In this paper we have explored the nature of the interaction between C3G and Bcr-Abl proteins. This interaction requires Abi1 and p130Cas, while Cbl seems not to contribute, albeit it forms complexes with C3G. In this regard, a connection between c-Cbl and the regulation of cell migration and spreading through CrkL-C3G-Rap1 and Rac has been described
. Likewise, the interaction between Bcr-Abl and p130Cas seems to be more stable in the presence of Abi1 and Cbl. In contrast, CrkL competes with Abi1 in its binding to the SH3 domain of Abl.
The Abl-SH3 domain interacts with C3G and Abi1, as previously published
[3, 8, 9]. In addition, we have uncovered a novel interaction between the Abl-SH3 domain and the Cbl-SH3-b domain. This has been demonstrated by i) pull-down assays and ii) far western analysis using arrays of purified SH3 domains, which indicate that this interaction is direct. A direct interaction between Cbl and the Abl-related protein Arg was also detected. Additionally, the Cbl-SH3-b domain can also establish an indirect interaction with Bcr-Abl, which involves Abl-Y177 and Grb2
. Therefore, we describe a novel direct interaction, between Cbl and Bcr-Abl through their SH3-b and SH3 domains respectively, in addition to the direct Bcr-Abl-Cbl interaction, involving the Abl SH2 domain and Cbl phospho-tyrosines
A direct association between p130Cas and the SH2 domains of Bcr-Abl or CrkL has been previously described
. Here we show that interactions involving the Bcr-Abl SH3 domain and the SH3 or SH3-b domains of p130Cas are also produced in CML cells. Moreover, the association between the p130Cas SH3-b and the Abl-SH3 domains could be direct, based on far Western experiments. Our results also illustrate a non-canonical association between p130Cas and p87C3G, which involves the proline-rich domain of p130Cas but not its SH3 domain. In this line, recent reports have assigned important roles to p130Cas SH3 and SH3-b domains in FA formation and sustained FA disassembling, respectively
[52, 53]. It is likely that the interaction between p87C3G and Bcr-Abl induces aberrant associations with FA molecules, thus, contributing to the adhesion defects observed in CML cells
In addition, we have uncovered the existence of a series of new interactions, not described previously, between C3G, Bcr-Abl and FA proteins, such as Cbl, p130Cas, Abi1 and CrkL: (i) interaction between p87C3G and Abi1 SH3 or SH3-b domains; (ii) interaction of Cbl SH3-b domain with p87C3G; (iii) interaction between Bcr-Abl SH3 domain and CrkL. All these novel interactions strongly suggest the existence of complex networks between these proteins with dynamic connections involving multiple different domains. This reflects the complexity and plasticity of the regulation of the FA contacts in CML cells.
Different from our results derived from Two-Hybrid assays (unpublished data), SH3 arrays hybridization showed a weak interaction between C3G-SH3-b and Abl-SH3 domain (lower than the positive controls). This weak interaction detected in vitro may be not strong enough to be detected in vivo.
It is noteworthy that the SH3-b domains of C3G, Cbl and p130Cas show similar hybridization patterns with the SH3 domain arrays, which suggests that these three proteins are involved in the regulation of common signaling pathways. In fact, C3G, p130Cas and Cbl directly interact with proteins involved in FAs dynamics, in agreement with its participation in these complexes. Among them, we find Cortactin and Vinexin (VINE), protein tyrosine kinases such as Abl2 and c-Src, and adapter proteins such as CrkL. Immunoprecipitation assays confirmed previously described interactions between p-Paxillin and CrkL or p38α MAPK
[44, 45] and of CrkL with Bcr-Abl
. In addition, we also found a strong interaction between Abi1 and p130Cas, FAK and p38α MAPK and an interaction of p38α MAPK with C3G or CrkL. To our knowledge, these interactions have not been described so far.
