3.1. BCAR3 regulates basal p130Cas phosphorylation in human breast cancer cell lines
In a panel of three estrogen-receptor alpha (ERα)-positive human epithelial breast cancer cell lines examined for BCAR3 and p130Cas expression, we noted that the line with the most abundant level of BCAR3, T-47D cells, contained predominantly a form of p130Cas that migrated more slowly on SDS-PAGE analysis, suggestive of higher basal phosphorylation (, left three lanes). The line with the lowest expression of BCAR3, MCF-7, had the smallest fraction of slowly migrating p130Cas protein. Similarly, among three ERα-negative mesenchymal breast cancer cell lines, the line with the most abundant BCAR3 expression, MDA-MB-231 cells, also contained the most of the slowly migrating form of p130Cas (, right three lanes), whereas the line with the least BCAR3, MDA-MB-435S, expressed the smallest fraction of this form of p130Cas. In contrast, BCAR3 did not correlate with slowly migrating p130Cas when epithelial and mesenchymal breast cancer cell lines were compared. Immunoprecipitation of p130Cas, followed by treatment with lambda protein phosphatase, a phosphatase that dephosphorylates serine, threonine and tyrosine residues, demonstrated that dephosphorylation eliminates the more slowly migrating p130Cas in these cell lines ().
BCAR3 regulates p130Cas phosphorylation in human breast cancer cell lines
In MCF-7 cells, BCAR3 levels are low and endogenous p130Cas runs as a doublet with the majority of the protein in the faster migrating lower band (). Over-expression of hemagglutinin (HA) epitope-tagged BCAR3 markedly shifted the migration of endogenous p130Cas to the slowly migrating upper band (, upper panel). Comparable results were obtained when HA-tagged p130Cas was examined in the presence or absence of co-transfected HA-BCAR3 (, lower panel). Transfection of the BCAR3 homologue NSP1 increased the fraction of both endogenous and co-transfected HA-tagged p130Cas running as the slowly migrating band to a more modest extent. Transfection with NSP3 did not alter p130Cas migration (). The reduction in PAGE migration of HA-p130Cas in BCAR3-transfected MCF-7 cells was due to phosphorylation as phosphatase treatment of the immunoprecipitates resulted in a single lower band (data not shown).
To assess the role of BCAR3 in basal p130Cas phosphorylation in mesenchymal breast cancer cell lines, we isolated nine MDA-MB-231 cell line clones stably transduced with two lentiviruses, one constitutively expressing tetracycline repressor and the other, in a tetracycline-repressor-regulated manner, an shRNA sequence we had previously determined could down-regulate BCAR3 expression. Following addition of doxycycline, expression of BCAR3 was markedly diminished (, lower panel). When the same MDA-MB-231 cell lysates were assessed for p130Cas PAGE migration, it was apparent that reduction in BCAR3 levels resulted in a loss of the slowly migrating phosphorylated p130Cas species and enrichment of the rapidly migrating p130Cas species (, upper panel). Treatment of MDA-231 cells infected with a lentivirus targeting GFP with doxycycline had no effect on p130Cas PAGE migration (). In aggregate, these results demonstrate that BCAR3 expression regulates a form of basal phosphorylation of p130Cas in breast cancer cell-lines that results in reduced PAGE migration.
BCAR3 over-expression results in EGFR-independent p130 Cas tyrosine phosphorylation
p130Cas is best known as an adapter protein that coordinates adhesion and growth factor receptor signaling pathways though Src family kinase-mediated tyrosine phosphorylation of the p130Cas substrate domain. In order to determine whether NSP family members induce p130Cas tyrosine phosphorylation, MCF-7 cells were transfected with vector alone or HA-NSP1, BCAR3 or NSP3, followed by immunoprecipitation of endogenous p130Cas and Western blotting with the anti-phosphotyrosine antibody PY100. BCAR3 over-expression induced tyrosine phosphorylation of both components of the p130Cas doublet to a greater degree than that observed following over-expression of NSP1 (). p130Cas tyrosine phosphorylation was also observed following over-expression of NSP3, although to a lower level than that observed with either other NSP family member. Treatment of the p130Cas immunoprecipitates with lambda phosphatase eliminated detectable tyrosine phosphorylation ().
