(Received for publication, June 8, 1995; and in revised form, July 12, 1995)
From the
The deregulated tyrosine kinase activity of the Bcr/Abl protein has been causally linked to the development of Philadelphia (Ph) chromosome-positive leukemia in mice and man. Abnormally tyrosine-phosphorylated substrates of the Bcr/Abl kinase in Ph-positive cells are likely to contribute to leukemogenesis by interfering with normal signal transduction pathways. We have previously shown that the adaptor molecule Crkl is a major in vivo substrate for the Bcr/Abl tyrosine kinase, and it is thought to connect Bcr/Abl with downstream effectors. In the current study, a tyrosine-phosphorylated protein with a molecular mass of approximately 120 kDa was identified which binds only to the Crkl Src homology 2 (SH2) domain in cells, including Ph-positive patient material, containing an active Bcr/Abl protein. We demonstrate here that this protein is Cbl, originally discovered as an oncogene which induces B-cell and myeloid leukemias in mice. The Crkl SH2 domain binds specifically to Cbl. The Src homology 3 (SH3) domains of Crkl do not bind to Cbl, but do bind Bcr/Abl. These findings suggest the existence of a trimolecular complex involving Bcr/Abl, Crkl, and Cbl and are consistent with a model in which Crkl mediates the oncogenic signal of Bcr/Abl to Cbl.
It is generally accepted that the Bcr/Abl protein is responsible
for the development of chronic myeloid leukemia (CML) ()and
a subset of acute lymphoblastic leukemia (ALL). Leukemic cells of such
patients have a common molecular defect. This is the Philadelphia (Ph)
chromosome, a hybrid chromosome consisting of fused parts of
chromosomes 9 and 22, which produces a chimeric Bcr/Abl protein at the
breakpoint on chromosome 22. The Abl segment of this protein has a
deregulated tyrosine kinase activity (reviewed in (1) and (2) ).
Proteins which can be directly or indirectly
tyrosine-phosphorylated by Bcr/Abl include p93,
p160
, p120
, p68
,
p95
, p52
, p46
,
p67
, p120
, p190, and
p62(3, 4, 5, 6, 7, 8, 9, 10) .
Substrates of Bcr/Abl which are likely to be important to the
leukemogenic process should be expressed in hematopoietic cell types
and should be abnormally tyrosine-phosphorylated. The adaptor molecule
Crkl meets these criteria(11) .
Crkl is clearly expressed
in hematopoietic cells but is not constitutively
tyrosine-phosphorylated in bone marrow ()or peripheral
blood(11, 12, 13) . In addition, stimulation
of neutrophils with GM-CSF, tumor necrosis factor, lipopolysaccharide,
12-O-tetradecanoylphorbol-13-acetate, interleukin 1,
fibroblast growth factor, epidermal growth factor, platelet-derived
growth factor, or insulin fails to evoke Crkl tyrosine phosphorylation (12, 13) . However, in the CML cell line K562, Crkl is
one of the most prominent tyrosine-phosphorylated
proteins(14) , and we have shown that it complexes with and is
tyrosine-phosphorylated by Bcr/Abl(14) . Significantly, Crkl is
only tyrosine-phosphorylated in peripheral blood cells of Ph-positive
patients with an active Bcr/Abl
protein(11, 12, 13) , which suggests a
distinctive role for this adaptor in leukemogenesis.
Crkl consists
of an SH2 and two SH3 domains in the absence of any catalytic
domains(15) . Adaptor proteins are thought to link tyrosine
kinases with downstream effectors, and Crkl could be an important
mediator of the leukemogenic activity of Bcr/Abl. Since SH2 and SH3
domains serve as docking sites for other proteins, it is critical to
identify the binding partners of Crkl in Ph-positive cells. In the
current study, we have identified a tyrosine-phosphorylated p120
protein which binds the Crkl SH2 domain but not its SH3 domains.
Binding is restricted to cells containing a Bcr/Abl protein and
apparently is the result of Bcr/Abl kinase activity. The identification
of p120 as p120, originally isolated as an oncogene
associated with myeloid and lymphoid leukemias in mice(16) ,
sheds light on signal transduction pathways likely to be affected in
Ph-positive leukemia.
