Using the Paxillin LD motifs to interfere with interactions targeting FAK to FAs, as a strategy to inhibit FAK
Our main aim was to develop and test a new strategy that would block both enzymatic and scaffolding functions of FAK, specifically at FAs, as a possible new approach for FAK inhibition. To achieve this, we set out to interfere with interactions responsible for FA targeting. The FAT domain is both necessary and sufficient to drive FAK at FAs. Previous work from our group and others revealed that the two hydrophobic pockets (HPs) formed by the FAT domain are essential for FA targeting [33, 34, 43]. The best characterized interaction of the HPs is with the LD2 and LD4 motifs of Paxillin [30, 35]. We postulated that a peptide encoding these LD motifs, but lacking FA targeting sequences (LIM domains), would interfere with interactions responsible for FAK FA targeting. We also took into account the fact that several cancer-linked Paxillin mutations have been mapped to the intrinsically disordered regions between LDs and not on the motifs themselves, such as P30S, G105A and A127T that lie between LD1 and LD2 and P233L and T255I that lie between LD3 and LD4 [44, 45]. Given the significance of this intermediate linking region, in LD interactions with binding partners and in LD scaffolding functions, we decided that it should be included in the construct [46]. We therefore generated a construct containing LD2-LD3-LD4 and intermediate linking regions, fused to GFP, hereunto referred to as LD2-LD3-LD4 (Fig. 1a). This construct led to expression of a stable protein, at the expected molecular weight, which localized primarily in the cytosol (Fig. 1b and c).
We went on to examine if LD2-LD3-LD4 interacted with FAK, in co-immunoprecipitation experiments, using extracts of HeLa cells transiently transfected with GFP LD2-LD3-LD4 or GFP (negative control). As shown in Fig. 1d, a band corresponding to co-precipitated FAK (at 125kD), was detected only in the precipitates from cells expressing GFP LD2-LD3-LD4, showing that LD2-LD3-LD4 specifically interacts with FAK, as expected. Overall, these experiments show that LD2-LD3-LD4 interacts with FAK directly and given its cytosolic localization it could potentially prevent FAK localization at FAs.
Expression of LD2-LD3-LD4 leads to the specific and dose-dependent displacement of FAK from FAs
In order to examine if LD2-LD3-LD4 expression could specifically disrupt FAT domain interactions and displace FAK from FAs, HeLa cells were transiently transfected with LD2-LD3-LD4, seeded on FN coated coverlips for two hours, fixed and immuno-stained for FAK and Talin. Talin was selected as a stable marker of mature FAs, given the fact that its recruitment to the complex relies on direct binding to β integrin cytoplasmic tails. As shown, expression of LD2-LD3-LD4 led to the clear displacement of FAK from FAs, while Talin localization was unaffected (Fig. 2a). This effect was confirmed using a second FAK antibody and, in addition, exogenous mKate FAK (Additional File 2: Fig. S1 a-c). We quantified this displacement, by calculating the ratio of FAK in the cytosol to FAK at FAs, revealing that LD2-LD3-LD4 expression led to a 4-fold reduction of FA-localized FAK (Fig. 2b). Interestingly, a similar quantification for Talin showed that LD2-LD3-LD4 expression leads to an increase in FA localized Talin, possibly due to enlargement of the FA complexes (Fig. 2c). In order to account for this, we calculated the FAK to Talin ratio at FAs, which revealed a dramatic 5-fold reduction in LD2-LD3-LD4 expressing cells, suggesting that LD2-LD3-LD4 is very effective in displacing FAK from FAs (Fig. 2d), unlike expression of GFP, which was used as a negative control (Additional File 2: Fig. S1d). We then examined how the levels of LD2-LD3-LD4 affected displacement efficiency, and revealed a clear dose response relationship; in cells expressing relatively high levels of GFP, we observed complete loss of FAK from FAs while in cells expressing moderate or low levels of GFP, we could still detect FAK at FAs, albeit at significantly reduced levels (Fig. 2e).
