(Received for publication, September 6, 1995; and in revised form, September 25, 1995)
From the
The Philadelphia chromosome translocation generates a chimeric
oncogene, BCR/ABL, which causes chronic myelogenous leukemia
(CML). In primary neutrophils from patients with CML, the major novel
tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein
which has an overall homology of 60% to CRK, the human homologue of the
v-crk oncogene product. Anti-CRKL immunoprecipitates from CML
cells, but not normal cells, were found to contain p210 and c-ABL. Several other phosphoproteins were also detected
in anti-CRKL immunoprecipitates, one of which has been identified as
paxillin, a 68-kDa focal adhesion protein which we have previously
shown to be phosphorylated by p210
. Using
GST-CRKL fusion proteins, the SH3 domains of CRKL were found to bind
c-ABL and p210
, while the SH2 domain of CRKL
bound to paxillin, suggesting that CRKL could physically link
p210
to paxillin. Paxillin contains three
tyrosines in Tyr-X-X-Pro (Y-X-X-P)
motifs consistent with amino acid sequences predicted to be optimal for
binding to the CRKL-SH2 domain (at positions Tyr-31, Tyr-118, and
Tyr-181). Each of these tyrosine residues was mutated to a
phenylalanine residue, and in vitro binding assays indicated
that paxillin tyrosines at positions 31 and 118, but not 181, are
likely to be involved in CRKL-SH2 binding. These results suggest that
the p210
oncogene may be physically linked
to the focal adhesion-associated protein paxillin in hematopoietic
cells by CRKL. This interaction could contribute to the known adhesive
defects of CML cells.
Chronic myelogenous leukemia (CML) ()is characterized
by the production of an active tyrosine kinase fusion protein,
p210
. In cell lines and primary cells,
p210
translocates to the cytoskeleton (1) and causes the tyrosine phosphorylation of several cellular
proteins, including the SH2-SH3-SH3 adaptor protein
CRKL(2, 3) . In cell lines either derived from
patients with advanced phase CML or generated by transfecting the BCR/ABL oncogene, there are a number of cellular proteins
which are constitutively tyrosine-phosphorylated, such as
p120
(4) ,
p120
(5) ,
p52
(6) ,
p93
(7) ,
p95
(8) ,
p68
(9) , and
p72
(10) . In contrast, in the early
(stable) phase of CML, there are only a few proteins which either
interact with BCR/ABL or are phosphorylated by BCR/ABL(3) . In
earlier studies, we and others identified a 39-kDa tyrosine
phosphoprotein complexed with BCR/ABL in CML stable phase cells as CRKL (2, 3, 11) .
The CRKL protein has an overall homology of 60% to c-CRK, the human homologue of the v-crk oncogene(12) . v-crk is the oncogene in the CT10 avian retrovirus and has a deletion of the C-terminal SH3 domain(13, 14) . v-Crk, c-CRK, and CRKL each have one SH2 domain, and two SH3 domains, without other known functional motifs (13) . The functions of c-CRK and CRKL in normal signaling are unknown, although recent studies have linked c-CRK to signaling in normal T-cells(15) . Since several proteins in v-Crk-transformed cells are heavily phosphorylated on tyrosyl residues, it has been suggested that v-Crk and c-Crk may serve as regulatory subunits of a tyrosine kinase (16) . Recently, c-Abl has been identified as a possible Crk-associated tyrosine kinase(17) . c-Abl binds in vitro to the first Crk SH3 domain and phosphorylates Crk on tyrosine 221. The phosphorylation on Crk (Tyr-221) creates a binding site for the Crk SH2 domain, possibly inhibiting its binding to other proteins(13, 17) . It is not known if Crk and Abl interact in vivo. Interestingly, despite the structural similarities of c-CRK and CRKL and the apparent interaction of v-Crk and c-Abl, our preliminary studies indicated that CRKL is phosphorylated in CML cells, while c-CRK is not.
In an
effort to determine if CRKL functions to link p210to other cellular proteins, we looked for proteins which
coprecipitated with CRKL in p210
-containing
cell lines. A 68-kDa protein precipitating with CRKL was identified as
paxillin, a focal adhesion protein we have previously shown to be
phosphorylated by p210
(9) . The
interaction between p210
, CRKL, and paxillin
could contribute to the pathogenesis of CML.
