J Biol Chem, Vol. 274, Issue 53, 37525-37532, December 31, 1999

The Adapter Protein Crkl Links Cbl to C3G after Integrin Ligation and Enhances Cell Migration^*

Naoki Uemura and James D. Griffin^§

From the  Department of Adult Oncology, Dana-Farber Cancer Institute, and the Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Crkl, an SH2-SH3-SH3 adapter protein, is one of the major tyrosine phosphoproteins detected in cells from patients with chronic myelogenous leukemia. Crkl binds to BCR/ABL through its N-terminal SH3 domain and is known to interact with several signaling proteins that have been implicated in integrin signaling, including Cbl, Cas, Hef-1, and paxillin. We have previously shown that overexpression of Crkl enhances adhesion to extracellular matrix proteins through ₁ integrins. In this study, the effects of Crkl on spontaneous and chemokine-directed migration of the hematopoietic cell line Ba/F3 were examined. Full-length, SH2-, and SH3(N)-domain deletion mutants of Crkl were expressed transiently as fusion proteins with green fluorescent protein. Successfully transfected cells were isolated by fluorescence-activated cell sorting. The ability of these cells to migrate across a fibronectin-coated membrane, either spontaneously or in response to the chemokine stromal-derived factor-1, was determined. Cells expressing green fluorescent protein alone were not distinguishable from untransfected or mock transfected Ba/F3 cells. However, Ba/F3 cells overexpressing full-length Crkl were found to have an increase in spontaneous migration of 2.8 ± 0.6-fold in seven independent assays. The enhancement of migration required both the SH2 domain and the N-terminal SH3 domain. Migration in response to stromal-derived factor-1 was not significantly enhanced by overexpression of Crkl. Overexpression of Crkii also augmented spontaneous migration but to a lesser degree than did Crkl. Because the SH2 domain was required for enhanced migration, we looked for changes in phosphotyrosine containing proteins coprecipitating with Crkl, but not Crkl SH2, after integrin cross-linking. Full-length Crkl, but not CrklSH2, coprecipitated with a single major tyrosine phosphoprotein with an M_r of approximately 120 kDa, identified as Cbl. The major Crkl SH3-binding protein in these cells was found to be the guanine nucleotide exchange factor, C3G. Interestingly, overexpression of C3G also enhanced migration, suggesting that a Cbl-Crkl-C3G complex may be involved in migration signaling in Ba/F3 cells. These data suggest that Crkl is involved in signaling pathways that regulate migration, possibly through a complex with Cbl and C3G.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Crkl is a 39-kDa adapter protein containing one SH2 and two SH3 domains. The Crkl gene was originally identified by ten Hoeve et al. (1) and found to have an overall amino acid homology of 60% to Crkii, one of two proteins generated through alternative splicing of the human Crk proto-oncogene. Both Crkl and Crk are related to v-Crk, the oncogene in the avian retrovirus CT10. Crkl has been shown to be involved in a number of signaling pathways, including pathways activated by T cell stimulation, integrin cross-linking, and hematopoietic cytokine receptor activation (2-8). Also, we and others have shown that Crkl is a target of the BCR/ABL oncogene in chronic myelogenous leukemia cells (9-17). Although the function of Crkl remains unknown, several investigators have shown that Crkl interacts with signaling proteins previously linked to the control of cytoskeletal functions. In particular, both the Crkl and Crk SH3 domains bind to an overlapping set of cellular target proteins in vitro, including c-ABL, C3G, EPS15, DOCK180, and SOS, and the Crkl and Crk SH2 domains also share in vitro binding to c-Cbl, Cas, Hef-1, and paxillin (18-24, 26-28). Overexpression of Crkl in hematopoietic cells enhances integrin-mediated cell adhesion to fibronectin-coated surfaces (2, 29). These observations led us to examine the role of Crkl in cell migration, an event that requires coordination of cellular polarization, membrane extension, formation of cell-substratum attachments, contractile force, traction, and release of rear attachment sites. Although cell adhesion to the substratum is required for migration it is not sufficient, and it is likely that important signals regulating migration come from many different sources including integrins, cytokine receptors, chemokine receptors, and others. The results indicate that Crkl is involved in the regulation of migration, likely involving an interaction with the guanine nucleotide exchange factor, C3G.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Plasmids-- A human Crkl cDNA was obtained from Dr. John Groffen (Children's Hospital, Los Angeles, CA). In-frame deletions of amino acids 14-64, 131-179, 238-290, and 131-290, respectively, were made by polymerase chain reactions to create an SH2-, an N-terminal SH3-, a C-terminal SH3-, and both SH3-domain(s) deletion mutants (hereafter termed CrklSH2, CrklSH3(N), CrklSH3(C), and CrklSH3(N+C), respectively) (29). Crkl and Crkl deletion mutants were cloned into mammalian expression vectors, pEBB and pEGFP-C (CLONTECH, Palo Alto, CA), resulting in the expression of green fluorescent protein (GFP)¹-Crkl fusion proteins. A human Grb2 cDNA was obtained from Dr. Akio Yamakawa (Dana-Farber Cancer Institute), and was also cloned in frame in the pEGFP-C vector. A murine c-ABL cDNA was obtained from Dr. Richard A. Van Etten (Center for Blood Research, Harvard Medical School, Boston, MA). A human C3G cDNA and a human DOCK180 cDNA were obtained from Dr. Michiyuki Matsuda (International Medical Center of Japan, Tokyo, Japan) and cloned into the pEBB vector. A hemagglutinin-tagged human Cbl cDNA in the pALTER-MAX expression vector was obtained from Dr. Hamid Band (Harvard Medical School).