Fluorescent proteins, despite mutations to reduce their ability to dimerize, still maintain some capacity to do so. Additionally, given their globular nature and relatively large size (27kD) they tend to stabilize fused peptides. Furthermore, GFP displays inherent accumulation to the nucleus and could thus be influencing peptide function, by affecting cellular distribution. To ensure that the LD2-LD3-LD4 peptide is stable and can be used effectively in the absence of GFP, we generated a FLAG- tagged peptide which is much smaller in size (1kD). As shown in Fig. S1e and S1f, the FLAG-tagged peptide can efficiently interact with FAK and lead to effective displacement from FAs., This confirms that the LD2-LD3-LD4 is sufficiently stable and functional in the absence of a large globular protein.
A previously generated inhibitor of the interaction of FAK with VEGFR3 (C4), was also reported to displace FAK from FAs [47]. This interaction, as characterized by docking studies, takes place through binding of C4 to His 1025 on Helix 4 of the FAT domain of FAK, adjacent to HP1, to which it may sterically hinder access [17]. We thus decided to compare the efficiency of C4 with that of LD2-LD3-LD4, to displace FAK from FAs. We examined the distribution of FAK and Talin in HeLa cells, following treatment with high concentrations of C4 (50 μM) for 48 h. Surprisingly, C4, failed to visibly displace FAK from FAs, compared to controls. Quantification of FAK to Talin signal ratios on FAs of control and treated cells, confirmed that C4 did not affect FAK FA localization. However, after inhibitor treatment, some cells appeared to have smaller FAs, with low FAK and Talin signals, because they were detaching from the substrate. These data show that C4 does not specifically block FAK targeting to FAs and suggest that in order to efficiently displace the protein, disruption of interactions taking place at the HPs is necessary (Additional File 2:Fig. S1g).
Given the potent displacement of FAK from FAs induced by LD2-LD3-LD4, we wanted to examine the specificity of this effect and possible consequences on FA composition. We therefore examined the localization of additional core FA proteins including Integrins (av), Paxillin, Tensin and Vinculin. As shown, similarly to Talin, FA localization of these proteins was not reduced by LD2-LD3-LD4 expression, suggesting that the effect of LD2-LD3-LD4 is specific to FAK and that the composition of FA complexes is broadly unaltered in expressing cells (Fig. 2f-m). Overall, these data provide evidence that LD2-LD3-LD4 could serve as an effective, site-specific inhibitor of interactions at the HP sites within the FAT domain of FAK and prevent FAK localization at FAs in a dose-dependent manner.
LD2-LD3-LD4 inhibits both kinase-dependent and scaffolding functions of FAK at FAs
FAK is a major transducer of integrin signaling and becomes phosphorylated and activated in response to integrin-dependent adhesion. Given that LD2-LD3-LD4 interacts with FAK directly, leading to its displacement from FAs, we went on to address its effects on FAK activation. To do so, we examined the phosphorylation state of a) Tyr397, the major FAK auto-phosphorylation site required for activation, b) Tyr576, which resides in the activation loop of the kinase domain and has been shown to lead to full activation upon phosphorylation and c) paxillin Tyr31, one of the major FAK/Src downstream targets [37, 48]. As shown, LD2-LD3-LD4 expression led to a significant reduction of phosphorylation at these sites, suggesting that LD2-LD3-LD4 expression blocks FAK activation and downstream signaling (Fig. 3a). Importantly, this reduction becomes even more significant, since transient transfection efficiency is never 100% and thus what we observe represents an underestimation of the effect. In order to examine the effects of LD2-LD3-LD4 on FAK phosphorylation in individual cells, we carried out Immunofluorescence (IF) using phospho-specific antibodies. As shown, LD2-LD3-LD4 expression led to a dramatic drop in FAK phosphorylation (on Tyr397) at FAs, suggesting that it effectively eliminates FAK activation at these complexes (Additional File 2: Fig. S1h).
