INTRODUCTION

Several experimental mouse models have been developed to investigate the oncogenic action of Bcr/Abl, which is causative of the development of chronic myeloid leukemia (CML)¹ and Philadelphia-positive (Ph⁺) acute lymphoblastic leukemia (ALL), in vivo. Transgenic P190BCR/ABL mice reproducibly develop lymphoblastic leukemia/lymphoma involving cells of pre-B-cell origin or their progenitors (1, 2). Established transgenic mice strains expressing P190Bcr/Abl provide an unlimited source and unique opportunity for studying the signal transduction pathways affected by the Bcr/Abl oncoprotein in vivo.

The tyrosine kinase activity located in the Abl segment of Bcr/Abl is dramatically increased (3), and this is critical for its transforming capacity. Although it remains unclear which intracellular signaling pathways are crucial to the in vivo oncogenic activity of Bcr/Abl, numerous signaling proteins have been implicated by studies involving patient-derived cell lines or stably transfected cells transformed by Bcr/Abl. However, only few signaling proteins are tyrosine-phosphorylated by Bcr/Abl or in complex with Bcr/Abl in leukemic cells isolated directly from Ph⁺ patients, indicating the importance of studying the molecular mechanisms behind Bcr/Abl-induced leukemogenesis in an in vivo context. Signaling proteins shown to be affected by Bcr/Abl include p130^Cas (4), p120^CBL (5), p68^paxillin (6), Grb-2 (7), p62^Dok (8) and p39^Crkl (9-11).

The human Crkl protein has a high degree of homology with the adaptor protein Crk (12, 13). Crkl consists of an SH2 and two SH3 domains in the absence of other functional motifs. It is ubiquitously expressed and tyrosine-phosphorylated abundantly during early embryogenesis, but tyrosine phosphorylation is very restricted in adult tissues (14). We and others have demonstrated that Crkl is constitutively tyrosine-phosphorylated in CML and ALL patient material containing an active Bcr/Abl protein (e.g. chronic and blast phase samples) but not in remission-stage samples or in several types of leukemia lacking Bcr/Abl (9-11). Moreover, it is also abnormally tyrosine-phosphorylated in leukemic samples of P190- and P210BCR/ABL transgenic mice (14). This indicates that Crkl is a signaling protein likely to be of significance in vivo to the development of Ph⁺ leukemia.

The N-terminal SH3 domain of Crkl interacts specifically with proline-rich sites present in the proto-oncoprotein Abl and in Bcr/Abl (15, 16), while the Crkl SH2 domain recognizes specific phosphotyrosine binding sites in its target proteins. We and others have identified p120^Cbl as a 120-kDa tyrosine-phosphorylated protein, that specifically interacts with the Crkl SH2 domain in cells expressing the Bcr/Abl protein, including Ph⁺ patient material (5, 17). Furthermore, Crkl is also associated through its SH2 domain with phosphorylated cytoskeletal proteins paxillin (6) and Crk-associated substrate p130^Cas (4) in Bcr/Abl-transformed cell lines and in Ph⁺ patient samples. Previously, analogous interactions have been described for family members Crk and v-Crk, which can form stable complexes with phosphorylated p130^Cas and paxillin through their SH2 domains (18-23). Both paxillin and p130^Cas are normally tyrosine-phosphorylated after integrin-mediated cell adhesion, are localized to focal adhesions, and are constitutively phosphorylated in v-src- and v-crk-transformed cells (18-25). Thus, Crkl might link different signaling proteins to Bcr/Abl through its SH2 and SH3 domains. In addition, these data implicate a role for cytoskeleton reorganization and integrin-mediated signaling pathways in the development of Ph⁺ leukemia, which correlates with the aberrant cell adhesion properties of Ph⁺ cells (26) (reviewed in Verfaille et al. (27)).

