p140Cap protein suppresses tumour cell properties, regulating Csk and Src kinase activity
2007; Springer Nature; Volume: 26; Issue: 12 Linguagem: Inglês
10.1038/sj.emboj.7601724
ISSN1460-2075
AutoresPaola Di Stefano, Laura Damiano, Sara Cabodi, Simona Aramu, Luca Tordella, Alice Praduroux, Roberto Piva, Federica Cavallo, Guido Forni, Lorenzo Silengo, Guido Tarone, Emilia Turco, Paola Defilippi,
Tópico(s)Cell Adhesion Molecules Research
ResumoArticle24 May 2007free access p140Cap protein suppresses tumour cell properties, regulating Csk and Src kinase activity Paola Di Stefano Paola Di Stefano Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Laura Damiano Laura Damiano Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Sara Cabodi Sara Cabodi Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Simona Aramu Simona Aramu Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Luca Tordella Luca Tordella Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Alice Praduroux Alice Praduroux Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Roberto Piva Roberto Piva Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Federica Cavallo Federica Cavallo Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Guido Forni Guido Forni Molecular Biotechnology Center, University of Torino, Turin, Italy Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Lorenzo Silengo Lorenzo Silengo Molecular Biotechnology Center, University of Torino, Turin, Italy Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Guido Tarone Guido Tarone Molecular Biotechnology Center, University of Torino, Turin, Italy Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Emilia Turco Emilia Turco Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Paola Defilippi Corresponding Author Paola Defilippi Molecular Biotechnology Center, University of Torino, Turin, Italy Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Paola Di Stefano Paola Di Stefano Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Laura Damiano Laura Damiano Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Sara Cabodi Sara Cabodi Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Simona Aramu Simona Aramu Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Luca Tordella Luca Tordella Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Alice Praduroux Alice Praduroux Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Roberto Piva Roberto Piva Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Federica Cavallo Federica Cavallo Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Guido Forni Guido Forni Molecular Biotechnology Center, University of Torino, Turin, Italy Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Lorenzo Silengo Lorenzo Silengo Molecular Biotechnology Center, University of Torino, Turin, Italy Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Guido Tarone Guido Tarone Molecular Biotechnology Center, University of Torino, Turin, Italy Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Emilia Turco Emilia Turco Molecular Biotechnology Center, University of Torino, Turin, Italy Search for more papers by this author Paola Defilippi Corresponding Author Paola Defilippi Molecular Biotechnology Center, University of Torino, Turin, Italy Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy Search for more papers by this author Author Information Paola Di Stefano1, Laura Damiano1, Sara Cabodi1, Simona Aramu1, Luca Tordella1, Alice Praduroux1, Roberto Piva2, Federica Cavallo1, Guido Forni1,2, Lorenzo Silengo1,2, Guido Tarone1,2, Emilia Turco1 and Paola Defilippi 1,2 1Molecular Biotechnology Center, University of Torino, Turin, Italy 2Center for Experimental Research and Medical Studies (CERMS), University of Torino, Turin, Italy *Corresponding author. Molecular Biotechnology Center, University of Torino, Via Nizza 52, Turin 10126, Italy. Tel.: +39 011 670 6422; Fax: +39 011 670 6432; E-mail: [email protected] The EMBO Journal (2007)26:2843-2855https://doi.org/10.1038/sj.emboj.7601724 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We recently identified p140Cap as a novel adaptor protein, expressed in epithelial-rich tissues and phosphorylated upon cell matrix adhesion and growth factor treatment. Here, we characterise p140Cap as a novel Src-binding protein, which regulates Src activation via C-terminal Src kinase (Csk). p140Cap silencing increases cell spreading, migration rate and Src kinase activity. Accordingly, increased expression of p140Cap activates Csk, leading to inhibition of Src and downstream signalling as well as of cell motility and invasion. Moreover, cell proliferation and ‘in vivo’ breast cancer cell growth are strongly impaired by high levels of p140Cap, providing the first evidence