- Open Access
C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion
© Maia et al; licensee BioMed Central Ltd. 2013
- Received: 31 July 2012
- Accepted: 18 January 2013
- Published: 23 January 2013
Previous studies by our group and others have shown that C3G interacts with Bcr-Abl through its SH3-b domain.
In this work we show that C3G and Bcr-Abl form complexes with the focal adhesion (FA) proteins CrkL, p130Cas, Cbl and Abi1 through SH3/SH3-b interactions. The association between C3G and Bcr-Abl decreased upon Abi1 or p130Cas knock-down in K562 cells, which suggests that Abi1 and p130Cas are essential partners in this interaction. On the other hand, C3G, Abi1 or Cbl knock-down impaired adhesion to fibronectin, while p130Cas silencing enhanced it. C3G, Cbl and p130Cas-SH3-b domains interact directly with common proteins involved in the regulation of cell adhesion and migration. Immunoprecipitation and immunofluorescence studies revealed that C3G form complexes with the FA proteins paxillin and FAK and their phosphorylated forms. Additionally, C3G, Abi1, Cbl and p130Cas regulate the expression and phosphorylation of paxillin and FAK. p38α MAPK also participates in the regulation of adhesion in chronic myeloid leukemia cells. It interacts with C3G, CrkL, FAK and paxillin and regulates the expression of paxillin, CrkL and α5 integrin, as well as paxillin phosphorylation. Moreover, double knock-down of C3G/p38α decreased adhesion to fibronectin, similarly to the single silencing of one of these genes, either C3G or p38α. These suggest that C3G and p38α MAPK are acting through a common pathway to regulate cell adhesion in K562 cells, as previously described for the regulation of apoptosis.
Our results indicate that C3G-p38αMAPK pathway regulates K562 cell adhesion through the interaction with FA proteins and Bcr-Abl, modulating the formation of different protein complexes at FA.
- p38α MAPK
- Cell adhesion
C3G is a guanine nucleotide exchange factor (GEF) for Rap1 and R-Ras, two members of the Ras family of small GTPases. C3G is a 140 kDa protein, build up with several modular domains clearly differentiated, both structurally and functionally. These comprise a REM-CDC25-H domain, which contains the catalytic or “GEF” domain, and a large proline-rich domain or SH3-binding (SH3-b) domain that interacts directly with Crk isoforms and other SH3-containing proteins such as p130Cas, Hck and c-Abl[1–3]. C3G-mediated Rap1 activation plays critical roles in adhesion. In fact, C3G-Rap1 pathway is essential during early mouse embryogenesis, due to its role in integrin- and paxillin-mediated cellular adhesion and spreading. Moreover, C3G is required for the formation and stabilization of β1-integrin and paxillin-positive FAs. C3G is also an essential activator of Rap1 during cell junction formation, both in epithelial and endothelial cells (revised by). In addition, C3G has been implicated in Rap1-dependent adhesion in many hematopoietic-cell types[6, 7].
We have previously identified a truncated C3G isoform, named p87C3G, which is abundantly expressed in chronic myeloid leukemia (CML) cells. p87C3G interacts with Bcr-Abl and is phosphorylated through a Bcr-Abl-dependent mechanism. Pull-down and immunoprecipitation studies revealed that the interaction between p87C3G and Bcr-Abl involves the SH3-b domain (mainly the second poly-proline stretch) of p87C3G and the SH3 domain of Bcr-Abl. However, this interaction is not direct, as assessed by Two-Hybrid analysis (C. Guerrero, unpublished results). In addition, recent reports have described an interaction between C3G and c-Abl, which involves the C3G SH3-b domain, as demonstrated by in vitro experiments the involvement of the C3G SH3-b domain in this interaction[3, 9]. The existence of an interaction between C3G and Bcr-Abl through CrkL has also been suggested, although this interaction would involve the SH3-b domain of Abl[10, 11].
