p130CAS forms a signaling complex with the adapter protein CRKL in hematopoietic cells transformed by the BCR/ABL oncogene.

The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph+ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210BCR/ABL. CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120CBL. Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130CAS. p130CAS was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130CAS was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125FAK, and paxillin constitutively associated with p130CAS. However, in BCR/ABL transformed cells, the interaction between p130CAS and tensin was disrupted, while the associations between p130CAS, p125FAK, and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130CAS in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130CAS containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130CAS with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells.

Chronic myelogenous leukemia (CML) 1 and some acute lym-phoblastic leukemias (ALL, Ph ϩ ) are caused by the activated tyrosine kinase fusion protein BCR/ABL. There are multiple proteins which are tyrosine phosphorylated in response to BCR/ABL transformation (1)(2)(3)(4). We and others have shown that one of the most prominent and consistently tyrosine-phosphorylated proteins is an adapter protein termed CRKL (CRKlike) (5)(6)(7). CRKL has a 60% homology to c-CRK-II (CRK), the human homologue of v-Crk (8,9). The v-crk oncogene was originally cloned from the CT10 virus and can transform fibroblasts and other cells. The v-Crk oncoprotein contains both one SH2 and one SH3 domain, while c-CRK-II and CRKL contain one SH2 and two SH3 domains (10,11). A second product of the c-CRK gene, c-CRK-I, has one SH2 and one SH3 domain, but to date, only one form of CRKL has been observed and no oncogenic forms have been described. v-crk transformation in 3Y1 fibroblasts leads to tyrosine phosphorylation of multiple proteins (12). In the v-crk transformed fibroblasts, c-Abl has been shown to bind to v-Crk and is believed to be activated by this interaction (13).
Another prominent binding protein for the v-Crk oncoprotein has been shown to be p130 CAS (Crk associated substrate) (14). p130 CAS was originally cloned from the v-crk transformed 3Y1 cell line and shown to be heavily tyrosine phosphorylated in these cells (14,15). Analysis of cDNA for p130 CAS reveals multiple domains. The N terminus contains an SH3 domain, followed by a "substrate domain" consisting of 14 Tyr-X-X-Pro (Y-X-X-P) motifs, each of which are potential CRK/CRKL-SH2 binding sites (15). There is also a pp60 c-Src binding site in the C terminus of the molecule (16). p130 CAS also has been shown to be tyrosine phosphorylated in response to v-Src (14,17,18). In v-src transformed cells, p130 CAS is localized to focal adhesions (18,19), and recently, several laboratories have shown that p130 CAS is tyrosine phosphorylated in response to ␤1 integrin cross-linking (17,18,20,21). Although the function of p130 CAS in either normal or transformed cells is unknown, these observations suggest that p130 CAS may be involved in integrin signaling.
Since p130 CAS apparently plays a prominent role in transformation by the v-crk oncogene, we examined the interaction of p130 CAS with c-CRK and CRKL in hematopoietic cells transformed by BCR/ABL and in normal hematopoietic cells. We find that p130 CAS is tyrosine phosphorylated in most cell lines transformed by BCR/ABL and associated with the SH2 domain of CRKL, but not with c-CRK. This potentially links p130 CAS to BCR/ABL since CRKL is constitutively associated with BCR/ABL through its SH3 domain. This interaction was unique to BCR/ABL transformed cell lines and was not observed in normal cells. Furthermore, in primary cells from patients with CML and Ph ϩ acute lymphocytic leukemia (ALL), p130 CAS co-immunoprecipitates with CRKL. In both normal and BCR/ABL transfected cells, we found that p130 CAS localized in focal adhesion-like structures, as did BCR/ABL. Our results are of interest as they extend the known interactions between BCR/ABL and cellular signal transduction molecules mediated by CRKL.

