In this work, we describe the identification and characterization of p140Cap, a novel protein associating with the adaptor molecule p130Cas. p140Cap is highly expressed in brain, testis, and epithelial-rich tissues such as lung and mammary gland. Its immunolocalization into ECV304 cells plated on fibronectin showed that p140Cap codistributes with actin filaments, is absent from focal adhesion structures and colocalizes with p130Cas in membrane ruffles. We also show that p140Cap is tyrosine phosphorylated after integrin-dependent adhesion or EGF treatment and that its expression in NIH3T3 and ECV304 cells inhibits early phases of cell spreading on fibronectin. Therefore, p140Cap is a new molecule involved in integrinand growth factor-mediated signaling that modulate the ability of cells to spread on matrix proteins likely by its association with p130Cas and actin stress fibers.
p140Cap is expressed both in normal epithelial cells, such as bladder ECV304 and FRT, in transformed breast carcinoma T47D, and in cervix carcinoma HeLa cells. It is also expressed in cells of neuroectodermal origin such as human SH-SY5Y (our unpublished data) and mouse NIE115 neuroblastoma cells, indicating that the molecule is widely distributed. In vivo, the p140Cap protein is detectable in mouse brain, testis, kidney, lung, and mammary gland. Interestingly, the mouse protein found in the brain and in the epithelial tissues migrates at 140 kDa, whereas that from testis at 116 kDa. The brain, but not the testis protein, is selectively recognized by a mAb raised against the carboxyterminal region of the protein, suggesting the existence of tissue-specific isoforms probably derived from an alternative splicing in this region. Therefore, the p140Cap protein is expressed in both human and mouse cells and tissues. Indeed, sequence comparison between human, mouse, rat, fish, and Drosophila
genomes revealed the existence of ortholog genes, coding for highly homologous proteins in all these species (www.ensembl.org
). The comparison of the recently published mouse and human genomic sequences allowed us to identify two alternative exons located in the 5′ region of the gene. These exons named 1a and 1b, once spliced on exon 2, give rise to two distinct amino-terminal domains. The 1a-2 spliced isoform corresponds to the unpublished mouse sequence p140, whereas the 1b-2 version is the mouse homolog of the rat SNIP protein (Chin et al., 2000
). Thus, it is possible to conclude that the p140Cap proteins are evolutionary conserved and exist in at least two different N-terminal alternative spliced forms. The recently identified rat SNIP protein, selectively expressed in brain and testis, was shown to be resistant to extraction with nonionic detergents such as NP-40, Triton X-100, or caotropic agents such as urea (Chin et al., 2000
). In contrast, in our experimental conditions, we were able to immunoprecipitate endogenous p140Cap from distinct tissues and cell types. Even if the different properties of the antibodies used to immunoprecipitate SNIP and p140Cap are taken into account, the solubility observed for p140Cap might also be related to different localization of specific isoforms.
The association between p140Cap and p130Cas detected by affinity chromatography was confirmed in ECV304 cells by coimmunoprecipitation and pull-down experiments. By using recombinant p140Cap and p130Cas fragments, we demonstrated that the interaction between p140Cap and p130Cas is mediated by the last 217 amino acids of the p140Cap carboxy-terminal region. Conversely, p130Cas associates with p140Cap in the region encompassing amino acids 544-678. By ProScan domain prediction program, we identified in the p140Cap carboxy-terminal region a charged amino acid-rich domain and a proline-rich region, putative binding sites for the SH3 domains of Abl, Crk, and Itk proteins. Our analysis showed that both the proline and the charged amino acid-rich regions are highly conserved among vertebrates. The fact that during evolution these sequences have been specifically selected suggests that they could be relevant for the function of the p140Cap protein, possibly by mediating interaction with other molecules such as p130Cas. Similarly, the region of p130Cas that associates with the carboxy-terminal fragment of p140Cap in the pull-down experiments is characterized by a proline-rich region that can bind to the SH3 domains of several transducing molecules (Bouton et al., 2001
) and by the presence of a tyrosine in position 668 that mediates binding to the Src SH2 domain (Nakamoto et al., 1997
). The domains that lie on the associating portions of the p140Cap and p130Cas molecules do not provide evidence of a direct binding between the two proteins. Preliminary data of in vitro binding studies indicate that the Cap 5 fragment of p140Cap and the interacting regions on p130Cas do not bind to each other, suggesting that other interacting molecules might mediate the association between p140Cap and p130Cas. However, the presence of Crk, a known interactor of p130Cas, in the p140Cap immunoprecipitates indicates that additional molecules are also recruited in the complex together with p130Cas and p140Cap. In addition to Crk, several proteins directly interacting with p130Cas have been already described, among which the most extensively studied are p125Fak, PTP-PEST, PTP-1B (Polte and Hanks, 1995
; Harte et al., 1996
; Garton et al., 1997
; Manie et al., 1997
), and the Src family kinases (Astier et al., 1997
; Manie et al., 1997
; Tachibana et al., 1997
). Their relevance, if any, to the organization of the p130Cas-p140Cap complex has yet to be defined.