Over-expression of BCAR3 induces p130Cas tyrosine phosphorylation but inhibition of such phosphorylation does not alter the BCAR3-mediated reduction in p130Cas PAGE migration
EGF receptor (EGFR) signaling has been reported to induce tyrosine phosphorylation of p130Cas [32
]. To determine whether BCAR3-mediated p130Cas tyrosine phosphorylation occurred by an EGF receptor-dependent signaling pathway, we examined immunoprecipitated p130Cas in wildtype MCF-7 cells, MCF-7 cells transiently transfected with BCAR3 and MCF-7 cells stably transfected with HA-BCAR3 (II-6 cells) in the presence or absence of the EGFR tyrosine kinase inhibitor AG1478. Tyrosine-phosphorylated p130Cas was detected in MCF-7 cells either transiently or stably transfected with HA-BCAR3, but not in wildtype MCF-7 cells (). Treatment of the transiently or stably BCAR3-transfected MCF-7 cells with AG1478 failed to alter BCAR3-mediated p130Cas tyrosine phosphorylation. To establish that the MCF-7 cells were responsive to EGF and that AG1478 blocked EGFR signaling, we treated the same three cell types with EGF (10 min, 10 pM) in the presence or absence of AG1478. Although EGF-induced p130Cas tyrosine phosphorylation was not detected in untransfected MCF-7 cells under these experimental conditions, EGF treatment resulted in robust ERK phosphorylation in all three sets of cells that was completely abrogated by AG1478 pre-treatment (). We conclude from these studies that among NSP family members, BCAR3 over-expression preferentially induces p130Cas tyrosine phosphorylation in an EGFR-independent fashion.
3.2 Inhibition of Src kinase reduces BCAR3 over-expression-induced p130Cas tyrosine phosphorylation but not altered p130Cas PAGE migration
The experiments described above suggested that Src family kinase-mediated p130Cas tyrosine phosphorylation might account for the accumulation of a slowly migrating form of p130Cas in BCAR3-expressing cells. To address this hypothesis, we transfected MCF7 cells with HA-BCAR3 and/or HA-p130Cas in the presence or absence of the Src family kinase inhibitor PP2 (10 μM for 20 h), followed by immunoprecipitation with an anti-HA antibody. While transfected HA-p130Cas had no detectable tyrosine phosphorylation, BCAR3 over-expression induced tyrosine phosphorylation of the upper, more slowly-migrating p130Cas band as well as a yet more slowly migrating band that could represent another tyrosine phosphorylated protein that associates with either HA-p130Cas or HA-BCAR3 (, third panel, lane 7). Pre-treatment of the transfected MCF-7 cells with PP2 eliminated the BCAR3-mediated induction of either tyrosine phosphorylated band. In contrast, PP2 treatment had no effect whatsoever on the BCAR3-mediated reduction in migration of HA-p130Cas (, fourth panel, lanes 7 and 8). MCF-7 cells were also transfected with BCAR3 alone and the tyrosine phosphorylation and PAGE migration of endogenous p130Cas in anti-HA BCAR3 immunoprecipitates was examined in the presence and absence of PP2 (first four lanes of ). PP2 treatment did not significantly alter tyrosine phosphorylation of the upper, more slowly migrating endogenous p130Cas band in BCAR3-transfected cells (, third panel, lanes 3 and 4). Once again, PP2 had no effect on the fraction of endogenous p130Cas that migrated as the upper band (, second panel, lanes 3 and 4). These experiments suggest that, while BCAR3 over-expression induces modest Src family kinase-mediated tyrosine phosphorylation of p130Cas, such tyrosine phosphorylation does not account for the BCAR3-mediated and phosphorylation-associated reduction in p130Cas migration.
