Evidence That DOCK180 Up-regulates Signals from the CrkII-p130Cas Complex*

DOCK180 is one of the two principal proteins bound to the SH3 domain of the adaptor protein CrkII. Here, we have studied the involvement of DOCK180 in integrin signaling. DOCK180 was neither phosphorylated nor bound to CrkII in quiescent NIH 3T3 cells and 3Y1 cells. We found that DOCK180 was phosphorylated and bound to CrkII in NIH 3T3 cells stimulated with integrin and also in 3Y1 cells transformed by v-src or v-crk. The binding of DOCK180 to CrkII correlated with the binding of CrkII to p130Cas, which is a major CrkII SH2 domain-binding protein at focal adhesions. In a reconstitution experiment, expression of DOCK180 induced hyperphosphorylation of p130Cas and a concomitant increase in the amount of CrkII bound to p130Cas. Similarly, binding of DOCK180 to CrkII was also enhanced by the coexpression of p130Cas. Finally, we found that coexpression of p130Cas and CrkII with DOCK180 induced local membrane spreading and accumulation of DOCK180-CrkII-p130Cas complexes at focal adhesions. These findings suggest that DOCK180 positively regulates signaling from integrins to CrkII-p130Cas complexes at focal adhesions.

implicate CrkII in the signaling pathways of both cell adhesion and cell proliferation.
It has been shown that the SH3 domain of CrkII binds to proteins of 135-145, 160, and 180 kDa. We have isolated the cDNAs of the 135-145-and 180-kDa proteins and designated them as C3G and DOCK180, respectively (12,13). C3G is a guanine nucleotide exchange factor for Rap-1, which was originally reported to be a suppressor of Ras (14). Activation of C3G reverses the Ras-induced transformation of NIH 3T3 cells, as does Rap-1 (15). DOCK180 did not show any significant homology to any known protein at the time the cDNA was isolated, except that it contains an SH3 domain at its amino terminus. To study the biological function of DOCK180, we constructed a DOCK180 mutant that is membrane-targeted by the addition of a farnesylation signal. The rationale for this approach is based on the observation that other proteins bound to the SH3 domain of adaptor proteins, such as murine Sos and C3G, are functionally activated by membrane targeting (15,16). Expression of the farnesylated DOCK180 induced the spreading of spindle-shaped NIH 3T3 cells, suggesting that DOCK180 may be involved in cytoskeletal reorganization (13).
p130 Cas was first identified as a 130-kDa protein that was heavily phosphorylated on tyrosine residues and bound tightly to v-Crk and v-Src in cells transformed by these oncogene products (17)(18)(19)(20). p130 Cas contains multiple binding motifs for SH2 and SH3 domains. p130 Cas localizes at focal adhesions and becomes tyrosine-phosphorylated upon integrin stimulation (6,21,22). Tyrosine phosphorylation of p130 Cas recruits CrkII to focal adhesions (23,24); however, the role of CrkII in the integrin signaling is not known. Here, we present data implicating DOCK180-CrkII-p130 Cas complexes in the integrin-mediated signaling at focal adhesions.
Integrin Stimulation-Cells were stimulated by integrin essentially as described previously (32). NIH 3T3 cells were grown in tissue culture dishes, labeled with 32 P i for 4 h, trypsinized, suspended in the same 32 P i labeling medium containing 0.5 mg/ml soybean trypsin inhibitor (Sigma), and kept at 37°C. After 1 h, the cells were plated onto dishes coated either with fibronectin for integrin stimulation or with poly-Llysine as a control. Cells were washed, lysed in lysis buffer, and cleared by centrifugation after various plating periods. In some experiments, 1 mg/ml cytochalasin D was incubated in the cell suspension 10 min prior to plating onto the fibronectin-coated dish. Thirty minutes after plating, cytochalasin D-treated cells were similarly processed. DOCK180 was immunoprecipitated from the lysates and analyzed by SDS-PAGE as described above. To demonstrate that equivalent amounts of DOCK180 were immunoprecipitated, we performed anti-DOCK180 immunoprecipitation from unlabeled cells, followed by immunoblotting with anti-DOCK180 serum.