Transient Transfection of Ba/F3 Cells-- The murine hematopoietic cell line, Ba/F3, was cultured in RPMI 1640 containing 10% fetal calf serum and 15% WEHI3B cell conditioned medium as a source of murine interleukin-3. Ba/F3 cells were transfected with plasmids by electroporation using a Gene Pulser apparatus (Bio-Rad). For migration assays GFP-expressing cells were purified by fluorescence-activated cell sorting 18 h after transfection using an EPICS Elite-EPS cytometer (Coulter Corp., Hialeah, FL).

Migration Assays-- Migration assays were performed using Transwell plates (0.33 cm² insert growth area, 8 µm pores; Corning Coster Corp. Cambridge, MA). Both sides of the porous polycarbonate membrane were coated with human fibronectin (5 µg/ml). The lower chamber contained 600 µl of RPMI 1640 medium containing 1% bovine serum albumin (BSA). Fluorescence-activated cell sorting (FACS)-purified cells were starved in RPMI 1640 containing 1% BSA for 6 h, placed into the upper chamber (1 × 10⁵ cells/100 µl of RPMI 1640 containing 1% BSA), and allowed to migrate into the lower chamber for 6-8 h. In some experiments, recombinant human SDF-1 (R & D Systems, Minneapolis, MN) (31) was added to the lower chamber.

Integrin Cross-linking-- 18 h after transfection cells were removed from interleukin-3 containing medium and placed in RPMI 1640 containing 1% BSA for 6 h. To induce integrin signaling, cells were suspended in phosphate-buffered saline (1 × 10⁷/ml), incubated for 10 min on ice with antibody against murine ₁ integrin (Ha2/5, PharMingen, San Diego, CA) or hamster IgM as a control, washed in phosphate-buffered saline, and stimulated by cross-linking using anti-hamster IgM at 37 °C for 5 min.

Immunoblotting and Immunoprecipitation-- Cells were lysed in buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM EDTA, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 20 mg/ml aprotinin, 1 mM sodium orthovanadate, and 40 mg/ml leupeptin. Immunoprecipitation and immunoblotting were performed as described previously (17). For immunodepletion studies, cell lysates were incubated with either anti-Cbl antibody or preimmune rabbit serum with protein A-Sepharose, and the resulting supernatants were used for immunoprecipitation.

Other antibodies used in this study were anti-Crkl monoclonal antibodies (#5-6 and #2-2), anti-Crkl polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphotyrosine monoclonal antibody (4G10; provided by Dr. Brian J. Druker, Oregon Health Science University, Portland, OR), anti-Cbl polyclonal antibody (Santa Cruz Biotechnology), anti-Cas monoclonal antibody (Transduction Laboratories, Lexington, KY), anti-C3G polyclonal antibody (Santa Cruz Biotechnology), anti-ABL monoclonal antibody (Ab-3; Oncogene Science, Manhasset, NY), anti-DOCK180 polyclonal antibody (Santa Cruz Biotechnology), and anti-GFP polyclonal antibody (CLONTECH).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Overexpression of Crkl and Crk, but Not Grb2, Increases Random Cell Migration on Fibronectin-coated Surfaces-- The hematopoieticU cell line Ba/F3 was selected for these studies because it is capable of migration on two-dimensional surfaces and across three-dimensional membranes, and because it expresses appropriate receptors for extracellular matrix proteins, hematopoietic growth factors, and chemokines such as SDF-1 (the CXCR4 receptor).² Migration assays were performed using modified Boyden chambers (Transwell membranes) coated with fibronectin on both surfaces. Crkl, mutants of Crkl, Crk, Grb2, or other test genes were expressed transiently along with appropriate controls in all cell migration experiments. To obtain cell populations in which the transgenes were overexpressed in more than 90% of cells, plasmids encoding GFP fusion proteins were transfected by electroporation, and positive cells were isolated by FACS. The protein expression of GFP fusion proteins was confirmed by immunoblotting using specific antibodies and an anti-GFP antibody. This technique had the advantage of allowing for purification of cell populations that expressed approximately equivalent amounts of each transgene, because cells were isolated that expressed approximately equivalent amounts of fluorescence by FACS.