It is known that Bcr-Abl induces abnormalities in the cytoskeletal function and alters normal interactions between FA proteins and their targets, thus disturbing normal adhesion. Specifically, Bcr-Abl interacts with FA proteins, such as p130Cas, paxillin, tensin and FAK. Bcr-Abl induces p130Cas phosphorylation and its constitutive binding to CrkL, disrupting the normal interaction between p130Cas and tensin. Additionally, Bcr-Abl is involved in the regulation of the leukemic cells adhesion to laminin, fibronectin and collagen through the complex formation with integrin α2β1, being the Abl-SH3 domain the responsible of these effects. As a result, CML cells have a reduced capacity to adhere to stromal layers and to fibronectin but show increased adhesion to laminin and collagen type IV[14, 15]. This is important since altered adhesion to extracellular matrix proteins could lead to premature release of CML cells from the bone marrow, resulting in a deregulated hematopoiesis.
Recently, we have described a functional relationship between C3G and p38α MAPK in the regulation of apoptosis in CML cells and in MEFs[16, 17]. Another common issue is that, similarly to C3G, p38MAPKs play important roles in the regulation of cell adhesion and migration processes[18, 19]. p38 MAPK is involved in the migration of mesoderm during the embryogenesis and mediates migration of several cell types, including tumor cells. p38 MAPK also regulates adhesion; cells lacking p38α showed increased adhesion to several ECM proteins[18, 22], which correlates with increased phosphorylation of the FA proteins FAK and paxillin. These results indicate that p38α negatively regulates cell adhesion.
The role of the adapter proteins CrkL, p130Cas and Cbl in CML is well documented[1, 12, 23, 24], and the association between Cbl and C3G, through CrkL, has been described in CML cells, fibroblasts, NK cells and T-cells[11, 25–28]. Direct interaction between C3G and p130Cas has also been reported. Interestingly, all these proteins contain SH3 and/or SH3-b domains and participate in cellular adhesion processes, being potential mediators of the Bcr-Abl/C3G interaction.
On the other hand, several Abl SH3 binding proteins have been identified, such as 3BP-1, Abi1, Abi2, AAP1, RIN1, and PAG. Remarkably, Abi1/2 has both, SH3 and SH3-b domains, which would allow its simultaneous interaction with Bcr-Abl and C3G. This arises the possibility that Abi1/2 may act as a mediator in the C3G/Bcr-Abl interaction.
In this work we have investigated possible mediators of the C3G-SH3-b/Bcr-Abl-SH3 domains interaction. Considering that the SH3 domain of Abl is the one involved in the regulation of the leukemic cells adhesive and invasive properties, one of the hallmarks of the pathogenesis of CML, and knowing the role of C3G in cellular adhesion, we hypothesize that C3G could modulate CML cells adhesiveness through its interaction with Bcr-Abl at the FAs. We have also evaluated the participation of p38α MAPK in the regulation of adhesion in CML and its functional interaction with C3G.
The Bcr-Abl SH3-domain interacts with C3G, Abi1, Cbl and p130Cas
Abi1-SH3/SH3-b, Cbl-SH3-b and p130Cas-SH3/SH3-b domains interact with C3G and Bcr-Abl
To study these interactions in depth, we performed pull-down assays, in K562 lysates, using the SH3 and SH3-b domains of Abi1, Cbl and p130Cas fused to GST as baits. Both Abi1-SH3 and SH3-b domains bound to Bcr-Abl, in agreement with the literature. In addition, both domains interacted with p87C3G and a slight interaction of the Abi1-SH3-b domain with p140C3G was also detected (Figure 1B). Regarding p130Cas, Figure 1C shows that the three tested domains (SH3-binding, P1 and P2) interact with Bcr-Abl, albeit with different affinities. Additionally, p130Cas-SH3 domain interacted with p140C3G, in agreement with other studies, while P1 and P2 domains associate preferentially with p87C3G. On the other hand, Cbl-SH3-b domain clearly interacts with both Bcr-Abl and p87C3G (Figure 1D). Association of Cbl with Bcr-Abl, as well as with CrkL or Grb2 (used as controls) in K562 cells has been described previously, although the interaction described by these authors involved the Bcr-Abl SH2 domain and Cbl phospho-tyrosines. The preferential interaction of p87C3G over p140C3G with most of the tested domains probably reflects its highest expression in CML cells (Additional file1).