Figure 1:
The Crkl SH2 domain
specifically binds to p120 only in cells containing an active Bcr/Abl
P210 or P190 protein. A, far-Western blot analysis using
GST-Crkl-SH2 as protein-binding probe. The presence or absence of an
active Bcr/Abl in the samples is indicated with a - or +,
respectively, beneath panel A. B, Western blot
analysis of tyrosine-phosphorylated proteins in patient material and
cell lines probed with -Tyr(P) antibodies (OSI; AB-2). Lysates
include those of the CML cell line K562, lane 1; Ph-negative
ALL patient A0018, lane 2; P210-expressing blast crisis CML
patient C1797, lane 3; Ph-positive, P190-expressing ALL
patient A0055, lane 4; P210-negative CML chronic phase patient
C2206, lane 5; P210-expressing CML chronic phase patient
C2283, lane 6, P210-expressing CML blast crisis patient C1316, lane 7; NIH 3T3 cells, lane 8; NIH 3T3 cells stably
transfected with a DNA construct encoding Bcr/Abl P210 (line 1F2), lane 9. The location of molecular weight markers is indicated
to the left, that of P190
,
P210
, and p120 to the right of the
panels.
Ph-positive (expressing P210 or P190) or -negative peripheral blood cells from leukemia patients expressed different tyrosine-phosphorylated proteins (Fig. 1B). Of these, the Crkl-SH2 domain bound almost exclusively to the p120 and only in cells expressing an active Bcr/Abl protein. This included patient samples of Ph-positive acute lymphoblastic leukemia expressing Bcr/Abl P190 (Fig. 1A, lane 4) and chronic myeloid leukemia samples expressing Bcr/Abl P210 (Fig. 1A, lanes 3, 6, and 7) (described in (11) ). Although the p120 was clearly detected in the Bcr/Abl expressing Ph-positive patient samples, the Crkl SH2 domain failed to detect the p120 in peripheral blood cells of patients not expressing Bcr/Abl (Fig. 1A, lanes 2 and 5). Similarly, the p120 was detected in involved spleens of BCR/ABL P190 transgenic mice but not in control spleens (not shown). K562, a CML cell line commonly used as a model for Ph-positive leukemia expressing P210, also contained the Crkl-SH2 domain-binding p120 phosphoprotein (Fig. 1A, lane 1).
To investigate whether
the Crkl protein binds to p120 in vivo,
tyrosine-phosphorylated proteins which associate with Crkl in K562 were
examined. Although some anti-phosphotyrosine antibodies (OSI AB-2)
detected Crkl as one of the most conspicuous phosphotyrosine-containing
proteins in K562(14) , the RC20 antibodies (Signal
Transduction) also detected additional prominent
phosphotyrosine-containing proteins and had a lower affinity for Crkl (Fig. 2, lane 1). However, only a very limited subset
of the tyrosine-phosphorylated proteins detected in K562 (Fig. 2, compare lanes 1 and 2) were
co-precipitated with Crkl. This included P210, in
agreement with previous results(14) , a group of
70-80-kDa phosphoproteins, and a p120 (Fig. 2, lane
2). We concluded that the tyrosine-phosphorylated p120 protein is
uniquely associated with Crkl in Ph-positive cells, and that this is
likely to be of relevance to leukemogenesis caused by Bcr/Abl.
Therefore, experiments were initiated to identify p120.
Figure 2:
The
Crk-associated p130 is not tyrosine-phosphorylated or in
complex with Crkl in the CML cell line K562. Lysates of K562 were
reacted directly with
-Tyr(P) antibodies (lanes 1 and 3) or with anti-Cas antibodies (lane 5). Extracts of
K562 were first immunoprecipitated with
-Crkl (lanes 2 and 7) or
-Cas (lanes 4 and 6)
antibodies, then probed with
-Tyr(P) or Cas antibodies as
indicated.
p120 is another good candidate for p120, since it
is tyrosine-phosphorylated in K562 cells(10) . However,
experiments similar to those described for p130
failed to
positively identify p120
as the p120 protein (not
shown).