Migration of GFP-Crkl-overexpressing cells was significantly increased (2.8-fold) compared with GFP-expressing cells (Fig. 1A). Overexpression of GFP alone had no significant effect on migration compared with untransfected cells or sham transfected cells (data not shown). To ensure that the observed effects were because of Crkl and unique to the fusion protein, Crkl and GFP were individually expressed in the same cells from different vectors, and migration experiments were repeated. Again, Crkl overexpression enhanced spontaneous migration, suggesting that the presence of the GFP tag did not alter Crkl function in this assay (data not shown). The Crkl-related adapter protein, Crkii, also increased migration but was reproducibly less effective than Crkl in these hematopoietic cells. The enhanced migration was likely to involve ₁ integrins, because there was no significant migration if the Transwell membranes were not coated with fibronectin (data not shown). Also, the effects of Crkl on Transwell migration were not a general property of adapter proteins, because overexpression of Grb2 did not have any significant effects on cell migration in this system (Fig. 1B).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Crkl enhances cell migration. A, Ba/F3 cells were transfected with pEGFP-C, pEGFP-C-Crkl, or pEGFP-C-CrklSH2. After 18 h, GFP-expressing cells were enriched to >90% fluorescence by FACS and then used for random migration assays as described under "Materials and Methods." The vertical axis shows relative migration expressed as the ratio of the number of GFP-Crkl transfected cells migrating to the number of GFP/control transfected cells migrating over the same time period. Shown is the mean ± S.D. of seven inde- pendent experiments; each experiment was performed using triplicate wells. B, Ba/F3 cells were transfected with pEGFP-C, pEGFP-C-Crkl, or pEGFP-C-Grb2, and the migration index was calculated as above. Shown is the mean ± S.D. of three independent experiments. C, Ba/F3 cells were transfected with pEGFP-C, pEGFP-C-Crkl (FL, full-length Crkl), pEGFP-C-CrklSH3(N), pEGFP-C-CrklSH3(C), or pEGFP-C-CrklSH3(N+C), and the migration index was calculated. Shown is the mean ± S.D. of three independent experiments.

Both SH2 and N-terminal SH3 Domains of Crkl Are Necessary to Increase Migration-- Overexpression of full-length Crkl was compared with deletion mutants in which the SH2 or SH3 domains had been deleted (Fig. 1, A and C). In contrast to full-length Crkl, overexpression of GFP-CrklSH2 did not alter cell migration, and overexpression of GFP-CrklSH3(N) significantly inhibited migration of Ba/F3 cells (Fig. 1C). Overexpression of GFP-CrklSH3(C) increased migration, although to a lesser degree than that of GFP-Crkl. Overexpression of GFP-CrklSH3(N+C) reduced migration. These results indicate that the SH2 and the N-terminal, but not the C-terminal, SH3 domains are required to enhance random migration of Ba/F3 cells. Further, because overexpression of GFP-CrklSH3(N) inhibited migration below the base-line value exhibited by control cells, this mutant may function as dominant-negative in a pathway required for regulating migration.

SDF-1 Overrides the Modulation of Migration by Overexpression of Crkl-- The above findings support the hypothesis that Crkl may play an important role in overall random migration of hematopoietic cells on fibronectin-coated surfaces. This raised the question whether overexpression of Crkl also affects cell migration induced by chemoattractant gradients. We have previously shown that Ba/F3 cells express the receptor for SDF-1, CXCR4, and migrate avidly along an SDF-1 gradient in vitro and in vivo.³ The effect of overexpression of Crkl on SDF-1-directed cell migration was evaluated. Adding SDF-1 into the lower chamber increased migration of all transfected cells tested. Overexpression of GFP-Crkl or GFP-CrklSH3(N) did not alter migration of transfected Ba/F3 cells in response to SDF-1 (data not shown).