that p140Cap is a novel negative regulator of tumour growth. Introduction Signalling originated from the integrin family of cell-matrix receptors is central to many physiological and pathological processes such as embryogenesis, inflammatory response, tissue repair and cancer cell progression. Early integrin signalling induces activation of Src family kinases, focal adhesion kinase (FAK) and Rho family GTPases (Damsky and Ilic, 2002; Frame et al, 2002; Miranti and Brugge, 2002; Schwartz and Ginsberg, 2002; Giancotti, 2003; Burridge and Wennerberg, 2004; Cabodi and Defilippi, 2006). Upon integrin-mediated adhesion, Src kinase regulates cell growth, spreading and migration through increased phosphorylation of FAK as well as other key adaptor molecules, like p130Cas (Mitra et al, 2005; Defilippi et al, 2006). As a result of the phosphorylation (Goldberg et al, 2003; Shin et al, 2004), p130Cas recruits other proteins such as Crk and DOCK180 that regulate Rac activation, which is crucial for actin cytoskeleton organisation and cell motility (Chodniewicz and Klemke, 2004). Cells derived from mice deficient in the three members of the Src family (Src/Yes/Fyn), FAK or p130Cas consistently exhibit impaired cell spreading and migration (Ilic et al, 1995, 1998; Klinghoffer et al, 1999). On the other hand, in transformed cells, increased Src activity induces reorganisation of epithelial adhesion systems and the actin cytoskeleton, leading to cell scattering and epithelial-mesenchymal transition (Boyer et al, 1997; Avizienyte et al, 2002). We recently identified p140Cap as a novel adaptor protein indirectly associated to p130Cas (Di Stefano et al, 2004). p140Cap co-distributes with cortical actin and actin stress fibres, but not with focal adhesions. Interestingly, expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin (FN). Moreover, this protein is tyrosine phosphorylated upon integrin-dependent adhesion or EGF treatment, indicating a potential role as a downstream effector of cell-matrix and growth factor signalling (Di Stefano et al, 2004). p140Cap is expressed in the mammary gland and in breast cancer cells such as MCF7, T47D, MDA-MB-231 and MDA-MB-435. By loss- and gain-of-function approaches, we show here that p140Cap affects breast cancer cell motility, invasion and ‘in vivo’ tumour growth and is a novel Src-interacting protein that acts as a negative regulator of Src via activation of Csk. Results p140Cap downregulation by shRNA enhances cell migration and integrin-dependent Src kinase activity Our previous data showed that p140Cap expression affects cell spreading in epithelial cells (Di Stefano et al, 2004). In this work, p140Cap expression was silenced in MCF7 cells by infection with pSuper Retro-p140-2 (p140-2) recombinant retroviruses or by transient transfection with siRNA oligos (siRNA2). Empty pSuper Retro, expressing GFP (Ctr) and scrambled siRNA oligos (siRNA1), were used as negative controls. Stably p140-2 expressing MCF7 cells and MCF7 cells transiently transfected with siRNA oligos showed a 70–85% reduction of p140Cap expression (Figure 1A). These cells were allowed to migrate towards 25 U/ml HGF chemoattractant stimulus. The mean of three experiments (Figure 1B) showed that MCF7 p140-2 cells migrated twofold more relative to Ctr cells, indicating that downregulation of p140Cap expression enhances HGF-stimulated cell migration. Transiently transfected siRNA oligos yielded similar results, confirming that the observed effects were not due to retroviral infection. In addition, MCF7 p140-2 cells transiently transfected with mouse p140Cap full-length complementary DNA to recover protein expression (p140-2/R) (Figure 1A), consistently migrated at a level comparable to control cells. Figure 1.p140Cap silencing enhances cells spreading and migration. (A) p140Cap expression was evaluated by Western blotting on SDS–PAGE in extracts of MCF7 p140-2, MCF7 Ctr, MCF7 cells transiently transfected with control (−), scrambled (siRNA1) or human p140Cap (siRNA2) siRNAs and in MCF7 p140-2/R, transiently transfected with murine pcDNA3.1 Myc-p140Cap to reconstitute p140Cap expression. The same blot was re-probed with antibodies to Src kinase. (B) Cells as in (A) were tested for migration in Transwell assays. Cells were seeded on the upper surface, left to migrate for 9 h in the presence or the absence of HGF (25 U/ml), then fixed, stained and counted. Numbers on the y-axes represent the rate of migration as the ratio between the number of cells migrated in response to the presence and the absence of HGF. (C) MCF7 Ctr, p140-2 and p140-2/R cells were plated on FN and on PL for the indicated times, fixed and stained with phalloidin (Phd). Left upper panel, the