In studying leukemic tissues isolated from transgenic mice expressing the transforming P190Bcr/Abl protein, we noted the presence of a group of 110-kDa tyrosine-phosphorylated proteins, collectively designated as pp110, that associated with the Crkl SH2 domain in vitro. We demonstrate here that pp110 is Hef1/Cas-L, a p130^Cas-related protein which is inducibly phosphorylated on tyrosine after ₁-integrin ligation in lymphoid cells. We show that Hef1 is hyperphosphorylated in leukemia/lymphomas induced by P190Bcr/Abl in transgenic mice, and, similar to p120^Cbl, is in complex with Bcr/Abl-bound Crkl. These findings lend support to an involvement of integrin-mediated signal transduction pathways during Bcr/Abl-induced leukemogenesis.

EXPERIMENTAL PROCEDURES

Chronic myelogenous leukemia cell line K562 was maintained in RPMI 1640 medium containing 10% fetal calf serum (Life Technologies, Inc.). Lysates of peripheral blood cells of patients have been previously described (9). Involved spleens and lymphoma tissues were obtained from terminally ill transgenic mice expressing P190Bcr/Abl (1, 2). Polyclonal rabbit anti-Cbl, anti-Cbl-b (C-20 and N-19), anti-Cas, and anti-Crkl were purchased from Santa Cruz Biotechnology. Crkl-specific antibodies (CH-16) recognizing the spacer region between the Crkl SH3 domains were affinity-purified (14). Anti-phosphotyrosine antibodies RC20H and PY20 and anti-Cas monoclonal antibodies, recognizing both Hef1 and p130^Cas, were obtained from Transduction Laboratory. Anti-Bcr (G6) was from Oncogene Science Inc.; anti-ABL monoclonal antibody 3F12 was a kind gift of Dr. Ravi Salgia (Dana-Farber Cancer Institute, Boston, MA).

GST-Crkl, GST-Crkl SH2, GST-Crkl SH33, and GST-Crkl SH23 have been described previously (5, 15). GST-Fer SH2 was constructed by ligating a 480-bp SstI-BamHI human FER cDNA fragment into pGEX-2T (Pharmacia) (28). To create GST-Abl SH2 and GST-Abl SH3 fusion proteins, a 290-bp HincII-HhaI and a 300-bp PvuII-HincII fragment, respectively, were excised and isolated from a human ABL cDNA clone isolated from K562,² and subsequently inserted into pGEX-2T. GST fusion proteins were expressed and purified as described previously (29), and glutathione was removed by dialysis against 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10% (v/v) glycerol.

Cellular and tissue extracts were prepared as described elsewhere (5, 15). In brief, cells were lysed in Triton-lysis buffer (25 mM Na-phosphate, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 µg/ml aprotinin and leupeptin, 1 µM pepstatin A, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 25 µM phenylarsine oxide) either directly (K562) or by disruption in a straight wall tissue grinder (leukemic mouse tissues). Per immunoprecipitation, 0.75-1 mg lysate proteins were precleared for 60 min at 4 °C with 25 µl of protein G-agarose (Life Technologies, Inc.), incubated with antibodies, and washed four times with lysis buffer without inhibitors as described previously (5). Proteins were separated on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and visualized with ECL (Amersham Corp.). Western blots were incubated with antibodies as described elsewhere (9).

Cellular or tissue extracts were precleared as described above. GST fusion protein precipitations were carried out as follows. Precleared extract (0.5-0.75 mg) was incubated with 10 µg of GST fusion protein and 25 µl of glutathione-agarose beads in 1 ml of 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM dithiothreitol, 0.1% Tween-20, 0.2% (w/v) bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, and 10 µg/ml each of aprotinin and leupeptin at 4 °C for 3 h. Beads were washed four times with ice-cold 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 0.1% Tween-20, 1% Nonidet P-40. Far Western assays were performed as described elsewhere (15).

RESULTS

Terminally ill P190Bcr/Abl transgenic mice typically show splenomegaly and lymphoblastic leukemia/lymphomas consisting to a large extent of clonal malignant lymphoblasts of predominantly pre-B cell lineage (1, 2). To investigate the presence of Crkl SH2-binding tyrosine-phosphorylated proteins in these leukemic tissues, total protein from involved lymph nodes was analyzed in a Far Western blot assay. The Crkl SH2 domain recognized several tyrosine-phosphorylated proteins with a molecular mass of approximately 110 kDa (pp110). These were most prominently present in lymph node tumors as compared with involved spleens (Fig. 1A and data not shown). The extent to which the pp110 was detected correlated with the amount of phosphotyrosine present in the different lysates. In a control experiment, the GST protein did not bind to proteins on a similar Far Western blot (data not shown).