MATERIALS AND METHODS
Cells, Cell Lines, and Cell Culture-The murine myeloid cell line 32Dcl3, murine pre-B cell line BaF3, human megakaryocytic cell line Mo7e, human T-cell line H9, human B-cell line Nalm6, and CML cell lines BV-173, and K562 were maintained in culture as described previously (22). The BCR/ABL expressing hematopoietic cell lines were generated by transfection with p210 BCR/ABL or p190 BCR/ABL cDNA and maintained as described previously (22). NIH-3T3 cells and NIH-3T3 expressing p210 BCR/ABL (courtesy of Dr. Warren Pear, MIT, Cambridge, MA) were maintained with 10% fetal calf serum in RPMI 1640. Normal neutrophils, thymocytes, mononuclear cells, and cells from CML, ALL, and chronic myelomonocytic leukemia patients were obtained with informed consent and approved IRB protocols, and isolated as described (23). A spleen sample was also obtained from a patient with CML and cells isolated by density gradient sedimentation.
Far Western Blotting-Immunoprecipitations were performed using anti-p130 CAS mouse monoclonal antibody as described previously and separated on 7.5% SDS-PAGE gels (24). The gels were then transferred as described previously (24). The blots were incubated at 4°C overnight in 5% dry milk and phosphate-buffered saline containing 0.1% Tween 20. The blots were washed for 1 h every 15 min at room temperature in phosphate-buffered saline, 0.1% Tween 20 between incubations. The blots were incubated in GST fusion proteins, GST, ABL-SH2, CRKL-SH2, and CRKL-SH3-SH3 for 2 h. The blots were washed and placed into anti-GST mouse monoclonal antibody (Santa Cruz Biotechnology) for 1 h. After washing, anti-mouse Ig, horseradish peroxidase (Amersham) was added for 1 h. The membranes were processed per established methods using enhanced chemiluminescense technique (Amersham) (22).

p130 CAS Associates with CRKL but Not c-CRK in BCR/ABL
Transfected Cells-Immunoblotting was used to investigate the expression and tyrosine phosphorylation of p130 CAS in hematopoietic cells. p130 CAS was detected in various hematopoietic cells (Fig. 1). The expression was compared to another protein, p120 CBL , in these cells and results are shown in Fig. 1. It was noted that an anti-p130 CAS rabbit polyclonal antibody from Santa Cruz detects a single band at 130 kDa by ECL; whereas an anti-p130 CAS mouse monoclonal antibody from Transduction Laboratories detects both a major band at 130 kDa and a minor band at 105 kDa. The minor 105-kDa band could be a related CAS form or a cross-reacting protein such as Hef1 (28).
In these experiments we have focused on the 130-kDa band identified by both antibodies as p130 CAS .
To determine if p130 CAS is tyrosine phosphorylated in BCR/ ABL transformed cells, cell lysates were immunoprecipitated with anti-p130 CAS antibody and membranes immunoblotted with anti-phosphotyrosine antibody. p130 CAS was tyrosine phosphorylated in 32Dcl3 and BaF3 cells transformed by BCR/ ABL and in the Philadelphia chromosome-positive human cell line BV-173, but only minimal tyrosine phosphorylation of p130 CAS was detected in K562 cells (2,29). The reverse experiment, using an anti-phosphotyrosine immunoprecipitation and p130 CAS immunoblot yielded similar results (Fig. 2). These data indicate that p130 CAS is tyrosine phosphorylated in most, but not all, cell lines transformed by BCR/ABL.
Previously, we have shown that CRKL, but not c-CRK, is tyrosine phosphorylated in BCR/ABL transformed cells (27). Coimmunoprecipitation studies were performed to check if CRKL could bind p130 CAS . As shown in Fig. 2, CRKL and p130 CAS are both present in the same immunoprecipitates in BCR/ABL transformed 32Dcl3 cells and not normal cells. Im- munoprecipitation studies in BCR/ABL transformed BaF3 cells also show coimmunoprecipitation between CRKL and p130 CAS (data not shown). The stoichoimetry of the association between CRKL and p130 CAS was difficult to discern because of the affinities of the antibodies are quite different. To determine if c-CRK could also bind to p130 CAS , we performed anti-CRK immunoprecipitations on unstimulated, interleukin-3 stimulated, and BCR/ABL transformed 32Dcl3 cells. There was no coimmunoprecipitation of c-CRK with p130 CAS in any of these cells. The reverse experiment also failed to detect any coimmunoprecipitation of p130 CAS to c-CRK (Fig. 3).
p130 CAS Associates with CRKL in Primary Cells from Patients with CML and ALL-To determine if CRKL can associate with p130 CAS by coimmunoprecipitation in primary cells expressing BCR/ABL, we examined cell lysates from samples obtained from normal, CML, and ALL (Ph ϩ , acute lymphocytic leukemia) patients. The expression of p130 CAS in neutrophils was approximately 10-fold less than BaF3 or 32Dcl3 cells (Fig.  1). p130 CAS immunoprecipitates from normal neutrophils did not contain CRKL. However, p130 CAS coimmunoprecipitated with CRKL in BCR/ABL positive samples (Fig. 4). As another control, we performed p130 CAS immunoprecipitations in cell lysates obtained from a patient with chronic myelomonocytic leukemia (a Ph negative chronic leukemia) and there was no apparent co-precipitation of p130 CAS and CRKL. The reverse experiment, anti-CRKL immunoprecipitation and p130 CAS immunoblot, showed p130 CAS coimmunoprecipitated with CRKL in BCR/ABL positive samples and not normal neutrophils. There was no difference in coimmunoprecipitation of CRKL with p130 CAS seen with the addition of growth factor granulocyte-macrophage colony-stimulating factor to the neutrophils.
Since the substrate domain of p130 CAS contains multiple, clustered, Tyr-X-X-Pro motifs, we sought to determine if the SH2 domain of CRKL associates with p130 CAS at these sites. It is shown that the phosphorylated substrate domain precipitated with CRKL in lysates from 32D.p210 BCR/ABL cells. As a control, the SH3 domain and unphosphorylated substrate domain did not precipitate with CRKL.
To show direct binding of CRKL-SH2 domain to p130 CAS , we also performed far Western blotting. As seen in Fig. 6, CRKL-SH2 but not CRKL-SH3 directly bound to p130 CAS in BCR/ABL expressing BaF3 cells. This was also true for 32D.p210 BCR/ABL cells (data not shown).
p130 CAS Associates with Focal Adhesion Proteins in Normal Myeloid Cells, but These Complexes Are Disrupted by BCR/ ABL Transformation-We have previously shown that BCR/ ABL is localized in focal adhesion-like structures in myeloid cells (4). p130 CAS cellular staining in myeloid cells expressing BCR/ABL also showed that p130 CAS is localized in focal adhesion-like structures (data not shown and Ref. 4). Since BCR/ ABL is known to alter focal adhesion structure, we examined the possible interaction of p130 CAS with normal focal adhesion proteins. Immunoprecipitations with anti-tensin, anti-p125 FAK , anti-vinculin, and anti-paxillin were performed and then immunoblotted with anti-p130 CAS antibody. p130 CAS immunoprecipitated with tensin, p125 FAK , and paxillin in normal cells; whereas, only p125 FAK and paxillin immunoprecipitated with p130 CAS in BCR/ABL expressing 32Dcl3 and BaF3 cells (Fig. 7). Vinculin, talin, and ␣-actinin did not coprecipitate with p130 CAS (Fig. 7, and data not shown). The reverse experiment, with p130 CAS immunoprecipitations and various focal adhesion  5-7), and immunoprecipitates of cell lysates with anti-CRKL antibody (I.P., 20 ϫ 10 6 cells, lanes 8 -10), 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 membranes were immunoblotted with anti-p130 CAS antibody, stripped, and reprobed with anti-CRKL antibody, and restripped and reprobed with antiphosphotyrosine antibody (4G10). The cells used are unstimulated 32Dc13 cells (32D(Ϫ)), 32Dc13 stimulated cells with interleukin-3 (32D(ϩ)), and 32D.p210 BCR/ABL .26 cells (32D.p210). Molecular weights are shown in kDa.
protein immunoblots yielded the same results (data not shown). DISCUSSION Chronic myelogenous leukemia was the first disorder identified with a specific abnormal chromosomal translocation, the Philadelphia chromosome, creating a fusion of two genes, BCR and ABL (exons b2a2 or b3a2), and thus generating the oncoprotein p210 BCR/ABL (30). In an alternative fusion of BCR and ABL (exons b1a2), there is production of the oncoprotein p190 BCR/ABL which can be seen in some acute lymphoblastic leukemias (30). BCR/ABL is a potent tyrosine kinase which translocates to the cytoskeleton and disrupts the actin cytoskeleton (31)(32)(33). There is growing evidence that CML cells have adhesion defects and that these defects contribute significantly to the clinical phenotype (34,35). There are multiple proteins which are phosphorylated by BCR/ABL in primary cells from subjects with CML or ALL and in cell lines generated by transfection of BCR/ABL (33). We and others have previously shown that a potentially key signaling intermediate is CRKL (5-7).
CRKL is a 39-kDa protein with one SH2 and two SH3 domains (9). It has a high homology to c-CRK-II, and belongs to the CRK family of adapter proteins (37). The CRK family consists of v-Crk, c-CRK-II, c-CRK-I, and CRKL. CRKL and c-CRK-II contain an NH 2 -terminal SH2 domain and two tan-dem SH3 domains at the COOH terminus. c-CRK-I and v-Crk both have a deletion of the most COOH-terminal SH3 (38). CRKL has been shown to have similar in vitro binding charac- FIG. 4. p130 CAS can associate with CRKL in BCR/ABL containing cells from patients with CML or Ph ؉ ALL. A, immunoprecipitates of lysates from cells from normal or leukemia patients (20 ϫ 10 6 cells) with anti-CRKL antibody 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-p130 CAS antibody, and thereafter stripped and reprobed with anti-CRKL antibody. Lanes 1 and 2 are normal neutrophils unstimulated (Ϫ) and stimulated (ϩ) with granulocyte-macrophage colony-stimulating factor GM-CSF, respectively. Lanes 3-8 are neutrophil samples from three separate patients with stable phase CML, unstimulated and stimulated alternately. B, immunoprecipitates of lysates from cells from normal or leukemia patients (20 ϫ 10 6 cells) with anti-p130 CAS antibody 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. Lanes 1 and 2 are neutrophils from two separate normal subjects, lanes 3 and 4 are blast samples from two separate ALL (Phϩ) patients, lanes 5 and 6 are neutrophils from two separate CML patients in stable phase, lane 7 is a sample from a spleen from a patient with CML, and lane 8 is a sample from a patient with chronic myelomonocytic leukemia (CMML). teristics as c-CRK (38). However, in BCR/ABL containing cells, it is CRKL and not c-CRK which is tyrosine phosphorylated (5)(6)(7). In order to explore the differences between c-CRK and CRKL in hematopoietic cells, we have tried to determine if they have a similar association with the recently cloned Crk-associated substrate p130 CAS (14,15). Our results indicate that p130 CAS is tyrosine phosphorylated, and associates with CRKL, but not c-CRK in BCR/ABL transformed cell lines and samples from patients with CML and Ph ϩ ALL. The timing of association between p130 CAS and BCR/ABL to CRKL has not been shown in this study and further experiments will be needed.
It is of interest that p130 CAS is not significantly tyrosine phosphorylated in K562 cells and the reasons are quite unclear (2,29). There is a possibility that K562 cells have other abnormalities which prevent p130 CAS from becoming phosphorylated. Classically, there is heterogeneity of tyrosine phosphorylation of various proteins in CML primary cells. This heterogeneity could also involve the tyrosine phosphorylation of p130 CAS by BCR/ABL. It has also been shown that K562 cells lack ␤2 integrins and this somehow is related to p130 CAS phosphorylation (39). Eventually, further experiments will need to be performed to explore the differences and localization of p130 CAS .
p130 CAS was initially identified as a prominent tyrosinephosphorylated substrate of the oncoproteins v-Src and v-Crk (17,40). It has also been shown that p130 CAS is tyrosine phosphorylated in response to adhesion and cross-linking of ␤1 integrins (17,18,20,21). p130 CAS has been shown to associate with the focal adhesion proteins p125 FAK and tensin in vivo and in vitro (19,41). p130 CAS may be important in transformation by ornithine decarboxylase (42). It has been shown that c-Abl may phosphorylate p130 CAS in vitro. In normal cells, p130 CAS is localized to both the cytoplasm and the nucleus; whereas in v-src and v-crk transformed fibroblasts, it is primarily localized to the focal adhesion (14,17,19).
p130 CAS was originally cloned from 3Y1 fibroblasts transformed with v-crk (14,15). There are two major tyrosine phos-phorylated proteins in v-crk transformed 3Y1 cells: paxillin and p130 CAS (43). Using isolated protein and peptide sequencing, the cDNA encoding p130 CAS has been characterized. p130 CAS contains an NH 2 -terminal SH3 domain which has been shown to bind to the proline-rich portion in the COOH terminus of p125 FAK . In the middle of the molecule is the "substrate domain" with nine Tyr-Asp-X-Pro motifs optimum for binding the c-CRK-SH2 domain (14). Mayer et al. (44) have shown that the c-Abl-SH2 domain binds to the substrate domain of p130 CAS in vitro. Lo et al. (19) have shown that the SH2 domain of tensin is also capable of binding p130 CAS in v-src transformed cells. We show here that the CRKL-SH2 domain binds directly to the substrate domain of p130 CAS .
We examined the association of p130 CAS with focal adhesion proteins in normal and BCR/ABL transformed hematopoietic cells. There was a constitutive association of p125 FAK and paxillin with p130 CAS . Interestingly, the focal adhesion protein tensin associated with p130 CAS in normal hematopoietic cells, but not in BCR/ABL transformed cells. Tensin is an SH2 containing protein which has three actin-binding domains, and is believed to be involved in linking integrins to the actin cytoskeleton (45). Tensin also is tyrosine phosphorylated in response to various mitogenic signals (36). In this study, we have shown that tensin and p130 CAS form a complex in normal cells and that binding is disrupted in BCR/ABL transformed cells. The reason for this possibly could be that tensin and/or p130 CAS could be located in different compartments, either of these molecules could be bound to another protein thereby not interacting with each other, or the tyrosine phosphorylation of either of these molecules could potentially inhibit their interactions. The alteration in binding between p130 CAS and tensin could be an important step toward BCR/ABL transformation, and this is currently being investigated.
In summary, we have shown that p130 CAS is expressed in hematopoietic cells, it is tyrosine phosphorylated in response to BCR/ABL, and associates with the unique adapter protein CRKL as shown by coimmunoprecipitation. p130 CAS also constitutively associates with focal adhesion proteins such as paxillin and p125 FAK . However, p130 CAS associates with tensin in normal but not BCR/ABL expressing cells. It is known that CML progenitor cells have abnormalities of adhesion through ␤1 integrins and potentially p130 CAS could be involved in mediating these abnormalities. with control antibody against ␥-interferon, and immunoprecipitates of cell lysates (I.P., 20 ϫ 10 6 cells, last four lanes) with various antibodies (anti-tensin, anti-p125 FAK , anti-vinculin, and anti-paxillin) 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 membranes were immunoblotted with anti-p130 CAS . The cells used are unstimulated 32Dc13 cells (32D(Ϫ)), 32Dc13 stimulated cells with interleukin-3 (32D(ϩ)), 32D.p210 BCR/ABL .26 cells (32D.p210), and K562 cells.