EGF Stimulation-NIH 3T3 cells expressing the human EGF receptor were serum-starved for 16 h before stimulation. Cells were labeled with 32 P i for 4 h and stimulated with 100 ng/ml EGF. After various incubation periods, DOCK180 was immunoprecipitated and analyzed as described above.
Binding of CrkII to DOCK180, C3G, and p130 Cas -Cells, with or without integrin stimulation, were lysed in lysis buffer. After centrifugation at 15,000 ϫ g for 10 min, the protein concentrations of the cleared lysates were determined using the micro-BCA kit (Pierce). Equal amounts of cell lysate were incubated with anti-Crk monoclonal antibody 3A8 (28) and protein A/G-Sepharose at 4°C for 2 h. After several washings, the immune complexes were separated by SDS-PAGE and probed with antibodies against Crk, DOCK180, C3G, and p130 Cas .
Transfection-293T cells were co-transfected with expression plasmids encoding CrkII, p130 Cas , and DOCK180. After 48 h, cells were lysed in lysis buffer. Cleared cell lysates were incubated with antibody against FLAG, Crk, His, or p130 Cas for 1 h at 4°C. The resultant immune complexes were precipitated with a mixture of protein A-Sepharose and protein G-Sepharose for 30 min at 4°C. After several washings, the immune complexes were separated by SDS-PAGE and probed with antibody against Crk, DOCK180, p130 Cas , or phosphotyrosine.
Microinjection and Cell Staining-NIH 3T3 cells were plated subconfluently onto fibronectin-coated glass dishes (Mattek Corp., Ashland, MA) 16 h before microinjection. Expression plasmids were injected into the nucleus by use of an automated microinjection system (Carl Zeiss, Oberkochen, Germany). After 16 h, cells were fixed with phosphate-buffered saline containing 4% paraformaldehyde and 0.33 M sucrose at 25°C for 15 min; permeabilized with phosphate-buffered saline containing 0.2% Triton X-100 for 3 min; preincubated with 1% bovine serum albumin for 10 min; and incubated with various combinations of anti-DOCK180, anti-Crk, anti-p130 Cas , and anti-vinculin antibodies for 2 h. The cells were further incubated with anti-rabbit or anti-mouse antibody conjugated to fluorescein isothiocyanate or Cy5 (Molecular Probes, Inc., Eugene, OR), mounted in 90% glycerol containing 1,4-diazabicyclo[2.2.2]octane (Sigma), and observed with a confocal laser microscope (Carl Zeiss).

Phosphorylation of DOCK180 upon Integrin Stimulation-In
a preliminary experiment, we found that DOCK180 was phosphorylated on serine residue(s) upon membrane recruitment, suggesting that phosphorylation could be used as an indicator of activation of DOCK180 (data not shown). Because two of the three major proteins bound to the SH2 domain of CrkII (p130 Cas and paxillin) have been shown to be involved in signaling from integrins (4, 6, 22), we examined whether DOCK180 was phosphorylated upon integrin stimulation. NIH

FIG. 1. Phosphorylation of DOCK180 by integrin stimulation and in cells transformed by various oncogenes.
A, NIH 3T3 cells were labeled with 32 P i for 4 h, trypsinized, and suspended in medium containing soybean trypsin inhibitor. After 1 h, cells were plated onto dishes coated with either fibronectin or poly-L-lysine. After the indicated periods, cells were lysed and immunoprecipitated with anti-DOCK180 serum. Immune complexes were analyzed by SDS-PAGE (upper panels). cytoD denotes samples that had been treated with cytochalasin D before plating onto fibronectin-coated dishes; cells were lysed 30 min after plating. In a parallel experiment, unlabeled cells were plated onto dishes, lysed after the indicated periods, and analyzed by anti-DOCK180 serum (lower panels). Bands corresponding to DOCK180 are depicted by arrows to the right. The 220-kDa molecular mass markers are indicated by arrows. B, NIH 3T3 cells were labeled with 32 P i for 4 h and stimulated with 100 ng/ml EGF for the indicated period. DOCK180 was immunoprecipitated with anti-DOCK180 serum and analyzed by SDS-PAGE. In a parallel experiment, unlabeled cells were plated on dishes, lysed after the indicated period, and analyzed by anti-DOCK180 serum (lower panels). Bands corresponding to DOCK180 are depicted by arrows to the right. The 220-kDa molecular mass markers are indicated by arrows. C, Crk-3Y1 and SR-3Y1 are rat 3Y1-derived cells transformed by v-crk and v-src, respectively. Cells were labeled with 32 P i for 4 h and lysed in lysis buffer. DOCK180 was immunoprecipitated with anti-DOCK180 serum and analyzed by SDS-PAGE (upper panel). The cells described above were lysed and analyzed by anti-DOCK180 serum (lower panel). Bands corresponding to DOCK180 are depicted by arrows to the right. The 220-kDa molecular mass markers are indicated by arrows.