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To establish whether pp110 is p120^Cbl, a leukemic tissue lysate was prepared from a typical pre-B lymphoma. Anti-phosphotyrosine immunoblotting of the lysate revealed only a limited set of very prominently tyrosine-phosphorylated proteins. This included proteins of 110-120 kDa and P190Bcr/Abl (Fig. 1B, upper panel, lane 1). Crkl co-immunoprecipitated P190Bcr/Abl and also brought down the 110-120-kDa proteins (lane 2). Preimmune serum of anti-Crkl (CH16) (15) did not precipitate any of the above proteins (lane 4). Cbl was tyrosine-phosphorylated in the P190Bcr/Abl-induced pre-B lymphoma (lane 3) and was also co-precipitated with Crkl (Fig. 1B, lane 2). It is clear that although Cbl is present in the Crkl immunoprecipitates, a substantial portion of the group of pp110 proteins is not Cbl.

We also reexamined whether P190Bcr/Abl-expressing peripheral blood cells from Ph⁺ leukemia patients contain tyrosine-phosphorylated proteins, in the 110-kDa range, that are capable of binding the SH2 domain of Crkl. The Crkl SH2 domain recognized the previously observed tyrosine-phosphorylated p120^Cbl in cells expressing an active Bcr/Abl protein (5), but, in addition, also bound to 110-kDa proteins in several ALL samples and in some CML samples expressing P210Bcr/Abl (data not shown).

These data indicated the involvement of unidentified tyrosine-phosphorylated proteins with a molecular size of approximately 110-kDa in Bcr/Abl-induced transformation in vivo, which bind Crkl and are likely to be of relevance to leukemia caused by P190Bcr/Abl.

To identify this pp110 protein, antibodies directed against proteins with a similar size, which could possibly be expressed in the hematopoietic cells transformed by P190Bcr/Abl in vivo, were tested. The Cbl-b protein was a likely candidate, since its structure and primary sequence are similar to that of Cbl, but it has a smaller size and is expressed in hematopoietic tissues and cell lines (30). The Cbl-b protein was expressed in leukemic tissues from P190BCR/ABL transgenic mice (Fig. 2B, lanes 1, 3, and 4). However, immunoprecipitated Cbl-b was not detectably tyrosine-phosphorylated, neither was it present in anti-phosphotyrosine precipitates (Fig. 2, A, lanes 3 and 4, and B, lane 2).

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During the course of these experiments, the Hef1 or Cas-L gene, hereinafter referred to as Hef1, was cloned. It encodes hematopoietically expressed proteins of approximately 110-kDa with a structure similar to the Crk-associated substrate protein p130^Cas (21, 31, 32). Although raised against p130^Cas, a monoclonal antibody recognizing both Hef1 and p130^Cas is available (Transduction Laboratory), and it efficiently recognized Hef1 in expression library screening, immunoprecipitations, and immunoblot analysis (32). To distinguish the tyrosine phosphorylation of Hef1 from that of p130^Cas, a lysate prepared from a P190Bcr/Abl-expressing lymphoma was depleted of Cas by one round of immunoprecipitation with Cas-specific antibodies, prior to subsequent immunoprecipitation of Hef1. Removal of Cas from the lysate was substantial (Fig. 3, A and C, lanes 1-3 and 5). Immunoprecipitated p130^Cas itself did not contain detectable phosphotyrosine residues (Fig. 3, A and B, lane 3). After Cas-depletion, no significant loss of any tyrosine-phosphorylated proteins in the lysate was observed (Fig. 3B, compare lanes 1 and 2). A group of proteins of around 110-kDa were precipitated by the dual Hef1/Cas monoclonal antibodies before and after Cas depletion, which correspond to Hef1 (Fig. 3B). These proteins reacted very strongly with the anti-phosphotyrosine antibodies and displayed a mobility indistinguishable from the pp110 identified in Fig. 1. In contrast, normal spleens from nontransgenic animals had very low levels of phosphorylated Hef1 (data not shown). These data show unambiguously that Hef1 proteins constitute a group of major tyrosine-phosphorylated pp110 proteins detectable in leukemia/lymphomas isolated from P190BCR/ABL transgenic mice.