Figure 1:
Expression and
tyrosine phosphorylation of CRKL in myeloid cells. Whole cell lysates (W.C.L., 5 10
cells, first four lanes) or
immunoprecipitates of cell lysates (I.P., 20
10
cells, last eight lanes) with anti-phosphotyrosine (p-Tyr
I.P.) or immunoprecipitates with control antibody against
-interferon, 3c11c8 (Cntrl I.P.) were processed as
described under ``Materials and Methods'' and applied to a
gradient SDS-PAGE gel (6-12%) and transferred to Immobilon-P
membrane. The membrane was immunoblotted with anti-CRKL antibody. The
cells used are unstimulated 32Dcl3 cells (32D(-)),
32Dcl3 cells stimulated with IL-3 (32D(+)),
32D.p210
.26 cells (32D.p210), and K562 cells (K562). Molecular masses are shown in
kilodaltons.
Figure 2:
c-CRK is expressed but not
tyrosine-phosphorylated or associated with paxillin in myeloid cells.
Whole cell lysates (W.C.L., 5 10
cells) or
immunoprecipitates of cell lysates (20
10
) with
anti-phosphotyrosine (p-Tyr I.P.) or immunoprecipitates with
anti-paxillin (Paxillin I.P.) were processed as described.
Membrane was immunoblotted with anti-CRK antibody. Shown are the bands
for CRK-I and CRK-II (c-CRK). The cells used are unstimulated 32Dcl3
cells (32D(-)), 32Dcl3 cells stimulated with IL-3 (32D(+)), 32D.p210
.26 cells (32D.p210), and K562 cells (K562). Molecular masses
are shown in kilodaltons.
Figure 3:
CRKL binds multiple
tyrosine-phosphorylated proteins in myeloid cells containing BCR/ABL.
Whole cell lysates (W.C.L., 5 10
cells) or
immunoprecipitates of cell lysates (CRKL I.P., 20
10
cells) with anti-CRKL rabbit polyclonal antibody were
processed as described. The membrane was immunoblotted with
anti-phosphotyrosine (4G10) antibody. An arrow marks the band
at 68 kDa. The cells used are unstimulated 32Dcl3 cells (32D(-)), 32Dcl3 cells stimulated with IL-3 (32D(+)), 32D.p210
.26 cells (32D.p210), and K562 cells (K562). Molecular masses
are shown in kilodaltons.
Figure 4:
CRKL binds paxillin in BCR/ABL-containing
myeloid cells. A, CRKL coimmunoprecipitates with paxillin in
BCR/ABL expressing cells. Whole cell lysates (W.C.L., 5
10
cells) or immunoprecipitates of cell lysates (15
10
cells) with control antibody using normal rabbit
pre-immune serum (Cntrl. I.P.) or anti-CRKL rabbit polyclonal
antibody (CRKL I.P.) were processed as described. The membrane
was immunoblotted with anti-paxillin antibody. The cells used are
unstimulated 32Dcl3 cells (32D(-)), 32Dcl3 cells
stimulated with IL-3 (32D(+)),
32D.p210
.26 cells (32D.p210), and K562 cells (K562). Molecular mass is shown in kilodaltons. B,
paxillin immunoprecipitates with CRKL in BCR/ABL expressing cells.
Immunoprecipitates of cell lysates (15
10
cells)
with anti-paxillin antibody were applied to a gradient gel and
transferred. The membrane was probed with anti-CRKL antibody. The cells
used are unstimulated 32Dcl3 cells (32D(-)), 32Dcl3
cells stimulated with IL-3 (32D(+)),
32D.p210
.26 cells (32D.p210), and K562 cells (K562).