Src family kinases have been reported to induce p130Cas phosphorylation in a FAK-dependent manner, with p130Cas binding by its SH3 domain to FAK, which in turn binds by a Tyr-397 autophosphorylation site to the SH2 domain of Src family kinases [3
]. Transfection of MCF-7 cells with wildtype Src led to striking tyrosine phosphorylation of p130Cas, far greater than that observed following over-expression of HA-BCAR3 (, lanes 2 and 5). While transfection of MCF-7 cells with FAK alone led to very modest p130Cas tyrosine phosphorylation, co-transfection with FAK and Src augmented such tyrosine phosphorylation and also accentuated the shift of p130Cas to a more slowly migrating species that co-migrated with that observed in BCAR3-transfected cells (, lanes 4 and 5). As previously reported by others, FAK/Src transfection also induced the appearance of a range of yet more slowly migrating p130Cas species that also appear to be tyrosine phosphorylated (, third panel, lane 4), suggesting that progressive substrate domain phosphorylation further alters p130Cas conformation [7
]. Such p130Cas species were not observed in MCF-7 cells transfected with BCAR3.
Dominant-negative forms of Src and FAK do not block BCAR3-mediated p130Cas phosphorylation
Given the importance of Src signaling to p130Cas biology, we sought a non-pharmacologic experimental approach to confirm that Src family kinases were not responsible for the BCAR3-mediated reduction in p130Cas PAGE migration. MCF-7 cells were co-transfected with p130Cas and BCAR3 alone or in combination with dominant negative forms of Src or FAK, followed by assessment of p130Cas PAGE migration. Transfection with dominant negative mouse Src (K296R Y528F) at two molar ratios reduced expression of co-transfected HA-p130Cas but did not reduce the ability of HA-BCAR3 to alter p130Cas migration (). Entirely similar results were obtained with dominant negative FAK (Y397F) () [23
]. These studies support the conclusion that Src-mediated signaling is not required for the BCAR3-regulated phosphorylation of p130Cas that results in altered p130Cas PAGE migration.
3.3. Mapping of BCAR3 and p130Cas domains required for BCAR3-mediated p130Cas phosphorylation
In order to identify the p130Cas structural determinants required for AND-34/BCAR3-mediated p130Cas phosphorylation, we transfected MCF-7 cells with HA-BCAR3 in combination with either wildtype p130Cas or a series of p130Cas mutants in which structural domains had been deleted [6
]. Deletion of the p130Cas SH3 domain (ΔSH3) reduced but did not eliminate the ability of BCAR3 to retard p130Cas migration (). Elimination of the p130Cas substrate domain (ΔSD) appeared to have relatively little effect on the BCAR3-mediated alteration in p130Cas migration; the interpretation of the data provided by this variant was complicated by the fact that under basal conditions, this protein runs as a doublet [25
]. Mutation of the carboxy-terminal p130Cas Src SH3 domain-binding motif from RPLP to RLGS (RPLP) had no effect on BCAR3-mediated reduction in p130Cas migration () [25
]. Similarly, replacement of the p130Cas tyrosine 762 shown to bind to the Src SH2 domain with phenylalanine (Y762F) also had no effect on BCAR3-mediated retardation of p130Cas migration (). These studies suggest that deletion of components of either of two domains previously implicated in Src binding to p130Cas or the principal region phosphorylated by Src do not abrogate the form of BCAR3-mediated p130Cas phosphorylation that is associated with a shift in p130Cas PAGE migration.