3T3 cells were trypsinized and plated onto dishes coated with either fibronectin or poly-L-lysine (Fig. 1A). Phosphorylation of DOCK180 was evident 15 min after plating, when most of the NIH 3T3 cells plated onto fibronectin began to spread, and persisted for 60 min after plating. Cells plated onto poly-Llysine-coated dishes attached to the dish after 15 min; however, they did not start spreading until several hours after plating. DOCK180 was not phosphorylated when cells were plated onto poly-L-lysine. Cytochalasin D, which inhibits actin polymerization and integrin-dependent p130 Cas activation (22), prevented the phosphorylation of DOCK180.
Recently, we showed that CrkII plays a pivotal role in EGFdependent transformation of normal rat kidney cells (33). This finding led us to examine whether EGF induces phosphorylation of DOCK180 in NIH 3T3 cells. As shown in Fig. 1B, phosphorylation of DOCK180 was not detected at any time upon EGF stimulation.
Phosphorylation of DOCK180 in Transformed 3Y1 Cells-Because integrins play a significant role in transformation (34,35), we examined the phosphorylation of DOCK180 in transformed rat 3Y1 cells. We used the Crk-3Y1 and SR-3Y1 cell lines, which express v-Crk and v-Src, respectively. DOCK180 is heavily phosphorylated in the SR-3Y1 and Crk-3Y1 cell lines. In contrast, DOCK180 was only slightly phosphorylated in the parental 3Y1 cell line (Fig. 1C).
Binding of DOCK180 to CrkII-We next examined the binding of DOCK180 to CrkII. In quiescent NIH 3T3 cells, the binding of CrkII to DOCK180 was undetectable (data not shown). To our surprise, integrin stimulation induced the binding of DOCK180 to CrkII in the same NIH 3T3 cells (Fig. 2, left  panel). In contrast, binding of C3G, the other major Crk SH3 domain-binding protein, was not changed by integrin stimulation. Binding of p130 Cas to CrkII was also induced by integrin stimulation, as reported previously (23).
The binding of DOCK180 to CrkII was compared in normal and transformed 3Y1 cells (Fig. 2, right panel). Binding of DOCK180 to CrkII was not detected in the parental 3Y1 cells, as we observed in NIH 3T3 cells. However, DOCK180 was co-immunoprecipitated with CrkII in 3Y1 cells transformed by v-Crk or v-Src. The anti-Crk antibody used for this experiment, 3A8, does not recognize chicken v-Crk protein 2 ; therefore, DOCK180 was co-immunoprecipitated with the endogenous rat CrkII protein, but not with v-Crk, in the v-Crk-transformed 3Y1 cells. Similar amounts of C3G were bound to CrkII in the parental, v-Crk-transformed, or v-Src-transformed cells. The amount of CrkII-associated p130 Cas was increased significantly in the cells transformed by v-Crk or v-Src compared with the parental 3Y1 cells. These results demonstrate that integrin stimulation induces the binding of DOCK180 to CrkII, whereas the other principal Crk SH3 domain-binding protein, C3G, constitutively binds to CrkII.