Identification of Cbl as the Major Crkl-SH2-binding Protein after ₁-integrin Ligation-- The above results suggest that Crkl is likely to enhance spontaneous migration by linking to other proteins using its SH2 domain and the N-terminal SH3 domain. To look for proteins interacting with the Crkl-SH2 domain, ₁ integrins were cross-linked using an anti-₁ integrin antibody, and cell lysates were subjected to anti-Crkl immunoprecipitation followed by immunoblotting with the anti-phosphotyrosine monoclonal antibody 4G10 (Fig. 2). Cross-linking  integrins in mock transfected cells resulted in a substantial increase in the coprecipitation of a 120-kDa phosphoprotein with Crkl. After overexpression of wild type Crkl, the association of Crkl with pp120 was increased before integrin activation. Several other proteins were detected in Crkl-overexpressing cells that were not observed in mock transfected cells, including bands at approximately 140, 95, 60, and 40 kDa. The interaction of each of these proteins with Crkl, and the enhanced binding to pp120, required the SH2 domain, because cells overexpressing CrklSH2 were indistinguishable from mock transfected cells (Fig. 2).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   1-integrin cross-linking induces tyrosine phosphorylation of Crkl and the formation of tyrosine phosphoprotein complexes containing Crkl. Ba/F3 cells were transfected with pEBB (mock), pEBB-Crkl, or pEBB-CrklSH2. 18 h after transfection, cells were made quiescent by culturing in RPMI 1640 medium with 1% bovine serum albumin for 6 h and then stimulated by 1 integrin cross-linking (+). Nonspecific immunoglobulin was used as a control (-) as described under "Materials and Methods." Cell lysates were used for immunoprecipitation with anti-Crkl monoclonal antibody 5-6 and immunoblotted with anti-phosphotyrosine Ab (4G10). Overexpression of the Crkl and CrklSH2 mutants was confirmed by anti-Crkl blotting. The immunoglobulin heavy chain is indicated as Ig.

The pp120 protein(s) coprecipitating with Crkl was found to be composed predominantly of Cbl (Fig. 3). Cbl coprecipitated with GFP-Crkl after the cross-linking of  integrins (Fig. 3A). This was not affected by mutation of tyrosine 207 to phenylalanine, but Cbl did not coprecipitate with GFP-CrklSH2, consistent with this interaction being mediated by the SH2 domain of Crkl (Fig. 3A). In contrast to the SH2 deletion, the integrin-induced association of Cbl with Crkl was only slightly reduced by deleting either SH3 domain (Fig. 3B). To determine whether Cbl was the major 120-kDa phosphoprotein induced to coprecipitate with Crkl after integrin ligation, immunodepletion using an anti-Cbl antibody was performed. Cbl immunodepletion significantly reduced the amount of pp120 coprecipitating with Crkl, suggesting that the Cbl is the major component of this band (Fig. 3C). An anti-Cas antibody was also used to determine whether Crkl coprecipitated with Cas. However, coprecipitation of Cas with Crkl in Crkl-overexpressing cells was at the lower limit of detection. Further, in these cells, cross-linking of  integrins induced only marginal tyrosine phosphorylation of Cas (data not shown). Thus, integrin signaling in Ba/F3 cells induces Crkl-SH2 to bind primarily to Cbl.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   CBL is the major Crkl binding tyrosine phosphoprotein after 1-integrin cross-linking. A, Ba/F3 cells were transfected with pEGFP-Crkl, pEGFP-Crkl Y207F, or pEGFP-C-CrklSH2. Cell lysates from cells transfected as shown were immunoprecipitated with anti-GFP Ab and immunoblotted with anti-Cbl Ab or anti-Crkl Ab 5-6. Starvation and 1 integrin cross-linking were performed as described in the legend for Fig. 1. B, Ba/F3 cells were transfected with pEBB (mock), pEBB-CrklSH3(N), or pEBB-CrklSH3(C). Cell lysates were immunoprecipitated with anti-Crkl Ab 5-6 and immunoblotted with anti-Cbl Ab. C, as in B, cell lysates were first immunodepleted with anti-Cbl Ab (+) or preimmune rabbit serum as a control (-) and then anti-Crkl immunoprecipitation was performed, followed by immunoblotting with phosphotyrosine (4G10) or anti-Cbl Ab as shown. D, Ba/F3 cells transfected with either pEBB (mock) or pEBB-Crkl were treated with anti-1 integrin Ab (+) or nonspecific immunoglobulin (-), washed, and then exposed to a secondary antibody for 5 or 60 min. Cell lysates were immunoprecipitated with anti-Cbl Ab and immunoblotted with anti-phosphotyrosine Ab (4G10). E, Ba/F3 cells were transfected with pEBB (mock) or pEBB-CrklSH3(N). Cell lysates from transfected cells were immunoprecipitated with anti-Crkl Ab 2-2, which recognizes only endogenous Crkl because the epitope recognized by this antibody is in the C-terminal SH3 domain, and immunoblotted with anti-Cbl Ab or anti-Crkl Ab 5-6. Appropriate expression of CrklSH3(N) was confirmed by immunoblotting of whole cell lysate with anti-Crkl Ab 5-6.