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To investigate if Hef1 is indeed the pp110 in complex with Crkl in vivo, we examined whether Hef1 was present in a Crkl immunoprecipitate of a P190Bcr/Abl-induced lymphoma. Several tyrosine-phosphorylated proteins were associated with Crkl in this lymphoma, as detected by anti-phosphotyrosine antibodies (Fig. 4A, lane 2). Clearly detectable (co-)immunoprecipitated proteins included p39, pp110, and P190, corresponding to Crkl, Hef1, and P190Bcr/Abl, respectively (Fig. 4, B, C, and E). No p130^Cas was detectable in the Crkl or anti-phosphotyrosine precipitate, confirming that p130^Cas is not phosphorylated in P190Bcr/Abl-induced leukemia/lymphoma (Fig. 4D).

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The pp110 was initially detected using the Crkl SH2 domain as a probe on a Far Western blot. To confirm the identity of the Crkl SH2-binding pp110 as Hef1, proteins precipitated by the Crkl SH2 domain were analyzed. Two major tyrosine-phosphorylated proteins associated in vitro with the GST-Crkl SH2 protein in the lymphoma extracts, including a 68-kDa phosphoprotein and the pp110 (Fig. 5A, lanes 6 and 9), which also constitute the major tyrosine-phosphorylated proteins detected in the whole cell lysates from these leukemic tissues (Fig. 5A, lanes 4 and 7). No proteins bound to the GST protein or the beads, indicating that the observed interactions are specific. As described previously, Crkl SH2 bound, besides several other proteins, to the 120-kDa phosphoprotein Cbl in the CML cell line K562, but no pp110 was detectable (Fig. 5A, lane 2). Immunoblotting with the Hef1/Cas antibody demonstrated that tyrosine-phosphorylated Hef1 was bound by the Crkl SH2 in both P190Bcr/Abl lymphoma samples (Fig. 5B, lanes 6 and 9). In contrast, no Hef1 was precipitated from K562 and this extract lacked Hef1, in agreement with previous expression studies (32). We have reported previously that in K562, p130^Cas is neither phosphorylated on tyrosine nor in complex with Crkl (5), which is consistent with the absence of p130^Cas in the GST-Crkl SH2 precipitate from K562 (Fig. 5C). No p130^Cas was observed in the Crkl SH2 precipitates of the two P190Bcr/Abl lymphoma extracts. As expected, the SH2 domain of Crkl bound to tyrosine-phosphorylated Cbl in both K562 and the P190Bcr/Abl lymphoma extracts (Fig. 5D).

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Although the binding of Crkl to Hef1 is mediated by the Crkl SH2 domain and phosphotyrosine residues on Hef1, it remains possible that Crkl also binds Hef1 via its SH3 domains. To investigate this, a P190Bcr/Abl leukemic tissue extract was incubated with different GST fusion proteins and subsequently blotted and probed with anti-Cas/Hef1 antibody. Although the Crkl GST fusion proteins containing the SH2 domain efficiently precipitated Hef1, the Crkl SH3 domains did not (Fig. 5E). The homologous SH2 domain of Abl was also capable of precipitating tyrosine-phosphorylated Hef1, albeit less efficiently than Crkl SH2 (Fig. 5E, lane 6). However, the unrelated Fer SH2 domain did not recognize Hef1. Thus, the SH2 domain of Crkl is responsible for the Hef1 binding.