Interaction of Bcr-Abl with CrkL
CrkL, the major substrate of the tyrosine kinase Bcr-Abl, interacts directly with Bcr-Abl through its amino terminal SH3 domain and the SH3-binding region of Abl[36, 37]. Here we show that the Abl-SH3 domain also interacts with CrkL (Additional file3A). One possible explanation is that CrkL, similarly to CrkII, has a putative SH3-b domain inside the SH2 domain that could mediate this interaction. Sequence alignment revealed that CrkL lacks the SH3-b domain present in CrkII, although it preserves a canonical SH3-b PXXP motif that itself is sufficient for binding to SH3 domains[29, 36] (Additional file3B). However, the CrkL-SH2 domain does not interact with Bcr-Abl, either by pull-down experiments (Additional file3C) or in Two-Hybrid analysis (Additional file4: Method 1 and Additional file3D), indicating that the interaction between CrkL and Bcr-Abl, involving the Abl-SH3 domain, is not direct. An indirect interaction between these two proteins independent of the Bcr-Abl proline rich domain has been previously suggested.
Abi1 and p130Cas knock-down alter the interaction between C3G and Bcr-Abl
C3G, Cbl and p130Cas bind directly to common adhesion proteins
To examine whether the interactions between the SH3 and/or SH3-b domains of the studied proteins were direct, we performed hybridizations with SH3 domain arrays (Panomics) containing 38 SH3 domains (Array I) and 36 SH3 domains (Array II) (Additional files5,6,7 and Additional file8: Table S1 and Additional file9:Table S2), using His-tagged-SH3-b domains from C3G, Cbl and p130Cas (P2). The C3G-SH3-b domain directly associates with the SH3 domains of LCK, SPCN, cortactin, Yes1, Abl2 (ARG), SLK, c-Src, Hck, VAV-D2, Y124, PEXD, BTK, and Stam and with less affinity to NOF2-D1, VAV-D1, Abl and PLCγ (Additional file5, Array I). Results with the array II confirmed the direct binding of C3G with c-Src and Abl2 and revealed its direct binding to NCK1-D2, OSF, Tec, PIG2, VINE-D3 and, with less affinity, PI3α. The known binding to CRKL-D1 was confirmed in this experiment and contrarily to what we expected, no direct interaction with the Abi2-SH3 domain (AbI2B) was found. Binding to p130Cas-SH3 domain (BCA1) was not detected because the C3G-SH3-b fragment used lacks the upstream sequence involved in this interaction.
Similarly to the C3G-SH3-b domain, the Cbl-SH3-b domain clearly binds to LCK, SPCN, cortactin, Yes1, Abl2, SLK, c-Src, Hck, VAV2-D2, NOF2-D1, VAV-D1, Y124, PEXD, BTK, Stam and Abl (array I), Abl2, CrkL-D1, NCK1-D2, OSF, PI3α, Tec, PIG2, VINE-D3 and c-Src (array II) (Additional file6). Interaction with PI3kα has been reported. Also, in agreement with previous findings, a direct, although weak, interaction between the Cbl-SH3-b domain and the CrkL-SH3 (CrkL-D1) domain was observed, although in CML cells Cbl interacts preferentially with the CrkL-SH2 domain. In contrast to C3G, Cbl also interacts directly with Itk, Dlg2, ITSN-D1 and TXK (array I) AbI2B (Abi-2), M3KA, SNX9, VAV3-D2, and SH3-1 (SH3-containing GRB2-like protein 1), being this last one in agreement with published data.
The p130Cas-P2 domain (SH3-b domain-2) renders a less specific hybridization, probably due to the smaller size of this fragment. Similarly to C3G and Cbl, it presents a clear direct association with LCK, Cortactin, Yes1, Abl2, SLK, c-Src, Hck, VAV2-D2, Y124, PEXD, BTK, ITSN-D1 (array I), Abl2, NCK1-D2, OSF, PI3α, Tec, PIG2, VINE-D3 and c-Src, (array II) and it binds with less affinity to Stam, BLK, Abl and CrkL-D1 (Additional file7). Similarly to Cbl, p130Cas-P2 binds with high affinity to Itk, ITSN-D1 (array I), AbI2B, SH3-1 and SNX9 and weakly to M3KA and VAV3-D2. p130Cas-P2 presents an exclusive strong interaction with MLPK3, PSD95, PI3-ß (array I), GRB2L-D1, NE-DLG and NOF2-D1 (array II), and binds with less affinity to amphiphysin, RasGAP (array I), CSKP, BIN1 and EFS. The direct interaction between p130Cas-P2 and CrkL-SH3 domain (CRKL-D1) contrasts with previous reports showing a direct association between CrkL and p130Cas through the CrkL-SH2 domain in Bcr-Abl expressing cells and CML patients.