In Crkl-overexpressing cells, a small amount of Cbl coprecipitated with Crkl even in resting, non-adherent, cells. This finding led us to examine whether overexpression of Crkl prolongs tyrosine phosphorylation of Cbl by blocking access of phosphatases to phosphotyrosine residues. Cbl was rapidly tyrosine-phosphorylated after integrin ligation, followed by nearly complete dephosphorylation within 60 min in either mock or Crkl-transfected cells (Fig. 3D). This result suggests that Crkl overexpression does not affect the kinetics of tyrosine phosphorylation or dephosphorylation of Cbl after integrin ligation. The increased association of Cbl with Crkl in resting, Crkl-overexpressing cells may be due to the fact that Cbl is relatively abundant in the cell and that the formation of a Cbl-Crkl complex is limited by the available Crkl. Interestingly, CrklSH3(N) competed with wild type endogenous Crkl for binding to phospho-Cbl (Fig. 3E). Anti-Crkl monoclonal antibody 2-2, which recognizes an epitope in the N-terminal SH3 domain of Crkl, was used to specifically detect binding of endogenous, full-length Crkl to Cbl. Overexpression of CrklSH3(N) reduced the integrin-induced association of Cbl with endogenous Crkl as compared with mock transfected cells (Fig. 3E). One explanation for these results is that both the level of Crkl and the level of Cbl tyrosine phosphorylation are important to migration signaling. The ability of CrklSH3(N) to compete with full-length Crkl for binding to phospho-Cbl may contribute to the dominant-negative effects this Crkl mutant had on spontaneous cell migration.

Because Cbl was the major protein interacting with the CrklSH2 domain in Ba/F3 cells after integrin cross-linking, the effect of overexpression of Cbl (approximately 3-fold) on cell migration was examined. Overexpression of Cbl with either GFP or GFP-Crkl did not have any additional significant effect on cell migration in multiple experiments (data not shown). It is possible that Cbl is not involved in regulating migration despite interacting directly with Crkl, that the amount of endogenous Cbl is not rate-limiting for migration, or that overexpressing Cbl does not increase the amount of phospho-Cbl available for binding to Crkl.

Some of the other tyrosine phosphoproteins that are coprecipitated with Crkl after integrin cross-linking were identified. The protein at 140 kDa was identified as SHIP (33) (data not shown). The protein at 40 kDa was tyrosine-phosphorylated Crkl (data not shown). In a longer exposure, a 70-kDa protein detected in Crkl immune complexes after integrin cross-linking in Crkl-overexpressing cells was identified as the SH2 containing tyrosine phosphatase SHP-2 (34) (data not shown). It is also possible that one or more of these proteins play a role in the regulation of migration, and further studies are underway to address this point.

C3G Cooperates with Crkl to Increase Cell Migration-- Because overexpression of Crkl enhanced migration and overexpression of CrklSH3(N) inhibited migration, it is likely that one or more CrklSH3 domain-binding proteins also contributes to the regulation of migration in Ba/F3 cells. Several molecules have been reported to bind to SH3 domains of Crk family proteins including c-ABL, SOS, C3G, EPS15, and DOCK180 (19, 20, 28, 35-37). Among these molecules, several have previously been linked to cytoskeletal regulation. The guanine nucleotide exchange factor C3G has been found to be abundant in Crkl complexes and is reported to play an important role in the signaling through Crk family proteins (2, 18, 20, 27, 35, 36, 38). The kinase activity and localization of c-ABL have been reported to be regulated by integrin activation (39). DOCK180 has been found to regulate cytoskeletal function by cooperating with the Crk-Cas complex (40, 41). However, DOCK180 is not expressed in hematopoietic cells, although a homologue may be.