To extend and confirm these results, anti-phosphotyrosine and anti-Cas/Hef1 precipitates were probed with Crkl SH2 on an immunoblot. Tyrosine-phosphorylated Hef1 was clearly and specifically detected by Crkl SH2 (Fig. 6A), whereas blotting to control GST protein detected no specific bands (Fig. 6D). Compared with the Crkl SH2 domain, the related Abl SH2 domain interacted less efficiently with Hef1 in the Far Western assay (compare Fig. 6, A and C, lanes 2). As a control, the Crkl SH3 domains were incubated with a duplicate filter. This domain bound to a 190-kDa phosphoprotein in the lysate and in the anti-phosphotyrosine precipitate, which was identified as P190Bcr/Abl (Fig. 6B). In addition, several other proteins including 145-155-, 170-, and 180-kDa proteins were recognized by the Crkl SH3 domains in the total cellular lysate. These Crkl-interacting proteins have been previously identified for Crkl or for the related Crk and correspond to, respectively, C3G (33), c-Abl, SOS (15), and DOCK180 (34).

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DISCUSSION

We previously reported that Crkl binds the tyrosine-phosphorylated proto-oncoprotein CBL in a CML cell line, and this corresponds to a 120-kDa protein also seen in Ph⁺ patient material (5). In the current study, we have demonstrated the presence of hyperphosphorylated proteins of approximately 110 kDa bound to the Crkl SH2 domain in lymphoma/leukemia tissue derived from P190BCR/ABL transgenic mice, in addition to the p120^Cbl. The pp110 was identified as Hef1/Cas-L, a p130^Cas-related protein. Interestingly, although some pp110 was also detectable in P210Bcr/Abl-expressing patient samples, preliminary data suggest that expression of Hef1 and its tyrosine phosphorylation might be more characteristic for P190Bcr/Abl-induced leukemia/lymphoma since pp110 was also prominently present in Ph⁺ blood samples of ALL patients (data not shown). This likely reflects the hematopoietic cell lineage transformed by the P190Bcr/Abl protein, since expression of Hef1/Cas-L proteins is restricted to isolated peripheral lymphocytes and lymphocyte cell lines (32). In concordance with this, the CML cell line K562 did not contain detectable (phosphorylated) Hef1. Taken together, the Crkl-binding protein Hef1 might represent a signaling mediator of Bcr/Abl in Ph-positive cells of the lymphoid lineage.

It has been reported that p130^Cas is phosphorylated in Bcr/Abl-expressing cells and is associated with Crkl in CML patient samples (4). In addition, the product of the Crkl-related gene Crk has been shown to interact with p130^Cas: in v-Crk-transformed cells, v-Crk is associated with constitutively phosphorylated p130^Cas (18, 19, 21), and integrin stimulation induces the phosphorylation of p130^Cas and its association with c-Crk (22, 23). Therefore, we investigated whether p130^Cas was also hyperphosphorylated in the leukemic tissues of P190BCR/ABL transgenic mice. In contrast to Hef1, p130^Cas was not detectably phosphorylated on tyrosine or complexed with Crkl in these samples. It has been suggested that these structurally similar proteins have a different function which depends on the differentiation state of the cell, since Cas appears to be phosphorylated only in more terminally differentiated B cells after B cell receptor or ₁-integrin stimulation (35). This concept would correlate with the observed lack of Cas tyrosine phosphorylation and the fact that preB cells or their progenitors are the target cell type transformed by P190Bcr/Abl in transgenic mice (2).

Hef1 was originally discovered as a p105 protein that becomes tyrosine-phosphorylated upon ₁-integrin ligation (36) and was subsequently cloned from a cDNA library expressed in yeast (31) and, independently, using the cross-reacting p130^Cas mAb from Transduction Laboratory (32). Hef1 and p130^Cas share a high overall sequence similarity and contain several similar protein structures. The SH3 domains of Hef1 and Cas recognize proline-rich sequences in the focal adhesion kinase FAK and the related RAFTK (31, 32, 35, 37). Similar as in p130^Cas, repeated YXXP motifs in Hef1 conform to the consensus SH2 binding site of the Crk family (38) are located in the central domain of Hef1. Analogous to the above mentioned Crk-p130^Cas interaction, an increased association between tyrosine-phosphorylated Hef1 and Crkl was recently demonstrated after B cell receptor or ₁-integrin stimulation in several different types of B cells (35). Our current results show that the association between Crkl and Hef1 is mediated by the Crkl SH2 domain and phosphotyrosine residues in Hef1, most likely involving the YXXP motifs.