In agreement with previous results
[4, 54], our data suggest that C3G plays a key role in the regulation of CML cell adhesion as (i) C3G silencing decreases adhesion to fibronectin, (ii) changes in C3G expression alters the levels of expression and activation of FA proteins, such as FAK, paxillin, CrkL, Cbl and integrin α5, and (iii) C3G silencing increases the interaction between CrkL and paxillin (difficult to observe in control cells) and decreases CrkL interaction with Bcr-Abl. This is relevant, since aberrant interaction between CrkL and paxillin is induced by Bcr-Abl in CML cells
. Therefore, p140C3G could act as a negative regulator of Bcr-Abl-induced abnormal adhesion. A role for C3G in the formation or stabilization of integrin β1- and paxillin-positive FAs has also been described in MEFs
In agreement with other studies
, Abi1 is a positive regulator of adhesion to fibronectin. In contrast, our results on Cbl are different from previous reports. Cbl negatively regulates cell adhesion in most systems by targeting α5-integrin, CrkL and FAK for ubiquitination
[56, 57]. However, our results reveal a positive role for Cbl in CML cell adhesion, as Cbl silencing impaired adhesion of CML cells. p130Cas seems to exert a negative effect in CML adhesion, in agreement with its role in migration and invasion
[58, 59]. Regarding p38α we have contradictory results as p38α knock-down decreased adhesion to fibronectin, but also increased the levels and/or activity of some FA proteins such as paxillin. The decreased adhesion observed in p38α silenced K562 cells point out to a positive regulation of CML adhesion by p38α, according to the role proposed for p38 in adhesion in human melanoma cells
 and in Karpas 299 lymphoma cells based on the effect of the p38α/ß inhibitor SB203580
. However, results derived from studies performed with p38α knock-out cells indicates that p38α plays a negative role in adhesion in mouse embryonic stem cells
 and in embryonic cardiomyocytes
. Differences between cell types might account for these distinct functions of p38α in adhesion. It would be also possible that p38 could play a different role in normal and tumoral cells as adhesion is altered in tumoral cells.
In contrast to the reduced adhesion found in p38α silenced K562 cells, either p38α knockdown or SB203580 treatment induced an increase in the expression of FA proteins, mainly paxillin, integrin α5 and CrkL, as well as an increase in phospho-paxillin, which is normally associated with increased adhesion. This effect was stronger in cells attached to fibronectin. Similar results were observed by Guo and coworkers
. A plausible explanation is that the increase in the expression of FA proteins induced by p38α silencing alters the stoichiometry of the FA complexes, leading to adhesion defects likely due to the impairment of assembly and disassembly of focal adhesion complexes. Additionally, it should be noted that adhesion experiments were performed under serum-deprivation, which induces the activation of p38α and other p38 isoforms (mainly ß)
[62, 63] and can alter the activity of other signaling molecules involved in adhesion such as Rac1
. All this would lead to a potential imbalance of different signaling pathways that could induce a decrease in adhesion. Finally, the fact that double C3G/p38α silenced cells present a similar decreased adhesion to fibronectin that the single knock-down clones, suggests that both proteins could participate in the same signaling pathway regulating cell adhesion. This is supported by the immunoprecipitation studies showing interaction between C3G and p38α MAPK. In addition, C3G and p38α MAPK interact with paxillin and FAK, indicating that they form complexes at the FA. Especially relevant is the interaction between p38α, paxillin and FAK, which strongly indicate that p38α stably interacts with these proteins.
The pathogenesis of CML is caused in part by disorders in the motility of CML cells as well as in their adherence to fibronectin and other substrates
. It has been suggested that Bcr-Abl interferes with the signaling normally induced by ß1 integrin activation, leading to a decrease in adhesion to fibronectin
[14, 64]. In fact, Bcr-Abl mimics integrin activation to establish aberrant interactions with paxillin
. There are evidences about the involvement of the SH3 domain of Bcr-Abl in the regulation of adhesion of leukemic cells through the formation of complexes with α2β1 integrin
. Moreover, interaction of Abi1/2 with the Bcr-Abl SH3 domain negatively regulates its kinase activity
[31, 32, 65]. In fact, Abi1 triggers a downstream pathway, involving WAVE2 that contributes to Bcr-Abl-induced abnormalities in the cytoskeletal and integrin function
. Results presented herein suggest that Bcr-Abl function and consequently CML cell adhesion, could also be regulated by C3G, Cbl, p130Cas, CrkL and p38α MAPK through interactions involving the SH3 domain of Bcr-Abl. Supporting this idea, silencing of Abi1, C3G, Cbl, p130Cas and p38α regulate the expression and/or activation of FA proteins in CML cells. Moreover, p140C3G silencing decreases the Bcr-Abl/CrkL association and reinforces the abnormal interaction between CrkL and paxillin induced by Bcr-Abl, indicating that p140C3G negatively regulates the defective adhesion induced by Bcr-Abl.