To examine the role of some of these proteins, we overexpressed C3G, c-ABL, or DOCK180 and determined if they cooperated with Crkl to increase cell migration. As expected, overexpression of C3G, c-ABL, or DOCK180 with Crkl led to the detection of increased complexes of Crkl with C3G, ABL, or DOCK180, respectively (Fig. 4A). Cotransfection of C3G and GFP-Crkl further increased cell migration significantly as compared with transfection of GFP-Crkl alone, whereas cotransfection of c-ABL or DOCK180 did not have any further effect on migration of Crkl-overexpressing cells (Fig. 4B). Interestingly, overexpression of C3G alone did not have any significant effects on cell migration. However, C3G cooperated with Crkl to enhance cell migration (Fig. 4C). These results indicate that the Crkl-C3G complex may play an important role in increasing cell migration and that Crkl, but not C3G, is rate-limiting for this effect.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   C3G cooperates with Crkl to increase cell migration. A, Ba/F3 cells were co-transfected with pEBB-Crkl (10 µg) and either pEBB (mock), pEBB-c-ABL, pEBB-C3G, or pEBB-DOCK180 (20 µg each). Cell lysates were prepared 18 h after transfection and were immunoprecipitated with anti-Crkl Ab 5-6 or preimmune mouse serum as a control and then immunoblotted with anti-ABL Ab, anti-C3G Ab, or anti-DOCK180 Ab. Immunoprecipitation and immunoblotting with anti-Crkl Ab (data not shown) detected a comparable amount of Crkl in each transfection. B, the migration ratio was determined in cells transfected with pEBB-Crkl and cotransfected with mock, c-ABL, C3G, or DOCK180 vectors as indicated. C, Ba/F3 cells were co-transfected with pEGFP-control or pEGFP-C-Crkl (20 µg each) plus either pEBB or pEBB-C3G (20 µg each). For B and C, GFP-expressing cells were enriched to >90% purity by FACS 18 h after transfection, cultured in serum- and growth factor-free medium for 6 h, and then tested for migration as described above. Shown are results from two independent experiments (mean ± S.D.). Each experiment was performed using triplicate wells.

The association of C3G with Crkl in cells overexpressing deletion mutants of Crkl was also examined. Overexpression of wild type Crkl, CrklSH2, and CrklSH3(C), but not CrklSH3(N), led to increased formation of Crkl-C3G complexes, consistent with the hypothesis that C3G binds to Crkl through the N-terminal SH3 domain, further suggesting that this complex is important in migration (Fig. 5A). Because we found that Cbl was the most abundant Crkl-SH2-binding partner and that C3G was the only SH3-binding partner of those tested that clearly altered migration, we looked for evidence of complexes that might contain both Cbl and C3G. After ₁ integrin cross-linking, C3G was detected in Cbl immunoprecipitates, and this was increased about 2-fold in cells overexpressing wild type Crkl, further supporting the notion that Crkl is the limiting factor in the formation of these complexes (Fig. 5B).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   C3G-Crkl complexes interact with Cbl after 1 integrin cross-linking. Ba/F3 cells were transfected with pEBB, pEBB-Crkl, pEBB-CrklSH2, pEBB-CrklSH3(N), or pEBB-CrklSH3(C) as indicated. Starvation and 1 integrin cross-linking were performed as described above. A, lysates from transfected cells were immunoprecipitated with anti-Crkl Ab 5-6 and immunoblotted with anti-C3G Ab. B, lysates from transfected cells were immunoprecipitated with anti-Cbl Ab and immunoblotted with anti-C3G Ab or anti-Cbl Ab.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell migration is a fundamentally important process that is not well understood at the level of signal transduction. During development, many precursor cells migrate in response to specific stimuli such as a gradient of chemoattractant molecules. In addition, numerous cells of the adult organism are also capable of extensive migration, particularly cells involved in wound healing and those of the hematopoietic and immune systems. Migration involves a carefully orchestrated series of events that allows cells to extend a cellular protrusion, attach to extracellular matrix proteins through integrins or other adhesive molecules, move in the direction of attachment, and simultaneously detach at the trailing edge (42). Whereas much is known about the events regulating changes in actin structure during cell movement, little is known about the signals from integrins or other receptors that initiate migration.