One of the best characterized downstream targets of FAK is Paxillin, which becomes phosphorylated on Tyrosines 31 and 118, in response to integrin activation in wild type but not in FAK null cells [49]. We thus went on using quantitative immunofluorescence and calculated the ratio of phosphorylated-Paxillin (pPax) to Paxillin, in order to assess the effects of LD2-LD3-LD4 on Paxillin phosphorylation, specifically at FAs. As shown, expression of LD2-LD3-LD4 led to a significant reduction of the levels of pPax, suggesting that it not only blocks FAK activation but also downstream signaling from FAs (Fig. 3b). In agreement with this result, staining of LD2-LD3-LD4 expressing cells with a well characterized pY antibody (pY20) revealed that overall tyrosine phosphorylation is dramatically reduced at FAs, suggesting that signaling is impaired due to FAK displacement (Fig. 3c). Given the dramatic reduction of tyrosine phosphorylation at FAs, we examined whether LD2-LD3-LD4 somehow prevents integrin activation. Quantification of the ratio of active Integrin β1 to Vinculin at FAs showed that LD2-LD3-LD4 has no effect on integrin activation (Fig. 3d). Therefore, the above data clearly show that LD2-LD3-LD4 expression blocks FAK kinase-dependent signal transduction events, downstream of integrin activation.
Upon recruitment at FAs, FAK is auto-phosphorylated on Tyr397, creating a high-affinity binding site for the SH2 domain of Src, which further phosphorylates FAK on Tyr576 and Tyr577 within the activation loop, leading to maximal enzymatic activity [50]. In order to examine the effects of LD2-LD3-LD4 expression on FAK-mediated Src recruitment to FAs we expressed the SH2 domain of Src fused to GFP (GFP Src_dSH2), previously shown to be necessary and sufficient for FA targeting of Src [51]. As expected, given the previous data indicating FAK FA displacement and abolishment of Tyr397 phosphorylation, expression of LD2-LD3-LD4 led to a significant reduction in Src_dSH2 FA localization, indicating an inability of Src to target FAs (Fig. 4 a and b).
FAK also has well-established scaffolding functions, including a kinase independent role in the recruitment of the FAK-Src substrate, p130Cas to FAs [27]. This is achieved through an SH3-dependent interaction with the C terminal proline-rich regions of FAK [27]. In order to determine if, unlike kinase inhibitors, LD2-LD3-LD4 could also suppress kinase-independent, scaffolding functions, we examined p130Cas localization. LD2-LD3-LD4 expressing and control cells, as well as cells treated with a previously characterized FAK kinase inhibitor (PF228) [52], were immunostained for Talin and p130Cas. There was a visible reduction of FA-localized p130Cas in LD2-LD3-LD4 expressing cells, unlike control and PF228-treated cells in which no change was observed as confirmed by quantification of the ratio of p130Cas to Talin (Fig. 4c and d). As expected PF228 treatment led to a clear reduction of tyrosine phosphorylated FAK at FAs but did not interfere with its localization; thus, as expected, p130Cas is maintained at the complex (Additional File 2: Fig. S2a-c). The above results show that unlike inhibitors of FAK’s enzymatic activity, expression of LD2-LD3-LD4 blocks both kinase-dependent and independent functions at FAs.
Expression of LD2-LD3-LD4 affects FA dynamics, and inhibits migration and invasion of tumor cells
It is well established that FAK is a critical regulator of FA assembly and disassembly, processes that are fundamental for efficient, directional cell migration [53,54,55]. Given that expression of LD2-LD3-LD4 displaces FAK from FAs, we initially examined whether this would elicit changes in FA dynamics. For this purpose, we evaluated the number and size of FAs in LD2-LD3-LD4 expressing vs control HeLa cells that were seeded on glass coveslips for 12 h. As shown in Fig. 5a, control cells formed a characteristic pattern of FAs, mainly found at the cell periphery. In contrast, LD2-LD3-LD4 expressing cells displayed a significant increase in both the number and size of FAs with prominent ventral FAs (Fig. 5b and c). This result, suggests a defect in FA turnover and is consistent with previous findings in FAK −/− cells [53]. We went on to directly examine the effects of LD2-LD3-LD4 expression on FA turnover. HeLa cells were transfected with RFP-Vinculin alone or co-transfected with RFP-Vinculin and LD2-LD3-LD4, seeded on fibronectin-coated chambered slides and time-lapse sequences were recorded, over a period of 35 min. Cells expressing the construct displayed markedly slower FA turnover compared to control cells (Fig. 5d and e). Therefore, these data show that LD2-LD3-LD4 expression elicits defects in FA turnover, leading to the appearance of more and larger FAs, in a similar manner to defects reported in FAK null fibroblasts [53].