Interestingly, in ECV304 cells plated on fibronectin in the presence of serum, p140Cap clearly localizes on stress fibers that extend throughout the cell body and on cortical actin (see examples in ), indicating an association of p140Cap with actin-enriched structures. In ECV304 cells spread on fibronectin, actin stress fibers end into focal adhesion structures, which by definition are the connecting point between the extracellular matrix and actin cytoskeleton (Burridge and Chrzanowska-Wodnicka, 1996
; Sastry and Burridge, 2000
). Although p140Cap strongly stains actin cables, it is completely excluded from focal adhesions (our unpublished data). The SNIP protein, the rat homolog of p140Cap, was also described to be tightly associated with actin cytoskeleton in differentiated PC12 pheochromocytoma cells (Chin et al., 2000
). Thus, both in epithelial and neuroectodermal cells, the p140Cap is strictly connected to the actin cytoskeleton, suggesting a role in actin cytoskeleton remodeling. In addition to the colocalization with actin stress fibers, p140Cap seems to colocalize with p130Cas in a cytoplasmic punctuate staining as well as in specific membrane ruffle structures induced by PMA treatment (Downey et al., 1992
; Chew et al., 2002
). The association of p140Cap and p130Cas in the cytoplasm and in ruffles and the codistribution of p140Cap with actin cables strongly suggest that p140Cap plays an important role either in the structural organization of the actin cytoskeleton, which affects cell spreading on extracellular matrix, or in functional regulation of cell motility.
Finally, our results show that p140Cap is tyrosine phosphorylated in response to adhesion or soluble mitogens such as serum and EGF. In HEK293, EGF treatment induces a rapid tyrosine phosphorylation of transfected p140Cap, which is dependent on the kinase activity of the EGFR itself, as demonstrated by using the specific EGFR inhibitor AG1478. These results demonstrate that p140Cap is a novel downstream effector of the EGFR in response to its ligand, opening a new perspective for analysis of possible functional roles of p140Cap. Integrin-mediated adhesion induces strong phosphorylation of cellular proteins that have been shown to play multiple functions affecting cell polarity, migration, growth, and survival (Defilippi et al., 1997a
; Giancotti and Ruoslahti, 1999
; Damsky and Ilic, 2002
; Giancotti and Tarone, 2003
). Plating HEK293 cells on integrin ligands stimulates p140Cap phosphorylation within 15 min of cell adhesion, demonstrating that p140Cap tyrosine phosphorylation is also modulated by integrin-dependent adhesion. We and others have previously shown that adhesion to matrix protein activates c-Src kinase, p125Fak, and EGFR and induces Src-dependent tyrosine phosphorylation of p130Cas (Moro et al
; for reviews, see Giancotti and Ruoslahti, 1999
; Damsky and Ilic, 2002
; Giancotti and Tarone, 2003
). Our data also indicate that c-Src kinase activity and p130Cas adaptor are required for integrin-dependent EGFR activation (Moro et al., 2002
). Both the c-Src kinase and the EGFR kinase could be responsible for integrin-dependent p140Cap tyrosine phosphorylation. Together, the phosphorylation of p140Cap by integrin-dependent adhesion or EGF and its association with p130Cas might represent a new example of cooperative signaling induced by growth factors and integrins in the organization of multiple cell responses. Intriguingly in different experimental conditions, p140Cap is associated to actin stress fibers or to ruffles. Therefore, the onset of p140Cap phosphorylation occurring upon cell-matrix adhesion or EGF stimulus and its association with p130Cas might be crucial in regulating distinct cell processes such as cell adhesion or membrane ruffling organization.
On integrin-mediated adhesion, cells attachment to extracellular matrix triggers a complex process of cytoskeleton remodeling that ultimately leads to cell spreading. Tyrosine phosphorylation has been demonstrated to be crucial in these adhesive events (Burridge and Chrzanowska-Wodnicka, 1996
; Panetti, 2002
). The functional role of p140Cap in regulating cell adhesion was studied by expressing the wild-type protein or the mutant lacking the carboxy-terminal domain that mediates the association with p130Cas. Our data showed that p140Cap plays a relevant role in the early phases of cell adhesion inhibiting NIH3T3 and ECV304 cell spreading on fibronectin. In fact, when cells were plated on fibronectin, p140Cap expression dramatically affects actin cytoskeleton organization, maintaining the cells in a round shape. This effect was abolished when the mutant p140-ΔCT was expressed, indicating that the region involved in p130Cas association is required for this effect. Therefore, these data led to the hypothesis that p140Cap might regulate cell spreading through sequestering p130Cas and/or outcompeting its interaction with other proteins involved in modulating spreading in the early phases of cell adhesion. Accordingly, it is well known that GTP loading of Rac is involved in cell spreading and membrane ruffles, through the formation of a CAS/Crk complex that activates DOCK180, an upstream effector of Rac (Klemke et al., 1998
; Cheresh et al., 1999
). Because both p130Cas and Crk participate in complex with p140Cap, a possible mechanism through which p140Cap affects cell spreading might account on inhibition of Rac activity during the early phases of cell adhesion. Alternatively, tyrosine phosphorylation of p140Cap and p130Cas that occur concomitantly with inhibition of cell spreading can represent an additional regulatory step in the ability of cells to spread on extracellular matrix. Together, these data indicate p140Cap as a mediator of integrin- and growth factor-dependent adhesion, involved in modulating cell shape through the association with p130Cas and the actin filaments.