Besides the prominently phosphorylated Hef1, we also observed the phosphorylation of the proto-oncoprotein p120^Cbl and its in vivo association with Crkl and P190Bcr/Abl in P190Bcr/Abl-induced leukemia/lymphoma. The association between Crkl and Hef1 or Cbl in leukemic tissue from P190BCR/ABL transgenic mice supports a model in which one of the important functions of Crkl in Ph⁺ leukemia is to act as a molecular link between Bcr/Abl and downstream signaling proteins. In this model, stable complexes exist in Bcr/Abl-induced leukemia, involving Bcr/Abl, Crkl, and either one of the Crkl SH2 binding partners Hef1, p130^Cas, p120^Cbl, or paxillin, depending on the Bcr/Abl protein and the transformed cell type. It is expected that individual Crkl proteins can only complex with one of the SH2-binding partners on an either/or basis. Crkl is constitutively bound to Bcr/Abl through its SH3 domain and tethers its tyrosine-phosphorylated SH2-domain targets to Bcr/Abl, which leads to a significant increase in their degree of tyrosine-phosphorylation because of the deregulated kinase activity of Bcr/Abl. Although this model favors trimolecular complexes, different Crkl SH2 targets might assemble in multimer complexes since Bcr/Abl contains an oligomerization domain (39). The initial association of Hef1 and Cbl with the Crkl SH2 domain in P190Bcr/Abl-induced leukemia requires a low level transient tyrosine-phosphorylation, which is likely provided by normal signaling events such as stimulation by growth factors or integrin signaling, after which tyrosine-phosphorylation would be constitutively switched on, leading to a continuous activation of the signaling pathways in which Hef1 and Cbl partake.

An analogous model for successive phosphorylation has been proposed for the c-Abl-associated Crk, which through its SH2 domain binds to phosphorylated Abl kinase substrates and enhances their processive phosphorylation in vitro and in cultured cells (40, 41). In addition to the indirect association mediated by Crkl, a direct interaction between Bcr/Abl and Hef1 might also occur, as was previously suggested for Cbl (17) and which might be the basis for the observed association between Hef1 and v-Abl (31). The Abl SH2 domain can bind directly in vitro to Hef1 in a Far Western assay, which is consistent with the resemblance between the Crk family SH2 and ABL SH2 domain binding preferences (38). This direct interaction could enhance the stability of the aforementioned complexes.

Our identification of another protein involved in integrin-mediated signaling that is connected with Bcr/Abl in vivo, substantiates the suggestion that Bcr/Abl transformation utilizes the intracellular components of the integrin signal transduction pathway. Previously, we reported that paxillin, a cytoskeletal protein involved in integrin signaling and cell adhesion, is tyrosine-phosphorylated and associated with the Crkl-SH2 domain in Bcr/Abl-transformed cells and in a CML cell line (6). Furthermore, in Bcr/Abl-expressing cells, Crkl is associated through its SH2 domain with phosphorylated p130^Cas (4), which is also implicated in integrin signaling. These data are consistent with the localization of Bcr/Abl to the actin cytoskeleton (42), and together with the observed increase in tyrosine phosphorylation of other focal adhesion proteins in P210Bcr/Abl-expressing myeloid cell lines (43), this suggests that actin cytoskeleton rearrangement might contribute to the aberrant adhesion properties of Ph⁺ leukemic cells. CML progenitor cells from patients are clearly abnormal in that they have defects in their adherence to bone marrow stroma (44). The present finding that P190Bcr/Abl phosphorylates Hef1, a protein normally involved in ₁- integrin signaling, provides a mechanism for how Bcr/Abl can modulate CML progenitor cell adherence to the stroma.