p120 was originally identified as a
retrovirally transduced oncogene which causes pre-B-cell lymphomas and
myeloid leukemias in mice(16) . Endogenous Cbl is
tyrosine-phosphorylated in NIH 3T3 cells expressing v-abl and
in K562(5) , suggesting it as a candidate for p120.
p120
had a mobility very similar to that of the p120
immunoprecipitated with two different
-Crkl antisera from K562 (Fig. 3A), and both contained phosphotyrosine (Fig. 3A, top). Immunoblotting of the Crkl
precipitates with
-Cbl antisera unambiguously identified the p120
as p120
(Fig. 3A, bottom). Mice
transgenic for BCR/ABL P190 reproducibly develop lymphoblastic
leukemia/lymphoma(20) . Tyrosine-phosphorylated p120
was also co-immunoprecipitated with Crkl antisera from the
spleens of leukemic transgenic Bcr/Abl mice (not shown).
Figure 3:
p120 is in complex with Crkl
and represents the 120-kDa tyrosine-phosphorylated protein band
detectable in Crkl immunoprecipitates. A,
co-immunoprecipitation of Cbl with Crkl antisera. Lane 1,
Western blot of K562 lysate; lanes 2-5,
immunoprecipitations from extracts of K562 using Crkl preimmune (lanes 2),
-Crkl CH-16 (lanes 3),
-Crkl crkL (lanes 4), and
-Cbl (lanes 5)
antisera. The blots were reacted with the antisera shown to the right of each panel. B, immunodepletion of
p120
abrogates the detection of the Crkl-associated
120-kDa phosphoprotein. K562 lysate was subjected to two rounds of
-Cbl immunoprecipitation, and the resulting Cbl-depleted lysate (lane 2) was immunoprecipitated with either
-Crkl (lane 5) or
-Cbl (lane 6), as indicated. For
comparison, control K562 lysate (lane 1) was
immunoprecipitated with
-Crkl (lane 3) or
-Cbl (lane 4) antisera. Lysates and immunoprecipitates were blotted
and incubated with
-Tyr(P) or
-Cbl antibodies, as indicated
to the right.
In similar
reciprocal experiments, we could detect Crkl in immunoprecipitates of
K562 generated by -Cbl antibodies, although Crkl co-precipitation
was not as efficient as co-precipitation of Cbl by
-Crkl antisera.
In addition, these Cbl precipitates also contained Bcr/Abl (data not
shown). These results identify Cbl as the p120 protein.
To determine
if all of the tyrosine-phosphorylated 120-kDa protein band in Crkl
immunoprecipitates represents Cbl, K562 lysates were subjected to two
rounds of immunoprecipitation with -Cbl (Fig. 3B).
This Cbl-depleted lysate was Western-blotted directly or was
immunoprecipitated with
-Crkl or
-Cbl antisera. The two
rounds of Cbl precipitation were sufficient to devoid the K562 lysate
of tyrosine-phosphorylated Cbl (lanes 2 and 6, top and bottom), resulting also in a selective loss
of all of the tyrosine-phosphorylated 120-kDa protein detected in the
lysate and in the
-Crkl precipitate (lanes 2 and 5, top). From these results we conclude that
p120
represents virtually all of the
tyrosine-phosphorylated p120 associated with Crkl.
Figure 4:
The Crkl SH2 but not its SH3 domain stably
binds to Cbl in K562. A, far-Western blot analysis of
immunoprecipitates of Cbl. Lanes 1 and 5 contain K562
total cellular lysates. Proteins immunoprecipitated with control
nonspecific antiserum (lanes 2 and 6), -Crkl
CH-16 (lanes 3 and 7), and
-Cbl (lanes 4 and 8) were blotted and reacted with GST (left
panel) or GST-Crkl-SH2 as shown. The location of p120
and the immunoglobulin heavy chain (Ig) is as indicated. B, binding of Crkl subdomains to Cbl. Extracts in lanes
2-5 were incubated with GST (fusion) proteins as indicated.
The locations of P210
and p120
are as
shown. Blots were reacted with
-Tyr(P) antibodies (top
panel) or
-Cbl antisera (bottom panel) as
indicated.