C3G, Cbl, Abi1 and p38α MAPK knock-down expression inhibits K562 adhesion to fibronectin, while p130Cas silencing increases it
C3G and p38α MAPK form complexes with focal adhesion proteins
Cell adhesion to the extracellular matrix is mediated by integrins through regulation of the formation of different FA complexes, being a bidirectional cross-talk between integrins and FA proteins. These complexes are constituted by protein kinases, such as FAK, and scaffold proteins, such as paxillin or p130Cas. In CML cells, it is known that paxillin interacts with protein kinases such as Src, p38 MAPK, c-Abl and FAK and with Bcr-Abl through CrkL. C3G also interacts with Bcr-Abl and p130Cas, which agrees with data presented here.
C3G modulates the expression and activation of focal adhesion proteins
To confirm these results through a complementary experimental approach, we analyzed the expression of these FA proteins in K562 C3G knock-down clones. Similarly to that observed in C3G overexpressed clones, paxillin expression decreased in C3G knock-down cells (Figure 6C). The decrease in paxillin expression was confirmed by immunofluorescence confocal microscopy of K562 cells attached to fibronectin (Figure 6D). In contrast, the levels of phospho-paxillin p33 and p68 isoforms decreased in C3G silenced clones, while they were increased by C3G overexpression, (Figure 6C). Additionally, a decrease in Cbl protein levels and a slight decrease in phospho-CrkL were also observed in the C3G knock-down clones, while no significant changes in p130Cas and CrkL levels were observed (Figure 6C). Fibronectin partially reversed the effect of C3G silencing on paxillin expression and phosphorylation, in contrast to the effect of fibronectin in C3G overexpressing clones. This is in agreement with the participation of C3G in the regulation of the outside-in pathway triggered by fibronectin.
We next analyzed the effect of C3G knock-down on FA proteins interactions by immunoprecipitation assays performed in control (shCT) and C3G knock-down K562 cells grown on fibronectin. Remarkably, C3G silencing produced a decrease in the association between CrkL and Bcr-Abl, while it promoted the CrkL-paxillin interaction (marked by arrows) characteristic of tumoral cells (Figure 6E).
Collectively, our data suggest that C3G plays a role in the regulation of the expression of FA proteins, and in their activation and association dynamics. Similarly to our previous published data on apoptosis, C3G seems to play a dual role in the regulation of cell adhesion, as both upregulation and downregulation of C3G expression have similar effect on the expression of proteins involved in the signaling pathways regulating cell adhesion.
p38α MAPK regulates FA protein expression and activation in a C3G antagonistic fashion
Cbl, Abi1 and p130Cas regulate the expression and activation of FAK and paxillin in K562 cells
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[24, 51].
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.
Our data indicate that C3G plays a relevant role in the regulation of adhesion in CML cells by interacting with Bcr-Abl, p38αMAPK, Cbl, p130Cas, Abi1, FAK and paxillin at the focal adhesions. It is likely that p140C3G levels in CML cells might be tightly controlled, as either its overexpression or downregulation induce a decrease in the protein levels of key FA proteins, such as paxillin and FAK. A similar behavior was observed in the regulation of apoptosis in CML cells. In this line, it is plausible to hypothesize that the C3G isoform, p87C3G, participates in the perturbation of the adhesive properties of the CML cells through interaction with the Bcr-Abl-SH3 domain (regulator of the adhesion) and the establishment of aberrant associations with FA proteins, thus avoiding their normal interaction with other proteins, including p140C3G. Further investigation is warranted to ascertain the relationship between p140C3G and p87C3G in the regulation of CML adhesion.
Our data also support a role for p38α in cell adhesion in CML as p38α knock-down decreases adhesion to fibronectin and changes the levels and/or phosphorylation state of some FA proteins. In addition, because silencing of either p38α and/or C3G induced a similar reduction in adhesion, p38α might be acting in the C3G pathway. Future studies will uncover the precise function of p140C3G, p87C3G and p38α in the regulation of all the different proteins involved in adhesion.
Cell lines and expression constructs
K562 (ATCC, CCL243), a human cell line derived from a patient with CML in terminal blast crisis, was maintained in RPMI 1640 containing 10% fetal bovine serum (FBS). C3G overexpressing construct, pLTR2C3G, and constructs containing shRNAs to target either human C3G or p38α genes using pSuper.neo+gfp vector (Oligoengine) have been described previously[16, 66].
K562 infection with shRNA lentiviral particles
Expression of C3G, Abi1 and Cbl was silenced in K562 cells by transfection of (h) Lentiviral Particles: C3G shRNA (sc-29863-V), Abi1 shRNA (sc-40306-V), Cbl shRNA (sc-29242-V) and control shRNA (sc-108080) from Santa Cruz Biotechnology, following the manufacturer`s protocol. For p130Cas silencing we used p130Cas shRNA (MISSION® Transduction particles NM_014567) and control shRNA (MISSION® pLKO.1-puro Empty Vector Control Transduction Particles) from Sigma. Puromycin-resistant clones were selected after 15 days in culture in 10%FBS/RPMI media supplemented with 2 μg/ml puromycin.
Antibodies against: C3G (C-19, G4 and H-300), CrkL (C-20), Bcr (G6), c-Abl (2411)–, Cbl (C-15), Abi1 (H-80), p130Cas (35B.1A4), Grb2 (C-23), GST (B-14), p38α (C-20) and integrin α5 (H-104), p-FAK (ser 722) were from Santa Cruz Biotechnology; β-Tubulin clone 2-28-33 and Actin, clone MM2/193 were from Sigma-Aldrich; FAK, phospho-p130Cas (Tyr249) and phospho-CrkL (Tyr207) were from Cell Signaling Technology; paxillin (clone 349) was from BD Biosciences; phospho-paxillin (Y118) was purchased from Life Technologies. Anti-His6-Peroxidase was from Roche. Anti-Abi1 antibody (SSH3BP1) is a monoclonal antibody from Abcam (#ab11222). C3G-1008 serum was used in the immunofluorescence experiments.
As secondary antibodies we used: Cy3 anti-rabbit, Cy3 anti-mouse, Cy5 anti-rabbit, Alexa Fluor 488 anti-goat, FITC anti-rabbit, and FITC anti-mouse from Jackson ImmunoResearch Laboratories, Inc. For F-actin detection we used Oregon Green® 514 phalloidin from Life Technologies.
Cell adhesion assay
Adhesion of K562 cells infected with shRNA lentivirus (knock-down) was performed as described. Briefly, 96-well plates were coated with fibronectin at 50 μg/ml in PBS and then blocked by the addition of BSA 1%. Cells were washed with PBS and resuspended in medium without serum at 5×106cells/ml. Then, cells were labeled with Calcein AM fluorescence dye (BD Biosciences) according to manufacturer`s instructions and added to each microplate well in 100 μl at 5×106 cells/ml. After 3 h at 37°C, plates were washed 3 times with medium and inverted onto filter paper to blot excess liquid. The remaining calcein-labeled cells were suspended in PBS and used to determine the total of cells added. Adhesion was measured in a fluorescence plate reader (ULTRA Evolution; Serial number: 12903200010; Firmware: E 1.03 02/03 EVOLUTION; XFLUOR4 Version: V 4.50). The percentage of adhesion was determined by dividing the corrected (background subtracted) fluorescence of adherent cells by the total corrected fluorescence of cells added to each microplate well.
Whole cell lysates were prepared using cell lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100 (or NP-40), 0.1% Na deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF, 10 μg/ml Aprotinin and 10 μg/ml Leupeptin. Cell debris was removed by spinning at 10000 g for 10 min at 4°C.
Immunoprecipitation was performed as described. Prior to the immunoprecipitation, lysates were precleared by incubation with washed GammaBind G Sepharose beads (GE Healthcare Life Sciences) for 30 minutes.
Constructs: Abi1-SH3, Abi1-SH3-b (SH3-binding), Cbl-SH3-b and p130Cas-SH3, p130Cas-P1 (proline-rich domain 1 or SH3-b1) and p130Cas-P2 (SH3-b2) domains were expressed as GST-fusion proteins. To do so, fragments were amplified by PCR and cloned into Eco RI-Xho I sites of pGEX-4T-1 (GE Healthcare Life Sciences). The oligos used were Abi1SH3-F: 5´-GGGGAATTCCCCAAGAATTATATTGAGAAAGTT-3´ and Abi1SH3-R: 5´-GGGCTCGAGTTAATCAGTATAGTGCATGATTGA-3; Abi1SH3b-F: 5´-GGGGAATTCCCCATTGCTGTGCCTACA-3´ and Abi1SH3b-R: 5´-GGGCTCGAGCAGCCTCCTCATCTTCAT-3´; CblSH3b-F: 5´-GAGGAATTCCCGCCTTCTCCATTCTCC-3´ and CblSH3b-R: 5´-GGGCTCGAGAGGTGGCAGTTTTGGCAC-3´; p130CasSH3-F: 5´-AGGGAATTCAACCACCTGAACGTGCTG-3´ and p130CasSH3-R: 5´-AGGCTCGAGGCCCACCAAGATCTTGAG-3; p130CasP1-F: 5´-AGGGAATTCGATAAGAAGCCAGCAGGG-3 and p130CasP1-R: 5´-AGGCTCGAGGTAGACGCTGTCTGGCTG-3´; p130CasP2-F: 5´-AGGGAATTCTCACTGCTCTTCAGACGG-3´ and p130CasP2-R: 5´-GGGCTCGAGGGTGAACTTAGGGGGTGA-3´. GST-Abl-SH3 and GST-C3G-SH3-b constructs have been described previously.
Pull-downs were carried out by incubating 1 mg of protein extract in lysis buffer with 12 –μg of GST-fusion proteins, bound to glutathione-sepharose 4 fast flow beads (GE Healthcare Life Sciences), for 2 hours at 4°C. Complexes were subjected to 3 washes in lysis buffer and boiled in loading buffer before SDS- PAGE.
Immunofluorescence was performed essentially as described.
SH3 domain arrays
Panomics´ SH3 domain Arrays are designed to determine whether a protein of interest binds to multiple SH3 domains (Panomics). Recombinant proteins of interest (C3G-SH3-b, Cbl-SH3-b or p130Cas-SH3-b domains) were engineered with a N-Terminal His-Tag by cloning the corresponding fragments into the pET15b-derived plasmids (Novagen-EMD Millipore) pETEV15b and pET15b-NBKSXa (Additional file11: Methods 2 and Additional file12: Method 3). Proteins were expressed in E. coli strain BL21 (DE3) and purified by affinity chromatography as described. Purified proteins were hybridized with the SH3 Domain Array I (Panomics, Cat #MA3010), and Array II (Panomics, Cat #MA3011), following the manufacturer`s protocol. Hybridizations were visualized with peroxidase-conjugated anti-His antibodies, followed by ECL plus (Amersham). Spots with intensities similar or stronger than positive controls (pos) were selected as ligands of the corresponding SH3-b domain.
Quantification of band intensity was performed by Image J version 1.24 software.
Data are represented as mean ± SEM. Statistical analysis was performed using an unpaired Student's t-test. Results were considered significant when p<0.05(*).
This work was supported by grants form the Spanish Ministry of Health (ISCIII) [FIS-PI070078 to CG and FIS-PI070071 to AP], by grants from the Spanish Ministry of Economy and Competitiveness [SAF2010-20918-C02-02 to CG, SAF2010-20918-C02-01 to AP and BFU2009‐08389 to JMP], by a grant for Research in Biomedicine from the Council of Health and Social Welfare of Junta de Castilla y León and by grants from the Council of Education of Junta de Castilla y León [CSI06A09 and SA157A12-1], Spain (to JMP and CG). All funding was cosponsored by the European FEDER Program.
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