p130Cas Src-binding domain mutants are robustly phosphorylated following over-expression of BCAR3
Transfection of MCF-7 cells with AND-34, the murine homolog of BCAR3, induces a shift in p130Cas migration identical to that observed following BCAR3-induced p130Cas phosphorylation (). In order to identify the domains in this family of proteins required for p130Cas phosphorylation, we took advantage of a series of mutant expression constructs we had previously created in which AND-34 structural domains had been deleted. MCF-7 cells were transfected with full-length or mutant AND-34 constructs, followed by analysis of both whole cell lysates and p130Cas immunoprecipitates. Transfection of ΔSH2, a construct in which the AND-34 amino-terminus including the SH2 domain (amino acid residues 1−256) is deleted, eliminated AND-34-induced p130Cas phosphorylation (, lanes 3 vs. 5). When the amino-terminus of AND-34 was expressed containing the SH2 domain but not the subsequent proline/serine-rich domain, no p130Cas phosphorylation was observed (, lanes 2 vs. 8)). In contrast, transfection of SH2-PS, a construct containing both the SH2 domain and the proline/serine-rich domain but lacking the carboxy-terminal 400 amino acids of AND-34 robustly induced p130Cas phosphorylation (, lanes 3 vs. 7). As expected from our prior studies demonstrating that AND-34 binds to p130Cas through AND-34's carboxy-terminal GEF-like domain, immunoprecipitation of p130Cas demonstrated association of p130Cas with full-length AND-34 and ΔSH2, whereas no association of p130Cas with SH2-PS was observed (, lanes 7 vs. 8) [24
]. Thus, at least as judged by these over-expression studies, stable association with p130Cas is not required for AND-34-induced p130Cas phosphorylation.
AND-34/BCAR3's SH2 and proline/serine-rich domains are required for induction of phosphorylation of p130Cas while its p130Cas-binding GEF-like domain is not
To further refine the region of AND-34 responsible for p130Cas phosphorylation, we generated a construct driving expression of human NSP3 in which NSP3's SH2 domain and serine/proline-rich region were replaced with the corresponding AND-34 sequence (SH2PS/N3). Unlike NSP3 itself that induces no shift in p130Cas migration, this chimeric construct induced phosphorylation of p130Cas indistinguishable from that obtained with full-length AND-34 (, lane 2 vs. 5). In order to determine whether it was AND-34's SH2 domain or its serine/proline-rich domain that conferred the ability to induce p130Cas phosphorylation, we generated two further NSP3/AND-34 chimeras in which NSP3's SH2 domain or serine/proline-rich domain were replaced with the corresponding region of AND-34 (SH2/N3 and PS/N3). Transfection of these constructs into MCF-7 cells demonstrated that in the presence of the AND-34 serine/proline-rich domain, the NSP3 SH2 domain could substitute for AND-34's SH2 domain to induce p130Cas phosphorylation (PS/N3, , lane 5). In contrast, the serine/proline-rich domain of NSP3 could not substitute for the corresponding AND-34 domain for induction of p130Cas phosphorylation (SH2/N3, , lane 4). Our studies suggest a model in which both the SH2 domain and serine/proline-rich domain of AND-34 are required to induce p130Cas phosphorylation but in which the specificity of the AND-34 effect resides in the serine/proline-rich domain.
3.4. BCAR3 over-expression leads to p130Cas serine phosphorylation
In order to determine the type of p130Cas phosphorylation induced by BCAR3 over-expression, we carried out 2-dimensional phospho-amino acid analysis. MCF-7 cells or their BCAR3-overexpressing counterparts, clone II-6 cells, were in vivo labeled with 32P for four hours, followed by p130Cas immunoprecipitation. An autoradiogram of the PAGE-separated and transferred immunoprecipitate demonstrated increased phosphate incorporation into p130Cas derived from II-6 cells relative to MCF-7 cells (). The two p130Cas bands were cut out of the membrane, hydrolyzed and analyzed by 2 dimensional thin-layer chromatography in combination with unlabelled serine, threonine and tyrosine standards. Relative to MCF-7 cells, a marked increase in serine phosphorylation was observed from the upper band of p130Cas derived from II-6 cells, with more modest serine phosphorylation noted in the lower band (: n = 2). Basal serine phosphorylation was noted in both forms of p130Cas derived from MCF-7 cells, but at markedly lower levels than that observed in II-6 cells. BCAR3 over-expression in II-6 cells also induced detectable tyrosine phosphorylation of both the upper and lower p130Cas bands, while basal phosphotyrosine was undetectable in MCF-7 cells. However, the level of phosphotyrosine observed in II-6 cells for either the upper or lower p130Cas band were far lower than phosphoserine. No clear evidence of phosphothreonine was observed in either cell line.