histogram represents the mean cell area for each time point in arbitrary units. The area of attached cells was calculated by Metamorph Software in 20 random fields (200 cells) at a magnification of × 60. Left lower panel, the histogram represents the mean ratio between cell length and width as a measure of cell shape at 4 h of adhesion on at least 200 cells. Right panels, representative fields at 30 min (upper) and 4 h (lower) of adhesion are shown at × 60 magnification. The results are representative of six independent experiments (*P<0.05). Download figure Download PowerPoint MCF7 Ctr, p140-2 and p140-2/R cells were allowed to adhere and spread on FN and poly-L-Lysine (PL) as negative control, for different times. Morphometric analysis showed that within 1 h of FN adhesion, MCF7 p140-2 cells, but not MCF7 Ctr and p140-2/R, had an increased area with extensions of large membrane protrusions (Figure 1C, upper panels), thus indicating that downregulation of p140Cap accelerates cell spreading in the early phases of cell adhesion. At 4 h, the difference in the extent of spreading was less evident; however, on FN, whereas Ctr cells showed a typical epithelial polygonal morphology, p140-2 cells presented a more elongated fibroblastoid phenotype (Figure 1C, lower panels). This phenotype was not observed in cells plated on PL (Figure 1C). Cell shape was calculated by the mean ratio between cell length and width on at least 200 cells and the variation was statistically significant. In the early phases of cell-matrix adhesion, cell spreading and migration might depend on Src kinase activation (Yeatman, 2004) and on Rac GTPase (Burridge and Wennerberg, 2004). Indeed, Src kinase activity analysed in MCF7 Ctr cells plated on FN for different times is upregulated within 30 min of adhesion to FN. Interestingly, in p140-2 cells, Src activity follows the same kinetics but is upregulated by four- to sixfold at the examined times (Figure 2A, left and middle panels), thus indicating that downregulation of p140Cap leads to enhanced integrin-dependent Src activation. For what concern Rac GTPase, its activity peaked within 30–60 min of adhesion on FN in Ctr cells and was downregulated at 90 min (Figure 2B, left and middle panels). Interestingly, in p140-2 cells, Rac activity was increased at 60 min and persisted over 90 min of adhesion, thus indicating that p140Cap downregulation induces sustained Rac activation. Both Src and Rac activities were decreased at the control levels in p140-2/R cells (Figure 2A and B, right panels). Therefore, silencing of p140Cap expression enhances the ability of breast cancer cells to respond to the extracellular matrix in terms of cell spreading, increased motility, Src and Rac activation. Figure 2.p140Cap silencing enhances Src and Rac activities. (A) Left panel, MCF7 Ctr or p140-2 cells were plated on FN for 30 and 60 min or kept in suspension (0). Right panel, MCF7 Ctr, p140-2 and p140-2/R were plated on FN for 60 min. Src kinase assay was performed as described in Material and Methods using Enolase as Src substrate. The amount of p140Cap and Src in cell extracts was evaluated by Western blot with specific antibodies. (B) Left panel, MCF7 Ctr or p140-2 cells were plated on FN for 30, 60 and 90 min or kept in suspension (0). Right panel, MCF7 Ctr, p140-2 and p140-2/R were plated on FN for 60 min. Activated Rac was pulled down from 1.5 mg of protein extract with the CRIB domain of PAK and detected by Western blot with anti-Rac mAb (upper panel). Total amount of Rac protein in cell extracts is shown in the lower panel. The histograms show the ratio between active and total protein levels in arbitrary units (*P<0.05). Download figure Download PowerPoint p140Cap overexpression affects cell spreading, migration and invasion of tumour cells For gain-of-function experiments, two independent populations of stable overexpressing cells (MCF7-p140/P9 and MCF7-p140/P23) were generated by transfecting pcDNA3.1-Myc-p140Cap full-length cDNA (Di Stefano et al, 2004) into MCF7 cells (see Supplementary Figure S1 for level of expression and localisation of the endogenous and exogenous p140Cap). MCF7-p140/P9, p140/P23 and the control Mock cells, transfected with the empty pcDNA3.1 vector, were allowed to adhere and spread on FN for different times in the absence of serum. The same number of MCF7-p140/P9, p140/P23 and Mock cells adhered to FN and beta1 integrin activation was not affected (Supplementary Figure S2 and data not shown). Morphometric analysis indicated that within 2 h of adhesion, MCF7-p140/P9 and p140/P23 cells exhibited a round morphology and a reduced area (Figure 3A, left panel), compared to Mock cells. Consistent with the RNA interference (RNAi) data, spreading was recovered at 4 h, suggesting that p140Cap interferes only with the early phases of cell spreading. When cells were plated on FN in the presence of serum, whereas the majority of the Mock cells spread on FN within 20 h, MCF7-p140/P9 and p140/P23 cells remained round although completely viable (Figure 3A, right panel, and Supplementary Figures S1C and S2B). The lack of spreading was also associated with the absence of membrane cell protrusions (Figure 3Bd–f). These results indicate that high levels of p140Cap causes a stronger inhibition of cell spreading in the presence of serum, implying that p140Cap is an effector of multiple and additive pathways. Figure 3.p140Cap overexpression inhibits cells spreading, migration and invasion. (A) MCF7-Mock, p140/P9 and p140/P23 cells were plated on FN for the indicated times in absence (left panel) or in presence of serum (right panel), fixed and stained with Phd. The histogram represents the mean cell area for each time point in arbitrary units. The area of attached cells was calculated by Metamorph Software in 20 random fields (200 cells) at a magnification of × 60. (B) MCF7-Mock (a–c) and p140/P9 (d–f) cells as in (A) (Right panel) were fixed and stained with Diff Quick kit and photographed at 20X magnification. (C) MCF7-Mock, p140/P9 and p140/P23 cells were tested for migration and invasion in Transwell assays. Cells were left to migrate for 9 h in the presence or the absence of HGF (25 U/ml), then fixed, stained and counted. For invasion, transwells were coated with Matrigel and cells left to invade for 48 h. Numbers on the y-axes represent the fold increase in migration in response to HGF compared to nonstimulated cells. The mean values were calculated on six independent experiments (*P<0.05). Download figure Download PowerPoint Transwell migration assays showed that, upon HGF stimulus, cells overexpressing p140Cap migrated four times less than MCF7-Mock cells (Figure 3C, upper panels), suggesting that these cells were strongly defective in migration. ‘In vitro’ invasion assays into Matrigel-coated Transwell showed that within 48 h of invasion, whereas Mock cells invaded the Matrigel properly in response to the stimulus, MCF7-p140/P9 and p140/P23 cells were significantly impaired in their ability to invade (Figure 3C, lower panel). Hence, p140Cap overexpression affects the ability of cells to move towards a chemoattractant stimulus and to invade extracellular matrix. p140Cap overexpression compromises integrin-dependent Src activation and downstream signalling As shown in Figure 4A (left panel), whereas in MCF7-Mock cells Src activity was upregulated within 30 min of adhesion to FN, Src activity was not induced in p140/P9 cells. Src kinase activity was also evaluated by phosphospecific antibodies against the critical tyrosine residue 416 in the Src kinase domain. Upon FN adhesion, tyrosine 416 was phosphorylated only in Mock cells, but not in p140Cap-overexpressing cells (Figure 4A, right panel). Taken together, these results demonstrate that high levels of p140Cap result in inhibition of integrin-dependent Src activation. Figure 4.p140Cap overexpression compromises integrin-dependent Src activation and downstream signalling. MCF7-Mock and p140/P9 were plated on FN or kept in suspension (S) for 30 min and extracted. (A) Left panel, Src kinase assay on Enolase was performed as described in Material and Methods. Right panel, in the same extracts, Src phosphorylation on tyrosine 416 was evaluated using phosphospecific antibodies (Y416). The total amount of Src was evaluated by Western blot. (B) FAK phosphorylation was evaluated in cell extracts using phosphospecific antibodies for the FAK autocatalytic tyrosine 397 (Y397) (Left panel) and FAK tyrosine 925 (Y925) (Right panel). The total amount of FAK was quantified using FAK mAbs. (C) Cell extracts from MCF7-Mock and p140/P9 plated on FN for 30 min were immunoprecipitated with Src antibodies and pre-immune serum as negative control. The amount of FAK co-immunoprecipitated with Src was detected with FAK antibody. The level of immunoprecipitated Src was quantified using Src polyclonal antibodies. (D) Cell extracts as in (A) were immunoprecipitated with anti phosphotyrosine antibodies (P-Tyr) followed by Western blot analysis with p130Cas mAbs. p130Cas was evaluated in cell extracts. (E) Cell extracts as in (A) were analysed for activated Rac as described in Figure 2B. (F) Cell extracts as in (A) were analysed for Akt phosphorylation using phosphospecific antibodies for the Akt Serine 473 Ser(473). The total amount of Akt was quantified using antibodies. The results are representative of three independent experiments. The histograms