the proteins that were bound to the CRKL SH3 domains were not affected by integrin cross-linking. The known CRKL SH3-binding proteins include c-ABL, C3G, and SOS. These proteins were first described as binding to the CRKII SH3 domain; however, we and others have shown that they also bind to the CRKL SH3 domain (14-19). SOS has known guanine-exchange factor activity for p21RAS; in contrast, C3G appears to have specific guanine exchange activity for p21RAP1. C3G does not have substrate specificity for p21RAS (31), but its substrate p21RAP1 appears to regulate, at least in part, the signal from p21RAS to the RAF kinase. C3G also shows sequence similarity to CDC25 and SOS family proteins (17) and preferentially binds to the N-terminal SH3 domain (16). The exact function of the tyrosine kinase c-ABL is unknown, although c-ABL has been shown to be involved in transcriptional activation (32) and possibly is activated in response to certain types of DNA damage (33). Interestingly, c-ABL can interact with the actin cytoskeleton through an actin binding site in its C terminus (34). During integrin signaling, c-ABL, C3G, or SOS could be linked to either p120CBL or p110HEF1 by CRKL, although no direct evidence of such multimeric proteins was demonstrated in this study.
The protooncoprotein p120CBL (for Casitas B-lineage lymphoma)
is a widely expressed 120-kDa protein. It is the cellular homolog of
v-Cbl, the oncoprotein in the CAS NS-1 retrovirus (35, 36) that induces
pre-B cell lymphomas and myelogenous leukemias in mice (37). The
p120CBL homolog Sli-1 in Caenorhabditis elegans is a
negative regulator of the epidermal growth factor receptor tyrosine
kinase homolog Let-23 (38). p120CBL is also known to be a
substrate of tyrosine kinases in response to T cell (39) and B cell
(40) activation, FC- receptor cross-linking (41, 42), and growth
factors (23, 43-45). In mammalian cells, the function of
p120CBL is not known, although several interactions with other
signaling proteins have been reported. For example, p120CBL has
been shown to associate with active phosphatidylinositol 3-kinase in
antigen receptor-stimulated cells or BCR/ABL transformed cells, and it
interacts with the SH3 domains of GRB2, NCK, or SRC kinases including
LYN and FYN (39, 42, 46). The H9 T cell line, which was derived from
the HuT 78 cell line, expresses both a full-size c-CBL protein and a
protein containing a C-terminal truncation of c-CBL (47). In H9 cells,
we observed tyrosine phosphorylation of full-length p120CBL
after CD3 stimulation, indicating that this p120CBL pathway is
intact.
p110HEF1 (for human enhancer of filamentation 1) is a tissue-specific protein first identified during cloning of human genes that induce morphological changes in Saccharomyces cerevisiae. Expression of the p110HEF1 C terminus induces pseudohyphae in S. cerevisiae. p110HEF1 shares 64% homology with p130CAS and similarly has an N-terminal SH3 domain. p110HEF1 is also a prominent substrate of oncogenic tyrosine kinases including v-ABL and may function as a docking protein. This protein is structurally related to p130CAS, it appears to be localized to the nucleus and the cell periphery (24). It is not known if p110HEF1 in mammalian cells is also involved in organization of the cytoskeleton. Interestingly, p110HEF1 has several Tyr-X-X-Pro motifs (24) that have been shown to be recognized by the CRKL SH2 domain (9, 48). This is consistent with our findings demonstrating coprecipitation of CRKL with p110HEF1.
Our data demonstrate that 1 integrin receptors in MO7e or H9 cells
are activating distinct signaling pathways. These differences are
probably not mediated through different expression of
1 integrins since we demonstrate that the major
1 integrin receptors (the fibronectin receptors
4
1 (VLA4) and
5
1 (VLA5)) are
expressed both in MO7e and H9 cells. Further, cross-linking
4
integrin chains with specific monoclonal antibodies in MO7e and H9
cells also resulted in selective phosphorylation of p120CBL and
P110HEF1, respectively. However, the
1 integrin family
consists of four known isoforms (A, B, C, and D) that have the same
extracellular domains but differ in their cytoplasmic domains. The
major isoform
1A is ubiquitously expressed but is substituted in
muscle cells by the
1D isoform and thus not expressed in
hematopoietic cells (49).
1B integrin is a minor isoform and is
coexpressed with
1A in some tissues and cells (50). The
1B
isoform might negatively regulate adhesion and mobility (51). The
1C
isoform is expressed in hematopoietic cells but does not appear to
colocalize to focal adhesions and has been shown to cause growth arrest
and inhibit DNA synthesis when transfected and expressed in fibroblasts
(52). It is possible that the cell is using these distinct signaling pathways involving p120CBL or p110HEF1 depending on the
differential expression of another regulatory signaling protein.
Interestingly, there is at least one additional pattern of CRKL-related
signaling in hematopoietic cells. We recently examined several
additional cell lines and found that after
1 integrin ligation in
the B cell line Nalm-6, CRKL binds to both tyrosine-phosphorylated
p120CBL as well as
p110HEF1.2 This suggests that the
mechanisms that activate either pathway are not mutually exclusive. The
differential activation of p120CBL and p110HEF1 could
be directly mediated through a process that leads to activation of
different tyrosine kinases that are specific for p120CBL or
p110HEF1, and we are currently investigating this
possibility.