BCAR3 over-expression leads to augmented serine phosphorylation of p130Cas
In order to identify specific sites of BCAR3-mediated p130Cas phosphorylation, we carried out tandem mass spectrometric analyses of tryptic digests of immunoaffinity-purified endogenous p130Cas isolated from wildtype MCF-7 cells and from stably BCAR3-transfected II-6 cells [33
]. Given that the purification was carried out with a commercial “high bis” (29:1) PAGE gel system, each preparation contained both slow and fast-migrating p130Cas species. Tryptic fragments corresponding to 88% of the II-6-derived p130Cas and 75% of MCF-7-derived p130Cas sequence were identified in this analysis (). In both p130Cas preparations, three tryptic peptides that had undergone serine phosphorylation were identified. In the first case, serine 139 was phosphorylated in a tryptic peptide encompassing residues 123−143. In the second case, the serine phosphorylation could be localized to one of three serines that are close to the amino terminus, residue 437, 438 or 440, in the p130Cas tryptic peptide SSQSASSLEVAGPGR (residues 437−451: ). In the third case, the phosphorylated, p130Cas residue, serine 639, is the sole serine in the observed tryptic peptide (residues 629−642) and lies within a proline motif, RPLPSPP, previously identified as the p130Cas binding site for the Src kinase SH3 domain [25
]. No tyrosine or threonine phosphorylated peptides were definitively identified in either p130Cas preparation.
To assess whether phosphorylation at serines 139, 639 or 437/438/440 might account for the altered migration of human p130Cas in cells expressing high levels of BCAR3, we carried out site-directed mutagenesis of the corresponding serines in the HA-tagged rat p130Cas expression construct. Mutation of rat p130Cas serine 138 (S138A corresponding to human residue 139) or 638 (S638A corresponding to human serine 639) to alanine did not alter the BCAR3-mediated shift in p130Cas. Among the three candidate human p130Cas serines 437/438/440, mutation of the rat p130Cas serine 436 (corresponding to human residue 437) to alanine (S436A) reduced the BCAR3-induced shift in p130Cas migration while mutation of the rat p130Cas residues 437 and 439 (S437A and S439A corresponding to human serines 438 and 440) had no reproducible effect (). Combined mutation of rat serines 138, 436 and 638 led to further reduction in the p130Cas PAGE shift relative to that observed with S436A alone (). Our results demonstrate phosphorylation of p130Cas serine residues 139, 437 and 639 and suggest that phosphorylation of Ser 437 contributes to reduced p130Cas PAGE migration in BCAR3-overexpressing cell lines.
3.5. BCAR3 regulates adhesion-associated and actin filament-dependent p130Cas serine phosphorylation
As p130Cas is known to play a role in regulating cell signaling pathways related to adhesion to extracellular matrix proteins, we examined BCAR3 regulation of p130Cas phosphorylation in breast cancer cell lines allowed to grow in a normal adherent state or maintained in suspension on HEMA-coated plates. In the mesenchymal MDA-231 cell line, culture on HEMA-coated plates for 24 h led to a pronounced reduction in the phosphorylated p130Cas species (). In the epithelial MCF-7 cell line that normally has only a modest fraction of such phosphorylated p130Cas, maintenance on HEMA essentially eliminated this species (). When MDA-231 cells previously maintained on HEMA were then transferred to a new set of either HEMA or fibronectin-coated plates, augmented autophosphorylation of FAK at tyrosine 397, a signaling event known to correlate with integrin-mediated adhesion, occurred immediately following plating of MDA-231 cells on fibronectin (). In contrast, the previously observed high basal ratio of slow to fast migrating p130Cas species in this cell line was reestablished only after four to six h of further culture on fibronectin but not HEMA (). Such late-phase adherence-dependent p130Cas phosphorylation proved to require BCAR3 as the accumulation of the slowly migrating form of p130Cas was markedly attenuated in an inducible BCAR3 shRNA MDA-231 clone in the presence of doxycycline (). As MDA-231 cells became adherent to the fibronectin-coated plates well before the four to six hours required for accumulation of the slowly migrating p130Cas, we conclude that adhesion is required but not sufficient BCAR3-regulated p130Cas serine phosphorylation.