show the ratio between active or phosphorylated and total protein levels in arbitrary units (*P<0.05). Download figure Download PowerPoint Upon integrin-mediated adhesion, Src phosphorylates downstream effectors such as FAK and p130Cas (Miranti and Brugge, 2002). To investigate whether p140Cap overexpression affects this signalling, both MCF7-Mock and p140/P9 cells were analysed for phosphorylation of FAK. As depicted in Figure 4B (left panel), p140Cap overexpression did not affect integrin-dependent autophosphorylation on FAK tyrosine 397. In contrast, tyrosine 925, a residue shown to be a specific substrate of Src kinase (Brunton et al, 2005; Mitra et al, 2005), was not phosphorylated in p140/P9 cells (Figure 4B, right panel). Moreover, the binding between FAK and Src is decreased in p140/P9 cells compared to Mock-transfected cells (Figure 4C). These results suggest that high levels of p140Cap do not modify integrin-dependent FAK autophosphorylation but rather, alter its ability to associate with Src, thereby affecting Src-dependent phosphorylation on specific residues. Moreover, integrin-dependent phosphorylation of p130Cas (Figure 4D), as well as its association with Crk (data not shown), were decreased in cells overexpressing p140Cap. As a consequence of decreased Src/FAK/p130Cas signalling Rac activity could also be affected. Active Rac was not present in p140Cap-overexpressing cells upon adhesion to FN (Figure 4E), indicating that p140Cap overexpression inhibits integrin-dependent Rac activation. In contrast, as shown in Figure 4F, Akt was equally phosphorylated on Ser 473 in MCF7 Mock and p140/P9-overexpressing cells plated on FN, thus suggesting that Src/FAK/p130Cas pathway is a specific target of p140Cap. p140Cap directly associates with Src kinase p140Cap has been identified for its ability to indirectly associate with the adaptor protein p130Cas (Di Stefano et al, 2004). As Src kinase is one of the major p130Cas-interacting molecules (Defilippi et al, 2006), we evaluated whether Src might also associate with p140Cap. Src and p140Cap reciprocally co-immunoprecipitated in MCF7 cells (Figure 5A), indicating that the two molecules are associated in a complex. p140Cap presents two proline-rich regions, potentially involved in binding to SH3 domains, as well as phosphorylated tyrosine residues (Figure 5B), which can associate with SH2 domains (Figure 5C, upper panel). GST-Src/SH3 or SH2 domains were used to affinity purify endogenous p140Cap from MCF7 cell extracts. As shown in Figure 5C (lower panel), only the GST-Src/SH3 fusion protein pulled down p140Cap, whereas the GST-Src/SH2 recombinant protein was ineffective. Recombinant GST-Cap3 and -5 fusion proteins (Di Stefano et al, 2004), which contain the first and the second proline-rich regions, respectively, and GST and GST-Cap4 fusion proteins serving as negative controls (Figure 5D, left panel), were incubated with the MBP-Src/SH3 fusion protein in an ‘in vitro’ binding assay. Only the GST-Cap5 protein was able to bind to the MBP-Src/SH3 domain (Figure 5D, right panel), indicating that p140Cap and Src directly interact through the binding of the Src SH3 domain with the most carboxy-terminal proline-rich region of p140Cap. Figure 5.p140Cap binds directly to Src SH3 domain. (A) MCF7 cell extracts were immunoprecipitated with mAbs to p140Cap or Src. The immunoprecipitates were blotted with Src antibodies. Molecular weights are indicated on the left. (B) MCF7 cell extracts were immunoprecipitated with mAbs to p140Cap or pre-immune serum. The immunoprecipitate were blotted with anti-p140Cap polyclonal antibodies and re-probed with anti-pTyr antibodies. The results are representative of six independent experiments. (C) Upper panel, a schematic representation of full-length p140Cap domains. Lower panel, GST, GST-Src/SH2 and GST-Src/SH3 fusion proteins were used to pull down p140Cap from MCF7 cell extracts. Bound proteins were immunoblotted using p140Cap polyclonal antibodies (left) or stained with Coomassie blue (right) to quantify the loading. The results are representative of two independent experiments. (D) A total of 2 μg of purified p140Cap GST-Cap3, -4 and -5 fusion proteins (left panel) were incubated in ‘in vitro’ binding assays with 2 μg of MBP-Src/SH2 or MBP-Src/SH3 proteins. Associated proteins were pulled down with Glutathione–Sepharose, eluted and immunoblotted with anti MBP antibodies. The results are representative of two independent experiments. Download figure Download PowerPoint p140Cap-dependent Src kinase inhibition is mediated by Csk activation Csk is a potent negative regulator of Src, due to its ability to phosphorylate the negative regulatory tyrosine 527 on the carboxy-terminal