Increased Binding of p130 Cas to CrkII by Expression of DOCK180 -To further study the binding of CrkII to p130 Cas and DOCK180, we overexpressed these proteins in 293T cells. Mutants of DOCK180 and CrkII were also used to determine the domains required for interaction (Fig. 3A). In contrast to the results obtained using NIH 3T3 cells, we found that FLAGtagged DOCK180 was bound to CrkII in 293T cells with or without the coexpression of p130 Cas (Fig. 3B, lanes 3 and 7). p130 Cas was bound to CrkII without the coexpression DOCK180; however, the amount of CrkII-associated p130 Cas was remarkably increased by the expression of DOCK180 (Fig.  3B, lanes 3 and 4). In addition, we observed the appearance of a slowly migrating form of p130 Cas , which is a hallmark of hyperphosphorylation, by the expression of DOCK180 (Fig. 3B,  lane 3). Association of DOCK180 with p130 Cas required the expression of CrkII (Fig. 3B, lanes 2 and 3). Mutation of the N-terminal SH3 domain of CrkII, CrkII W169L, abolished the DOCK180-dependent increase in the binding of CrkII to p130 Cas and also the appearance of the slowly migrating form of p130 Cas (Fig. 3C). The SH2 mutant of CrkII, CrkII R38V, did not bind to p130 Cas as expected. The DOCK180-delPS mutant, which lacks the Crk SH3 domain-binding domain, did not enhance the binding of CrkII to p130 Cas (Fig. 3D). By contrast, the DOCK180-delGS mutant, which consists of the carboxyl-terminal Crk SH3 domain-binding domain, did enhance the binding of CrkII to p130 Cas ; however, it did not induce the slowly migrating form of p130 Cas .
Tyrosine Phosphorylation of p130 Cas by Coexpression of DOCK180 -It has been shown that phosphorylation of p130 Cas on multiple tyrosine residues causes the retardation of its migration on SDS-polyacrylamide gel (29). Induction of the slowly migrating form of p130 Cas by DOCK180 urged us to examine the tyrosine phosphorylation of p130 Cas in the presence of CrkII and DOCK180 (Fig. 4A). Expression of CrkII alone induced a slight tyrosine phosphorylation of p130 Cas . Expression of DOCK180 alone did not increase the tyrosine phosphorylation of p130 Cas ; however, coexpression of DOCK180 with CrkII strongly enhanced the tyrosine phosphorylation of p130 Cas , concomitant with the appearance of the slowly migrating form of p130 Cas . Both the carboxyl-terminal SH3 domain-binding sites and the amino-terminal region of DOCK180 were required for the hyperphosphorylation of p130 Cas (Fig. 4B). In addition, we confirmed that both the SH2 and SH3 domains of CrkII were also required for the enhancement of p130 Cas tyrosine phosphorylation by DOCK180 (Fig.  4B). Finally, we found that the slowly migrating form of p130 Cas bound to CrkII and DOCK180 in the cells overexpressing all three proteins (Fig. 4C). This result further suggests that DOCK180-CrkII complexes are involved in the hyperphosphorylation of p130 Cas . confocal laser scanning microscope. DOCK180 expressed in NIH 3T3 cells localized diffusely in the cytoplasm (Fig. 5A). Expression of CrkII with DOCK180 induced local membrane spreading at the periphery of the cells. Both DOCK180 and CrkII were localized diffusely in the cytoplasm. Coexpression of p130 Cas and CrkII without DOCK180 also induced slight membrane spreading. Both p130 Cas and CrkII were detected mostly in the cytoplasm. The most prominent morphological change was observed when both p130 Cas and CrkII were coexpressed with DOCK180. These cells showed remarkable membrane spreading, and in these cells, all of DOCK180, CrkII, and p130 Cas were concentrated at the focal adhesions, particularly near the edge of membrane spreading. Because our antibodies against CrkII, DOCK180, and p130 Cas were derived from either mouse or rabbit, we could not detect all three proteins simultaneously. However, most of the DOCK180-expressing cells also expressed p130 Cas and CrkII; colocalization of DOCK180 with CrkII and of DOCK180 with p130 Cas indicates the existence of a DOCK180-CrkII-p130 Cas complex. To confirm that this DOCK180-CrkII-p130 Cas complex was concentrated at focal adhesions, cells were doubly stained with antibodies against DOCK180 and vinculin, which is a component of the focal adhesion complex. As shown in Fig. 5B, DOCK180 colocalized very well with vinculin, particularly at the periphery of the cells. We did not observe remarkable differences in the number and size of focal adhesions between the parental NIH 3T3 cells and the cells expressing DOCK180, CrkII, and p130 Cas .