Members of the Crk family have previously been shown to participate in signaling pathways activated by integrins and have been further linked to the control of adhesion and migration (2, 18, 29, 30, 41, 43).¹ For example, after integrin cross-linking in hematopoietic cells, Crkl is rapidly tyrosine-phosphorylated and binds through its SH2 domain to other cellular tyrosine phosphoproteins (29). The SH3 domains of Crk and Crkl bind constitutively to an overlapping set of cellular proteins that have also been linked in some cases to integrin signaling, including c-ABL, DOCK180, and C3G (17). Overexpression of either Crkii or Crkl has been shown to increase short-term integrin-mediated adhesion of 32D and Ba/F3 hematopoietic cells to fibronectin-coated surfaces (2, 29). Crkii, but not Crkl, has also been linked to migration (44). Klemke et al. (44) showed that Crkii-Cas complexes correlated with enhanced migration of the FG-M carcinoma cell line and that dominant-negative mutants of either CrkII or Cas inhibited spontaneous migration of this cell line. Thus, it is likely that both Crkl and CrkII participate in signal transduction initiated by  integrins and, as adapter proteins, may serve to link or transport critical signaling molecules that regulate adhesion and possibly migration.

The goal of the current studies was to develop a model system in which the effects of specific signaling proteins activated by ₁ integrins could be studied in hematopoietic cells. Compared with more commonly used fibroblastic or epithelial cell lines, hematopoietic cells display higher levels of motility and migration using in vitro assays. The Ba/F3 cell line was selected for this study because its expression of integrin receptors and other adhesion molecules is well characterized.² Cross-linking of ₁ integrins on Ba/F3 cells was found to cause rapid tyrosine phosphorylation of Crkl and association of Crkl with other tyrosine phosphoproteins, notably Cbl. Although Crkii is also expressed at high levels in Ba/F3 cells, compared with Crkl, Crkii was only minimally tyrosine-phosphorylated after integrin cross-linking and was less involved in forming new protein-protein complexes. Transient overexpression of intact Crkl enhanced spontaneous migration on fibronectin-coated surfaces but not on uncoated surfaces. The role of the SH2 and SH3 domains of Crkl were further explored by creating specific deletion mutants and transiently overexpressing these mutants as GFP fusion proteins in Ba/F3 cells. Interestingly, deletion of the SH2 domain resulted in loss of the migration-enhancing properties of intact Crkl but did not, at least at the levels of expression achieved in the present studies in Ba/F3 cells, generate a dominant-negative mutant. In contrast, deletion of the N-terminal, but not the C-terminal, SH3 domain both resulted in loss of the migration-enhancing effects of Crkl and reduced spontaneous migration below control levels. However, the effects of overexpressing Crkl were limited to spontaneous migration, because after the addition of a strong chemoattractant, the chemokine SDF-1, overexpression of Crkl or Crkl mutants had no additional affects.

The results presented here suggest that Crkl plays a similar role in Ba/F3 cells to that of Crkii in epithelial carcinoma cells (44). As shown here, overexpression of Crkii in Ba/F3 cells also enhanced migration, but to a substantially less degree than did Crkl. These results suggest that Crkl may be more significantly involved in integrin signaling and migration in hematopoietic cells, whereas Crkii is more important in non-hematopoietic cells. Interestingly, overexpression of Crkl, but not Crkii, has been reported to transform fibroblasts (45, 46). Overall, our results significantly extend the understanding of the functions of Crkl and provide additional evidence that Crkl is involved in signaling pathways that involve integrins.

Potential mechanisms to explain the migration-enhancing effects of Crkl were explored by identifying the major cellular proteins that associate with the CrklSH2 domain after integrin ligation and by overexpression studies of some of the known proteins that constitutively bind to the Crkl SH3 domains. The major Crkl SH2-binding phosphoprotein was identified as Cbl, a proto-oncogene with multiple YXXP phosphorylation sites that serve as CrklSH2-binding sites (47). v-Cbl is the transforming gene of the Cas NS-1 virus, which causes lymphomas in mice (48). Cbl is the mammalian homologue of the Sli-1 gene in Caenorhabditis elegans that is involved in negative regulation of the epidermal growth factor receptor (49). Targeted disruption of the Cbl locus in mice results in hyperplasia of T cells, extramedullary hematopoiesis in the spleen, and ductal hyperplasia of mammary tissue (50). Recent studies suggest that Cbl directs ubiquitination and degradation of ligand-internalized epidermal growth factor receptors in mammalian cells, whereas v-Cbl induces epidermal growth factor receptor recycling to the surface (51).

Cbl has also been associated with integrin signaling and the cytoskeleton. Cbl is prominently tyrosine-phosphorylated after integrin-mediated adhesion in many cell types (27, 52). It is localized to the cytoplasm and has been shown to interact directly with several cytoskeletal proteins, although the significance of these interactions is unclear (53). Oncogenic forms of Cbl abrogate anchorage dependence of fibroblasts for proliferation (52). After integrin ligation, Cbl forms complexes with other proteins involved in cytoskeletal functional regulation, including Src and PI3K (54). However, the role of Cbl in regulating migration is unclear. In our study, overexpression of Cbl in Ba/F3 cells did alter short-term adhesion or spontaneous migration, although Cbl is already a relatively abundant protein in these cells. The association and activation of PI3K by Cbl may provide a pathway that links Crkl to adhesion and migration, and further studies are warranted to explore this possibility.