domain of Src (Latour and Veillette, 2001). Densitometric analysis revealed that upon adhesion to FN, phosphorylation of Src tyrosine 527 was increased by 2.5-fold in MCF7-p140/P9 cells in comparison to Mock cells (Figure 6A), suggesting a potential role for p140Cap in regulation of Csk activity. Figure 6.p140Cap inhibition of Src kinase activity is dependent on Csk activity. (A) MCF7-Mock and p140/P9 cells were plated on FN for different times or kept in suspension (S). Phosphorylation of Src Tyr-527 was detected by Western blot with specific antibodies. The same blots were re-probed with antibodies to Src. The histogram shows the ratio between Tyr-527 phosphorylation and Src protein levels in arbitrary units. The results are representative of three independent experiments. (B) Left panels, cell extracts of MCF7-Mock and p140/P9 plated on FN for 30 min, were immunoprecipitated with antibodies to Csk or pre-immune rabbit immunoglobulins (PI) and subjected to kinase assay. The amount of immunoprecipitated Csk was evaluated by Western blot with specific antibodies. Right panel, densitometric analysis of Poly-Glu-Tyr signal is reported in arbitrary units. The results are representative of two independent experiments. (C) Mock-MCF7 and p140/P9 cells were infected with GFP or kinase-negative mutant of Csk (Csk-KD) recombinant adenoviruses. Infected cells were plated on FN for 30 min. Src activity was analysed by in vitro kinase assay as shown in Figure 2A. The level of Csk was detected by Western blot. Arrow indicates immunoglobulin. The results are representative of three independent experiments. (D) MCF7-Mock cell extracts were immunoprecipitated with mAbs to p140Cap, Csk or pre-immune serum (PI) as negative control. Immunocomplexes were analysed by Western blotting using antibodies to Src, Csk and p140Cap, respectively. The results are representative of four independent experiments. (E) Cell extracts from HEK293 transfected with p140Cap were immunoprecipitated with mAbs to p140Cap and GAPDH. Upper panel, Western blot was probed with recombinant Csk produced by in vitro transcription/translation followed by anti-Csk antibodies. Middle and lower panels, the blots were re-probed with anti-p140Cap and GAPDH antibodies. L, lysates; MW, molecular weight. Download figure Download PowerPoint ‘In vitro’ kinase assays showed a twofold increase in poly-Glu-Tyr phosphorylation in MCF7-p140/P9 cells relative to control cells (Figure 6B), demonstrating that p140Cap overexpression upregulates Csk activity. Infection with adenoviruses expressing a kinase-defective Csk mutant (Csk-KD) induced a strong activation of Src in MCF7-p140/P9 cells (Figure 6C), indicating that the presence of the kinase-defective Csk counteracts the ability of p140Cap to inhibit Src activity. These data were further supported by the analysis of Src activity in MCF7-Mock and MCF7-p140/P9 cells transiently transfected with Csk siRNA. As depicted in Supplementary Figure S3, silencing of Csk protein led to increased Src phosphorylation in cells overexpressing p140Cap. Taken together, these data show that p140Cap negatively regulates Src kinase through the activation of Csk kinase. Reciprocal immunoprecipitation of p140Cap and Csk from MCF7-Mock cells showed that the two molecules co-immunoprecipitated (Figure 6D), revealing the association of Csk and Src with p140Cap in a macromolecular complex, which could favour the regulation of kinase activities. To investigate the direct interaction between p140Cap and Csk, we performed Far-Western analysis on p140Cap and GAPDH (negative control) immunoprecipitates using recombinant Csk produced by in vitro transcription/translation as probe. As shown in Figure 6E, the Csk antibody detected a band at 140 kDa only in the p140Cap and not in the GAPDH immunoprecipitates, indicating that the Csk protein binds to p140Cap on the filter. Therefore, this experiment demonstrates that the p140Cap and Csk directly interact. The carboxy-terminal proline-rich region of p140Cap is required for inhibition of c-Src kinase, cell spreading, motility and invasion To assess the role of the carboxy-terminal proline-rich region PPPPPRR in cell signalling, MCF7 cells were transfected with cDNAs expressing either a large Myc-tagged truncated form of p140Cap (MCF7-p140Delta) or a small deleted mutant (MCF7-p140Pro) lacking amino acids 1000–1048, which include specifically the PPPPPRR sequence (Figure 7A). Co-immunoprecipitation experiments indicated that these mutants failed to bind to Src (Figure 7B), confirming the relevance of the proline-rich sequence in Src binding. By immunofluorescence experiments with anti-Myc anti
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