DISCUSSION
CrkII is involved in many signaling cascades, including those stimulated by integrins and the EGF receptor (36). However, the role of DOCK180, one of the two principal Crk SH3 domainbinding proteins, has not been studied in great detail. We have made several observations that DOCK180 is one of the downstream effectors of the integrin-p130 Cas signaling cascade. First, the phosphorylation of DOCK180 correlates very well with the tyrosine phosphorylation of p130 Cas , which occurs in integrin-stimulated cells as well as in cells transformed by v-src or v-crk (6,22,37). Second, cytochalasin D, which inhibits the integrin-dependent phosphorylation of 130 Cas (22), also inhibits the phosphorylation of DOCK180. Third, binding of DOCK180 to CrkII was detected only in the cells that contained the CrkII-p130 Cas complex. Fourth, expression of DOCK180 enhanced the binding of CrkII to p130 Cas and the tyrosine phosphorylation of p130 Cas . Finally, we found that the DOCK180-CrkII-p130 Cas complex was concentrated at focal adhesions. CrkII is tyrosine-phosphorylated and associates with the EGF receptor upon EGF stimulation (25). However, DOCK180 was neither phosphorylated nor bound to CrkII  upon EGF stimulation (Fig. 1B). 2 This result indicates that not all Crk SH3 domain-binding proteins are recruited equally to Crk SH2 domain-binding proteins, and various stimuli may result in the formation of the distinct Crk complexes.
By contrast to the phosphotyrosine-dependent binding of proteins to SH2 domains, it was initially believed that adaptor proteins and their SH3 domain-binding proteins form constitutive complexes (38). However, the binding of proteins to the SH3 domain of NADPH is regulated by a conformational change of NADPH (39). Furthermore, binding between Grb2 and the Sos exchange factor for Ras is decreased by insulin stimulation (9,40,41). Thus, SH3 domain binding is also subject to regulation. We have found that the binding of CrkII to DOCK180 upon integrin stimulation is another example of signal-mediated binding of an SH3 domain to its target molecule.
CrkII and Src have been suggested to play an important role in integrin signaling (42). It has been proposed that the association of CrkII with p130 Cas promotes further phosphorylation of p130 Cas by a mechanism termed processive phosphorylation (43). We have found that binding of CrkII to DOCK180 is required for this hyperphosphorylation of p130 Cas in 293T cells (Fig. 4). This hyperphosphorylation, which cause p130 Cas to migrate more slowly on SDS-polyacrylamide gel, may function to amplify signaling to CrkII, judging from the increased amount of p130 Cas -associated CrkII (Fig. 3B). Currently, we do not know the biochemical function of DOCK180; therefore, we do not know how DOCK180 contributes to the hyperphosphorylation of p130 Cas . The amino-terminal SH3 domain of DOCK180 may serve as a binding site for another tyrosine kinase.
Genetic studies in Caenorhabditis elegans and Drosophila melanogaster have shown that defects in their DOCK180 homologs, ced-5 and mbc, respectively, resulted in an abnormality of cell movement. ced-5 mutants are defective in the engulfment of cell corpses and the migration of the gonadal tip cells (44). The latter phenotype is rescued by the expression of human DOCK180, demonstrating that Ced-5 and DOCK180 are functionally conserved. The mbc mutant is defective in myoblast fusion, dorsal closure, and cytoskeletal organization (45). In this study, we have shown that DOCK180, coexpressed with p130 Cas and CrkII, localizes at focal adhesions and induces cellular spreading (Fig. 5). Thus, the abnormality observed in ced-5 or mbc mutants may be due to defective signaling from cell adhesion molecules including integrins.