A second well known target of the Crkl SH2 domain is Cas, which is likely to be involved in integrin signaling and regulation in some cell types. Cas was discovered as a Crk-binding protein, and has multiple YXXP motifs for binding Crk SH2 domains. Like Cbl, Cas functions as a docking protein after integrin ligation, interacting with 14-3-3 proteins (53, 55). In transient expression assays, Crk-Cas complexes have been shown to activate c-Jun kinase through activation of p21^rac (56). In these cells, activation of Rac was believed to occur through DOCK180, which was brought to Cas by Crkii (41). Homologues of DOCK180 in both Drosophila melanogaster (Myoblast City) and C. elegans (ced-5) have also been linked to the formation of cell extensions, migration, or both (57). Activation of Rac is frequently associated with enhanced migration and adhesion and would explain the enhanced migration of cells transiently overexpressing Crkl (41). However, DOCK180 is not expressed at detectable levels in Ba/F3 and many other hematopoietic cell lines, suggesting that either a related protein exists or that there are other pathways involved. Of note, v-Crk does not activate Rac in PC12 cells but does activate Rho, thereby inducing cell spreading and focal adhesion formation (58).

Overexpression of another Crkl SH3-binding protein, C3G, did enhance spontaneous migration but only when expressed with Crkl. C3G is a guanine nucleotide exchange factor for the small G protein Rap1, also known as smg p21 or Krev-1, originally identified as an anti-oncogenic protein capable of reversing the morphologic transformation of fibroblasts by v-Ki-Ras (59). Rap1 is likely to antagonize Ras by competition with the effector domain. Binding of either Crk or Crkl SH3 domains to C3G has been shown to activate the guanine nucleotide exchange activity of C3G (36), possibly by inducing translocation to the membrane or other sites. The functions of C3G and Rap1 are incompletely understood, but both have been associated with adhesion or migration. C3G is tyrosine-phosphorylated after integrin stimulation in normal cells (35). C3G can interact directly with Cas through the SH3 domain of Cas, in addition to an indirect interaction through Crk or Crkl (60). Rap1 has been shown to be activated during cell adhesion, and the basal Rap1 activity is cell density-dependent (32). However, direct links between C3G, Rap1, and the regulation of adhesion or migration have not been reported. The results presented here suggest that additional studies on the role of C3G and Rap1 and integrin signaling, adhesion, and migration are warranted.

Overall, the present report suggests that Crkl plays a prominent role in mediating integrin-mediated signals in hematopoietic cells and further suggest that the functions of Crkl and Crkii are, at least in part, distinct. In non-hematopoietic cells, integrin-induced formation of Cas-Crk complexes activates migration, possibly through bringing DOCK180 to the cytoskeleton or membrane (44). In hematopoietic cells, Cbl-Crkl complexes predominate, but downstream signals are unclear. C3G is proposed as one pathway that can induce migration through this complex. Crkl is likely to link C3G to Cbl after integrin cross-linking, because Cbl is the predominant target of the Crkl SH2 domain in hematopoietic cells. In cells transformed by tyrosine kinase oncogenes such as p210BCR-ABL, Crkl and Cbl are constitutively tyrosine-phosphorylated and form a permanent complex independent of integrin activation (17, 25). This complex is likely to contribute to the abnormal adhesion and migration exhibited by BCR/ABL transformed hematopoietic cells.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants CA36167 and DK560654 (to J. D. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Adult Oncology, Dana-Farber Cancer Inst., 44 Binney St., Boston, MA 02115. Tel.: 617-632-3360; Fax: 617-632-4388.

³ R. Salgia, E. Quackenbush, J. Lin, N. Souchkova, M. Sattler, D. Ewaniuk, K. M. Kulcher, G. Q. Daley, S. K. Kraeft, U. H. von Andrian, L. B. Chen, J.-C. Gutierrez-Ramos, A.-M. Pendergast, and J. D. Griffin, submitted.

² N. Uemura, R. Salgia, M. Sattler, and J. D. Griffin, unpublished data.

	ABBREVIATIONS

The abbreviations used are: GFP, green fluorescent protein; BSA, bovine serum albumin; FACS, fluorescence-activated cell sorting; SDF-1, stromal-derived factor-1; Ab, antibody.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES