Journal of Cell Science 113, 215-226 (2000) 215 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0951 c-Cbl localizes to actin lamellae and regulates lamellipodia formation and cell morphology Robin M. Scaife* and Wallace Y. Langdon Department of Pathology, University of Western Australia, QE II Medical Centre, Nedlands WA 6907, Australia *Author for correspondence (e-mail: [email protected]) Accepted 4 November 1999; published on WWW 13 January 2000 SUMMARY Adhesive and locomotive properties of cells have key roles lamellae, lamellipodia and membrane ruffles. The in normal physiology and disease. Cell motility and induction of lamellipodia and membrane ruffles are also adhesion require the assembly and organization of actin inhibited during cell spreading and migration, conditions microfilaments into stress fibers, lamellipodia and when these structures are normally most prominent. The filopodia, and the formation of these structures is mediated inhibitory effect of truncated c-Cbl expression on by signalling through Rho GTPases. Here we identify c-Cbl lamellipodia formation can be reversed by mutational (a multi-adaptor proto-oncogene product involved in inactivation of its divergent SH2 domain, by the co- protein tyrosine kinase signalling) as an important expression of constitutively active Rac or by the regulator of the actin cytoskeleton. By immunofluorescence overexpression of c-Cbl. This study therefore identifies a microscopy we have determined that c-Cbl co-localizes cytoskeletal role for c-Cbl which may involve the regulation with the adaptor protein Crk to submembranous actin of Crk and Rac, and which is dependent on targeting of c- lamellae in NIH 3T3 fibroblasts and that c-Cbl’s actin Cbl to actin lamellae and the ability to recruit signalling localization requires specific SH3-binding sequences. protein(s) associated with its divergent SH2 domain. Further, we have found that truncation of this SH3-binding domain in c-Cbl profoundly alters the morphology of NIH Key words: Cbl, Actin cytoskeleton, src-homology, Signal 3T3 fibroblasts by inhibiting the formation of actin transduction, Cell shape INTRODUCTION pathways. For example, developmental defects resulting from partial loss-of-function mutations of the Caenorhabditis The cytoskeleton is comprised primarily of microfilaments, elegans Let-23 receptor tyrosine kinase are reversed by microtubules and intermediate filaments, and is required for a mutation of the c-Cbl ortholog Sli-1 (Jongeward et al., 1995; plethora of cellular events. Among these are cell motility and Yoon et al., 1995). Also, engagement of a variety of plasma cell adhesion, which involve the organization of actin membrane receptors, including receptor tyrosine kinases, microfilaments into stress fibers, lamellipodia and filopodia. antigen receptors and integrin receptors results in tyrosine Stress fibers play important roles in the maintenance of cell phosphorylation of c-Cbl and its association with numerous shape and adhesion as a result of their contractile nature and cytoplasmic signalling proteins (Liu and Altman, 1998; their ability to interact with components of focal adhesion Miyake et al., 1998; Ojaniemi et al., 1997; Smit and Borst, complexes (Ben-Ze’ev, 1997; Hall, 1998). Lamellipodia and 1997; Thien and Langdon, 1998). membrane ruffles (sheet-like extensions of the plasma The c-Cbl protein contains many structural domains membrane that contain a meshwork of F-actin) and filopodia involved in protein interactions (see Fig. 1A). Firstly the N- (pointed actin-rich membrane protrusions) play important roles terminal v-Cbl region, which is highly conserved among all in cell spreading and motility (Lauffenberger and Horwitz, Cbl/Sli proteins, has been found to interact directly with 1996; Mitchison and Cramer, 1996). The critical importance of activated protein tyrosine kinases such as the EGF and PDGF the actin cytoskeleton in normal physiology and disease is receptors, ZAP-70 and Syk (Bonita et al., 1997; Bowtell and hence largely due to its central role in cell motility and Langdon, 1995; Galisteo et al., 1995; Lupher et al., 1996, 1998; adhesion. The assembly and organization of the actin Thien and Langdon, 1997). This interaction is dependent on cytoskeleton is subject to input from numerous signal the tyrosine phosphorylation of these kinases and a divergent transduction pathways involving Rho GTPases (Hall, 1998). Src homology (SH) 2 domain (designated as SH2*) in the N- A common component of many signalling pathways is the terminal region of c-Cbl (Meng et al., 1999). The maintenance multi-adaptor proto-oncogene product c-Cbl. Genetic and of SH2* binding to phosphotyrosine residues in protein biochemical studies have implicated c-Cbl in the attenuation tyrosine kinases is essential for fibroblast transformation and of receptor-coupled tyrosine kinase mediated signalling transcriptional activation in T cells by oncogenic forms of Cbl 216 R. M. Scaife and W. Y. Langdon (Bonita et al., 1997; Thien and Langdon, 1997; van Leeuwen Cells were grown to confluency in DME + 10% FCS prior to et al., 1999; Zheng et al., 1998). wounding of the monolayers with a plastic pipette tip. Migration of c-Cbl also contains a highly conserved Ring finger motif of cells into the monolayer wound was monitored using an inverted unknown function while the C-terminal portion contains phase contrast microscope (Leitz, ×20 objective lens) equipped for multiple SH3 domain-binding proline motif sequences as well capture of digital images (Optimas software). as several tyrosine residues which, when phosphorylated, form Immunofluorescence microscopy SH2 domain-binding sites (Andoniou et al., 1996; Blake et al., Cells were seeded at low density onto coverslips coated with 1991; Feshchenko et al., 1998). These Src homology domain- polylysine (0.1 mg/ml) and cultured at least 24-48 hours prior to binding sequences serve as association sites for specific signal fixation in 4% p-formaldehyde/PBS. The fixed cells were then transducing proteins such as Grb2, phosphatidylinositol (PI) 3- permeabilized for 2 minutes with 0.2% Triton X-100 in PBS kinase, CAP, Nck, Vav and Crk, however, it is not known how containing 2.5 mg/ml BSA. Coverslips were rinsed with PBS and these associations mediate, or affect, the biological activity of incubated for 60 minutes with either 0.5 µg/ml TRITC-phalloidin c-Cbl. In hematopoietic cells, attenuation of receptor induced (Sigma) or primary antibodies (2.5 µg/ml anti-c-Cbl mAb, signal transduction by c-Cbl appears to involve effects on Transduction Laboratories catalog number C40320; 100 ng/ml 3F10 anti-HA mAb, Boehringer; 8 µg/ml affinity-purified anti-c-Cbl R2 specific intracellular kinases such as Syk and ZAP-70 (Lupher µ et al., 1998; Murphy et al., 1998; Naramura et al., 1998; Ota polyclonal antibodies (Blake et al., 1993) or 2 g/ml anti-CrkII mAb, Transduction Laboratories catalog number C12620) at 37°C for 90 and Samelson, 1997; Thien et al., 1999) and/or modulation of minutes in PBS containing 2.5 mg/ml BSA. Following a PBS wash the activity of low-molecular mass GTPases such as Rap1 the coverslips were incubated with either 5 µg/ml biotin-SP-goat anti- (Boussiotis et al., 1997). The attenuation of receptor signalling mouse antibodies (Jackson Laboratories, catalog number 115-065- by c-Cbl also involves the enhanced ubiquitination and 003), 1 µg/ml biotin-SP-goat anti-rat antibodies (Jackson degradation of the EGF, PDGF and CSF-1 receptors (Lee et Laboratories, catalog number 112-065-003) or TRITC-conjugated al., 1999; Levkowitz et al., 1998; Miyake et al., 1998, 1999). swine anti-rabbit antibodies (Dako, catalog number R1560) at 37°C Recently, c-Cbl has also been implicated in PI 3-kinase for 60 minutes in PBS containing 2.5 mg/ml BSA. Biotin-labeled dependent macrophage spreading and migration (Meng and antigen-antibody complexes were then visualized by incubation for µ Lowell, 1998). Since PI 3-kinase and Crk are involved in Rac- 60 minutes with PBS containing 2.5 mg/ml BSA and 2 g/ml Alexa dependent effects on the actin cytoskeleton (Kiyokawa et al., 488-conjugated Streptavidin (Molecular Probes). Midbodies were visualized by staining with 7.5 µg/ml anti-Tubulin antibodies (Sigma 1998a; Rodriguez-Viciana et al., 1997) these results suggest mAb, catalog number T-4026) followed by FITC-conjugated anti- that c-Cbl, by binding to these proteins, could play a role in mouse secondary antibody (Silenus) diluted in PBS containing 2.5 cytoskeletal assembly and organization. In order to assay for mg/ml BSA (60 minutes at 37°C). Following a PBS rinse coverslips c-Cbl involvement in modulation of the cytoskeleton we have were mounted with SlowFade Light Antifade reagent (Molecular expressed full-length and truncated versions of c-Cbl in NIH Probes). Images of representative fields were obtained with Comos 3T3 fibroblasts. We report here that c-Cbl is targeted with Crk and Confocal Assistant software (Bio-Rad) following capture on a to membrane ruffle-associated actin lamellae. Deletion of c- Nikon Diaphot 300 microscope equipped for UV laser scanning Cbl SH3 domain-binding sequences prevents its localization confocal microscopy (Bio-Rad MRC 1000/1024). The 543 nm to these structures and as a result profoundly alters the excitation signal from TRITC and the 488 nm excitation signals from organization of the actin cytoskeleton and cell morphology. EGFP and Alexa 488 were collected sequentially with 580/32 and 522/35 nm emission filters, respectively. For each Cbl construct, Our results therefore suggest that by influencing components visualisation of the actin cytoskeleton by immunofluorescence of the cytoskeleton c-Cbl may regulate a number of microscopy (×400 magnification) permitted determination of the fundamental cellular properties. number of cycling cells that contained visually distinct lamellipodia. Triplicate counts of 50 cells each were scored for the presence of strong linear actin staining at the cell periphery. The short actin fragments (see Fig.