Given the central role of FA turnover in cell migration, we decided to examine how LD2-LD3-LD4 affected cell spreading and migration. Control and LD2-LD3-LD4 expressing HeLa cells were seeded on fibronectin-coated coverslips and monitored using time-lapse video microscopy over a period of 16 h. We used a motorized stage to image multiple areas simultaneously, so as to record and track large numbers of cells. Analysis of the recordings revealed that LD2-LD3-LD4 elicited dose-dependent defects in both cell spreading and migration (Fig. 6a and b and Additional File 3: Movie S1). In addition, analysis of the time-lapse images revealed that cells expressing high levels of LD2-LD3-LD4, displayed slightly increased apoptosis (16,9% compared to 6,2% in control cells). Similar effects were observed in other highly migratory and metastatic cell lines, namely MDA MB-231 (Additional File 2: Fig. S3a), H460 (Additional File 2: Fig. S3b) and HCT-116 (Additional File 2: Fig. S3c), in which FAK is effectively displaced from FAs, upon expression of LD2-LD3-LD4 (Additional File 2: Fig. S3d-f). Overall, these data show that LD2-LD3-LD4, not only elicits defects in cell spreading and FA turnover, consistent with phenotypes observed in FAK null cells, but is also an effective inhibitor of two-dimensional (2-D) cell migration.
Additional file 3: Movie S1. Expression of LD2-LD3-LD4 reduces the migration of HeLa cells.
Although active cell migration is a prerequisite for metastasis, there is strong evidence suggesting that 3-D culture and gel invasion assays better mimic the tumor microenvironment and predict therapeutic responses, in vivo, more accurately [56, 57]. To examine the effects of LD2-LD3-LD4 on tumor cell invasion, we developed a modified Boyden-chamber gel invasion assay, which allows live and end-point evaluation of cell invasion and permits imaging, tracking and quantification of both invading and non-invading cells. The highly invasive MDA MB-231 cells were used for these experiments and both end-point measurements, as well as time-lapse recordings were generated with mixed populations of LD2-LD3-LD4 expressing and control cells, in the same setup. As shown, LD2-LD3-LD4 expression, inhibited invasion of MDA MB-231 cells in a dose-dependent manner; in fact, high expression drastically reduced invasion of this highly metastatic cell line (Fig. 6c and Additional File 4: Movie S2). These results show that displacement of FAK from FAs is an effective strategy to block both cell migration, as well as tumor cell invasion. It could therefore form the basis for the development of anti-metastatic drugs.
Additional file 4: Movie S2. Expression of LD2-LD3-LD4 inhibits the invasion of MDA MB-231 cells.
The LD2 and LD4 motifs are sufficient for effective FAK displacement from FAs
Previous work revealed that LD2 and LD4 are responsible for the interaction with the HPs of FAK [30, 35]. In the work described above, the construct used to displace FAK from FAs also contained LD3, as well as intermediate linking regions (Fig. 1a). This was initially deemed necessary given the significant regulatory role assigned to these unstructured linking segments for the FAK-paxillin interaction [44,45,46]. However, these regions also bare numerous phosphorylation sites and binding sites for proteins other than FAK, thus their presence would be expected to be detrimental to the specificity of the polypeptide and lead to off target effects. In addition, the large size of the polypeptide containing these regions (24 kDa, 226aa) poses restrictions in its potential use as a metastatic inhibitor, in the form of a synthetic peptide since it is well beyond the size limit for effective peptide synthesis (100-120aa). Therefore, we decided to determine the minimum paxillin sequences required for efficient displacement of FAK from FAs. To this end we implemented a subtractive approach, removing individual linking regions in a stepwise fashion and assessing the activity of each construct.