The possible association of CRKL and paxillin was also investigated in freshly isolated, primary neutrophil samples from seven patients with CML and compared with neutrophils from four normal subjects (Fig. 5). Again, anti-CRKL immune complexes contained paxillin in all CML stable phase neutrophil samples tested, but not when neutrophils from normal individuals were examined. Since the neutrophils from the normal subjects were isolated in a quiescent state, we also stimulated normal neutrophils with the potent neutrophil-activating cytokine GM-CSF (10 ng/ml for 10 min) prior to repeating the immunoprecipitation experiments. GM-CSF stimulation did not induce CRKL and paxillin coprecipitation in normal neutrophils (Fig. 5). In contrast, paxillin coprecipitated with CRKL both before and after GM-CSF stimulation of CML neutrophils. After stimulation with GM-CSF, CRKL coimmunoprecipitated with both the slower and faster migrating forms of paxillin (phosphorylated forms of paxillin have slower migration(9) ). These results suggest that the interaction of CRKL and paxillin in myeloid cells is restricted to cells expressing an active BCR/ABL protein.
Figure 5:
CRKL binds to paxillin in neutrophils from
stable phase CML but not in normal neutrophils. A, CRKL
coimmunopreciptates with paxillin in CML neutrophils.
Immunoprecipitates of neutrophil lysates (20 10
cells) with anti-CRKL rabbit polyclonal antibody were applied to
a gradient gel and transferred. The membrane was probed with
anti-paxillin antibody. The lower panel shows the same
membrane stripped and reprobed with anti-CRKL antibody. IgH represents the immunoglobulin heavy chain. Shown are neutrophil
lysate immunoprecipitates from two different normal subjects (lanes
1 and 2) and four different CML stable phase subjects (lanes 3-6). B, GM-CSF stimulation of
neutrophils and coimmunoprecipitation of CRKL with paxillin.
Immunoprecipitates of neutrophil lysates unstimulated (-) or
stimulated with GM-CSF (10 ng/ml, 10 min, +) with anti-CRKL rabbit
polyclonal antibody were applied to a gradient gel and transferred. The
membrane was probed with anti-paxillin antibody. Shown are neutrophil
lysate immunoprecipitates from two different normal subjects (lanes
1 and 2) and three different CML stable phase subjects (lanes 3-5).
Figure 6:
Binding of CRKL subdomains to
tyrosine-phosphorylated proteins in BCR/ABL expressing 32Dcl3 cells.
Precipitations with various GST fusion proteins of CRKL and its
subdomains with 32D.p210.26 cell lysates were
processed as described. The top panel shows Western blot with
anti-phosphotyrosine antibody (4G10). The same membrane was stripped
and reprobed with anti-ABL antibody (middle panel) and
thereafter re-stripped and probed with anti-paxillin antibody (lower panel). Shown are precipitations with GST protein
alone, GST-CRKL (full length), GST-CRKL-SH2, GST-CRKL-SH3(N)-SH3(C),
and GST-CRKL-SH2-SH3(N). Molecular mass is shown in
kilodaltons.
Figure 7: In vitro binding showing amino acid tyrosines at positions 31 and 118, but not 181, of paxillin are required to bind to the SH2 domain of CRKL. Full-length paxillin (wild type, W.T.) and tyrosine to phenylalanine mutants at amino acid positions 31 (Y31F), 118 (Y118F), or 181 (Y181F) were all expressed in the vector pGEX-2TK. Fusion proteins were isolated as described under ``Materials and Methods'' and thereafter tyrosine-phosphorylated by v-Abl in vitro. The phosphorylated protein product was then thrombin-cleaved and applied to a gradient SDS-PAGE gel (A), transferred, and immunoblotted with anti-paxillin. The same amount of phosphorylated thrombin-cleaved product was then precipitated with 1 µg of SH2-CRKL-GST fusion protein and applied to another SDS-PAGE gradient gel, transferred, and immunoblotted with anti-paxillin (B).
BCR/ABL is a unique tyrosine kinase oncogene which
transforms hematopoietic cells in vivo and in vitro,
but does not effectively transform many nonhematopoietic cell lines.
The chimeric oncoprotein p210 produced from the
Philadelphia chromosome is known to bind the actin cytoskeleton through
a conserved C-terminal domain in ABL, and this interaction is believed
to be important for
transformation(1, 24, 25) . It has been
suggested that once p210
translocates to the
cytoskeleton, it recruits signaling proteins which result in aberrant
regulation of adhesion, proliferation, and viability(26) .
One of the potentially most interesting proteins that interact with
p210 is CRKL. Previous studies from three different
laboratories have identified CRKL as a major tyrosine-phosphorylated
protein in primary cells or cell lines derived from patients with
stable phase CML(2, 3, 11) . The CRKL gene
was initially identified independently in the process of generating a
long range physical map of the region between the BCR gene and
the centromere of chromosome 22q11(12) . An exon of an unknown
gene was found to encode a possible SH2 domain, and the complete cDNA
sequence revealed a novel crk-like gene, termed CRKL.
The CRKL gene product has about 60% amino acid identity to c-Crk. CRKL,
like c-CRK, has an SH2 domain at the N terminus of the protein, and two
tandem SH3 domains at the C terminus, with no other known functional
domains(13) . The structural similarity between the SH2 and SH3
domains of CRKL and c-CRK suggests that they could interact with an
overlapping set of proteins(27) . However, in vivo,
c-CRK and CRKL may or may not have related functions, and it is largely
unknown even if they are in the same subcellular compartment(s).
The
finding that CRKL is a prominent substrate for the BCR/ABL tyrosine
kinase and coprecipitates with p210, suggested that
it could function to link BCR/ABL to other cellular signaling proteins.
At present, however, very few binding partners are known for either
c-CRK or CRKL. Interestingly, Feller et al.(17) have
recently showed that both v-Crk and c-Crk could bind in vitro to c-Abl. This interaction was shown to occur at least in part
through the SH3 domains of v-Crk or c-Crk, and possible proline-rich
Crk-SH3 binding sites have been identified in c-Abl. In this study, we
looked for CRKL-associated proteins, in addition to p210
and c-ABL, in cells transformed by BCR/ABL and found
that CRKL coprecipitated with several additional cellular proteins. In
the current study, one of these proteins has been identified as the
focal adhesion protein paxillin. In vitro binding studies
indicated that CRKL bound to paxillin predominantly through the
CRKL-SH2 domain, but bound to p210
through the
CRKL-SH3 domain, the later finding confirming earlier
studies(20) . Taken together, these results suggest that CRKL
may indeed function as an adaptor protein, linking p210
to paxillin in CML cells, but not in untransformed cells. In
support of this hypothesis, we have shown that a small amount of
paxillin coprecipitates with p210
and c-ABL in
lysates from CML cell lines, but not from untransformed cell lines.
These immune complexes also contain CRKL.
The interaction of
p210 with paxillin is not unanticipated. Previous
studies have shown that p210
binds to actin in
transformed fibroblasts(1) , and we have recently shown that
p210
in hematopoietic cells is localized to punctate
cytoskeletal structures that contain paxillin and vinculin, two
proteins characteristic of focal adhesions(28) . Paxillin was
originally identified by a monoclonal antibody produced by Glenney and
Zokas (29) during a blind screen for potential substrates of
the v-Src tyrosine kinase. A cDNA encoding human paxillin has recently
been cloned by our group and predicts the presence of several
protein-protein interaction domains in this protein(9) . The
C-terminal half of paxillin is composed of four tandem LIM domains. A
number of transcription factors such as rhombotin-I and -II have LIM
domains(30) , and at least two other cytoskeletal proteins also
have LIM domains, zyxin, and cysteine-rich protein. These latter two
proteins have been shown to bind to each other through their LIM
domains, suggesting that LIM domains are capable of mediating protein
interactions(31) . Paxillin also contains a proline-rich domain
which is a potential binding site for SH3-containing proteins. The
binding sites in paxillin for talin, vinculin, and p125
have been partially localized. Paxillin has also been shown to
bind to the SH2 domain of v-Crk in vitro, and recent studies
indicate that phosphopeptides with pY-X-X-P (where pY
indicates the phosphorylated tyrosine residue) motifs can bind to
CRK-SH2 domains(23, 32, 33) . Human paxillin
has three of these motifs at tyrosine positions 31, 118, and 181.
Schaller and Parsons (33) have recently shown that tyrosines at
positions 31 and 118 of paxillin may be important in binding the
Crk-SH2 domain.