Although the binding of
Crkl to Cbl must involve the interaction of the Crkl SH2 domain and
phosphotyrosine residues on Cbl, it was possible that Crkl could also
interact with Cbl via its SH3 domains. This possibility is illustrated
by Fyn and Lck, of which both SH2 and SH3 domains bind to Cbl from
activated T-cells(21) . To test this, bacterially expressed
GST-Crkl and GST-Crkl SH2 and SH3 domains were used to pull down
proteins from K562 extracts. The Crkl SH2 domain but not the SH3
domains precipitated the tyrosine-phosphorylated Cbl p120 protein (Fig. 4B, top and bottom panels) with
an efficiency comparable to that of the entire Crkl protein (Fig. 4B, compare lanes 3 and 4). In
agreement with previous results, the entire Crkl protein also bound to
a P210 protein which was identified as P210 (not
shown), and this interaction was mediated by Crkl SH3 domains (Fig. 4B, lane 5).
Crkl is one of the most prominent tyrosine-phosphorylated
proteins in Ph-positive patient (11) and transgenic Bcr/Abl
mouse material. Because it is non-tyrosine-phosphorylated
in normal bone marrow, this modification is leukemia-specific, and we
are in the process of investigating the effect of tyrosine
phosphorylation of Crkl on its cellular interactions. The current study
demonstrates a second level of Crkl involvement: in Ph-positive
leukemia, Crkl is bound to tyrosine-phosphorylated Cbl through its SH2
domain.
Although the phosphorylation status of Cbl has not been examined in detail in hematopoietic cell types, our results show that it is not constitutively tyrosine-phosphorylated in peripheral blood cells and are in agreement with results of others(21, 22) . We therefore suggest that the constitutive tyrosine phosphorylation of Cbl in Ph-positive cells is abnormal. Transient tyrosine phosphorylation of Cbl has been demonstrated recently in normal hematopoietic signaling events: T-cell receptor activation of Jurkat cells and GM-CSF and Epo stimulation of UT-7 cells(21, 22) . This shows that Cbl tyrosine phosphorylation is also part of normal signal transduction pathways.
The specific activity of the oncogene v-cbl in causing myeloid leukemias and lymphoblastic leukemia/lymphomas in mice also is in concordance with a defined normal role for Cbl in hematopoietic signaling. In addition, since removal of a defined region of 17 amino acids from Cbl causes it to become phosphorylated on tyrosine and to transform cells(5) , a correlation appears to exist between myeloid/lymphoid cell types, Cbl phosphorylation, and leukemogenesis.
How does Cbl become phosphorylated? Andoniou et al.(5) showed that anti-Abl antiserum is capable of co-precipitating Cbl from K562 but did not show evidence for the mechanism through which this would occur. We have previously shown that Crkl and Bcr/Abl form complexes and demonstrate here that this is mediated through the Crkl SH3 domain. In the current study, we also demonstrate Crkl and Cbl binding through the Crkl SH2 domain. Although it remains a formal possibility that each of these molecules form independent bimolecular complexes with each other (Bcr/Abl + Crkl; Bcr/Abl + Cbl; Crkl + Cbl), we favor a model in which a trimolecular complex involving Bcr/Abl, Crkl, and Cbl is formed. In the most simple version of this model, Crkl is constitutively bound to Bcr/Abl via its SH3 domain. Low level transient tyrosine phosphorylation of Cbl through normal signaling events (e.g. GM-CSF stimulation) would cause an association of Crkl and Cbl and a significant increase of tyrosine phosphorylation of Cbl by Bcr/Abl. Because the kinase activity of Bcr/Abl is deregulated, Cbl tyrosine phosphorylation would be constitutively switched on, leading to a long-lasting activation of the signaling pathways normally controlled by cytokines. Interestingly, the results of Odai et al.(22) suggest that the GM-CSF signal transduction pathway in which Cbl appears to be involved is distinct from that of Ras.
Although some models of Bcr/Abl-associated leukemogenesis invoke several distinct signaling pathways, it remains very well possible that only a single pathway leads to leukemogenesis in vivo. The similarities between the types of malignancies associated with Bcr/Abl and v-cbl in animal models suggests that the pathways of these oncogenes converge, a possibility which can be currently investigated in complementation studies.