Among NSP family members, over-expression of AND-34/BCAR3/NSP2 (BCAR3) preferentially induces an adherence and actin microfilament-dependent phosphorylation-associated reduction in PAGE migration of p130Cas
Given that following adhesion of MDA-231 cells to fibronectin, there is a time-dependent accumulation of stress fibers and focal adhesions (data not shown), we next tested whether inhibition of actin polymerization with cytochalasin D would reduce BCAR3-dependent late-phase p130Cas phosphorylation. MDA-231 cells were cultured for 24 h on HEMA followed by re-plating on fibronectin-coated plates and treatment with vehicle or cytochalasin D (2 μM) for varying periods of time. The previously observed time-dependent p130Cas phosphorylation was greatly diminished by cytochalasin D treatment despite the fact that the cells adhered to the plate ().
3.6. BCAR3 expression regulates p130Cas location and breast cancer cell line growth pattern
Doxycycline-induced reduction in BCAR3 expression had a striking effect on the growth pattern of the shRNA MDA-231 clones. All nine of the untreated BCAR3 shRNA-transduced inducible clones grew similarly to wildtype mesenchymal MDA-231 cells as angular cells that frequently lacked cell-cell cohesion (). Following treatment with doxycycline, the MDA-231 clones developed a more epithelial-like morphology, growing as tightly coherent “islands” of cells with a smooth external border that lacked any cellular projections. In contrast to the elongated or angular morphology of the untreated MDA-231 cells, the doxycycline-treated cells grew in a “cobblestone” pattern (). Comparable morphologic changes were not observed in the doxycycline-treated MDA-231 cells stably transduced with a lentiviral shRNA construct targeting GFP (). The morphologic changes observed upon reduction of BCAR3 expression in MDA-231 cells were complementary to those we previously observed following over-expression of BCAR3 in MCF-7 cells [22
]. Stable over-expression of BCAR3 in this epithelial breast cancer cell line results in a marked reduction in homotypic adhesion and the acquisition of an invasive mesenchymal-like growth pattern ().
AND-34/BCAR3 regulates breast cancer cell line growth pattern as well as p130Cas localization
To assess whether the alterations in cell growth pattern observed following knockdown of BCAR3 expression were associated with changes in focal adhesions and the localization of p130Cas, we carried out immunofluorescence studies on the MDA-231 shRNA clones in the presence and absence of doxycycline. In untreated MDA 231 cells, mature focal adhesions were easily identified with anti-paxillin antibodies as peripherally-located structures oriented perpendicular to the cell membrane along actin stress fibers (). Following down-regulation of BCAR3 with doxycycline, stress fibers were lost, actin filaments were restricted to a cortical location and paxillin staining was now identified in plasma membrane-associated structures that were parallel to the axis of the cell membrane (). Analysis with fluorochrome-labeled antibodies against p130Cas and vinculin, two other focal adhesion-associated proteins, confirmed and extended these findings. In untreated MDA 231 clones, both p130Cas and vinculin were once again associated at the edges of cells with radially-distributed focal adhesions. Upon doxycycline treatment, p130Cas and vinculin were now found in complexes parallel to and associated with the plasma membrane (). Doxycycline treatment of the control GFP shRNA MDA 231 cells had no effect on actin filament, paxillin, vinculin or p130Cas distribution ( and data not shown). These studies demonstrate that, in mesenchymal breast cancer cell lines, BCAR3 is required for both basal p130Cas serine phosphorylation and localization of p130Cas to stress fiber-associated focal adhesion complexes.