We investigated the binding of CRKL-SH2 to paxillin in vitro using wild type and tyrosine mutants of paxillin. Each of the tyrosines with the Y-X-X-P motif (at tyrosines position 31, 118, and 181) in paxillin was mutated to phenylalanine for in vitro binding assays. Decreased binding of CRKL-SH2 to paxillin was observed with mutant tyrosines at positions 31 and 118, but not 181. It is not clear why the data suggest that both tyrosine 31 and 118 need to be intact for binding. These two tyrosines could both be important in binding either a single CRKL protein or two different CRKL proteins. Overall, CRKL could link BCR/ABL and paxillin and thereby contribute to the adhesion defects of CML progenitor cells(34, 35) .
The mechanism of transformation of
avian cells by v-crk may have some parallels in the
transformation of hematopoietic cells by BCR/ABL. v-Crk
activates a cellular tyrosine kinase, now believed to be
c-Abl(17, 36) . v-Crk has lost the C-terminal SH3
domain through deletion and with it, a potentially important
phosphorylation site at Tyr-221 of c-CRK. It has been suggested that
Tyr-221 is phosphorylated in resting, nontransformed cells and forms a
binding site for the c-CRK-SH2 domain(13) . This intramolecular
interaction would be predicted to make the SH2 domain unavailable for
binding to other cellular phosphoproteins and could also potentially
interfere with the availability of one or both of the c-CRK-SH3
domains. In contrast, the SH2 domain of v-Crk may constitutively be
available for binding, while the SH3 domain interacts with one or more
proline-rich domains in c-ABL, linking c-ABL to other cellular
proteins. In BCR/ABL-transformed cells, CRKL coprecipitates
with p210, and as shown here, binds p210
through one or both CRKL-SH3 domains. Since anti-CRKL immune
complexes contain a number of proteins in addition to p210
and CRKL, the SH2 domain of CRKL appears to be available for
binding. In this study, we have identified one of these binding
proteins as paxillin. Interestingly, we did not detect tyrosine
phosphorylation of either CRKL or CRK in resting, untransformed,
myeloid cell lines, and it is not yet known if the CRKL-SH2 domain will
bind through an intramolecular interaction with the tyrosine residue
comparable with Tyr-221 of c-CRK (Tyr-207 of CRKL). Also of interest
was the finding that CRK-I and -II were not detectably
tyrosine-phosphorylated in cells transformed by BCR/ABL.
Interestingly, paxillin is also known to be prominently phosphorylated
on tyrosine residues in cells transformed by
v-crk(23) , and it is quite possible, based on the
current studies, that paxillin is linked to c-Abl by v-Crk, functioning
as an adaptor protein in v-crk-transformed cells.
Although most of the studies reported here were performed in cell lines transformed by BCR/ABL, the coprecipitation of CRKL and paxillin was confirmed by studying primary neutrophils from patients with CML stable phase. Paxillin and CRKL were found to coimmunoprecipitate in lysates from CML neutrophils but not from normal neutrophils. This is noteworthy since there are only a few proteins in primary CML cells which contain more phosphotyrosine than in normal cells, and CRKL is one of the two most prominent of such proteins(3) . Further studies will be necessary to determine if these interactions lead to aberrant adhesive properties or other biological effects.
The available data suggest a model in which
p210 in the cytoskeleton binds to the CRKL-SH3
domain. The SH2 domain of CRKL is free to interact with other proteins
such as paxillin. The tyrosines at positions 31 and 118 of paxillin
appear to be important in binding to the CRKL-SH2 domain. This trimeric
complex may be able to transduce signals which are normally regulated
by either integrin or growth factor receptor activation. One testable
hypothesis is that p210
mimics or interferes with the
signaling normally induced by integrin activation. There are multiple
potential biological consequences of such interactions, including
altering adhesive properties mediated by integrins, and it has been
shown previously that CML cells have characteristically decreased
adhesion to fibronectin(34, 35) . Also, interfering
with integrin signaling could have effects on cell viability. For
example, in some hematopoietic cells, it has been shown that adherence
to fibronectin leads to increased apoptosis(37) . Interference
with the appropriate signaling pathway could therefore lead to
resistance to apoptosis, another property of CML cells. We suggest that
the interaction of p210
with paxillin, through CRKL,
could contribute to these signaling abnormalities.