We initially deleted the region upstream of LD2 (LD1-LD2 linking region-LR), previously reported to be necessary for optimal binding to FAK [23, 24, 30] and examined how it affected the peptide’s capacity to displace FAK from FAs (Fig. 7a). This construct led to expression of a stable polypeptide, at the expected molecular weight, hereunto referred as LD2-LD3-LD4 ΔLR (Fig. 7b). As shown, expression of LD2-LD3-LD4 ΔLR led to clear displacement of FAK from FAs, while Vinculin localization (used as an FA marker) was unaffected as expected (Fig. 7c). Quantification of the FAK to Vinculin ratio showed that LD2-LD3-LD4 ΔLR displaced FAK with the same efficiency as the original peptide, suggesting that the linking segment upstream of LD2 does not play a pivotal role for efficient binding of the LD2 and LD4 motifs to the FAT HPs in the cell (Fig. 7d).
Next, we went on to examine whether the intermediate linking region between LD2 and LD4, containing the LD3 motif, plays a role in the ability of the polypeptide to displace FAK. Using the DNA encoding for LD2-LD3-LD4 ΔLR as template, we replaced the region between the LD2 and LD4 motifs with a flexible standard linker (GGGGS). Optimization of the length of the linker was performed by evaluating the efficiency of LD2- GGGGSn-LD4 polypeptides, containing linkers of different sizes (15, 25 and 30 amino acids), to displace FAK from FAs (Fig. 7e). At the end, a 30 amino acid-long linker containing 6 (GGGGS) repeats was selected, leading to the generation of a new construct, hereunto referred to as LD2-LD4 (Fig. 7a and b). We went on to quantify the ability of LD2-LD4 to displace FAK from FAs in comparison to the original construct. As shown, LD2-LD4 is as efficient as the original in displacing FAK from FAs (Fig. 7c and d), suggesting that the sequence of the intermediate (LD2-LD4) linking region and LD3 are not essential for binding to FAK. The new polypeptide lacks critical phosphorylation sites present in the original and is devoid of any paxillin sequences other than the two LD motifs, ensuring improved specificity. Importantly the 30 amino acid linker is significantly shorter than the 99 amino acid linker contained in the original polypeptide; coupled with the removal of LD2 upstream sequences, this peptide is much smaller, only 69 amino acids (6 kDa), compared to the original peptide that was 226 amino acids (24 kDa) and thus well within the limits of solid-phase peptide synthesis. This effectively raises the possibility of using a synthetic polypeptide as an anti-metastatic agent.
Inducible expression of LD2-LD4 interferes with the interaction of FAK with endogenous paxillin
Having determined the minimum LD motif and linker length requirements of the polypeptide, we decided to confirm the molecular mechanism of action, which we postulated is the disruption of interactions between endogenous paxillin and FAK. Use of a transient expression system imposes limitations on using a biochemical approach, given the inability to attain 100% efficiency within a single transfection, uneven expression levels between transfected cells and variation in efficiency between transfections; we thus generated a stable HeLa cell line to inducibly express LD2-LD4 using a lentiviral vector system. As indicated in Fig. 8a, induction using Doxycycline, leads to the expression of a stable polypeptide at the expected molecular weight (~35kD), which interacts with FAK, similarly to the transiently expressed polypeptide (Fig. 8b). Furthermore, inducible expression of LD2-LD4 led to clear displacement of FAK from FAs, while Vinculin localization (used as an FA marker) was unaffected as expected (Fig. 8c). Quantification of the FAK to Vinculin ratio showed that the inducible expression of LD2-LD4 displaced FAK with the same efficiency as the transiently expressed peptide (Fig. 8d). To validate the IF results we performed biochemical fractionation to isolate FAs followed by Western blot analysis, so as to determine resident protein levels. As shown in Fig. 8e the levels of FAK at FAs are markedly reduced, upon inducible expression of the LD2-LD4 polypeptide, and this is further supported by quantification of FAK/Paxillin ratio (both normalized to actin expression levels) (Fig. 8e). In contrast, Vinculin and paxillin levels are increased, in agreement with the immunofluorescence results described above. Collectively, these results provide firm confirmation that LD2-LD4 expression displaces FAK from FAs.
As extensively discussed in previously published work (from our group and others), targeting of FAK to FAs depends on interaction of the LD motifs of endogenous paxillin with the FAT domain of FAK. We postulated that the overexpressed LD2-LD4 polypeptide, shown to bind FAK (Fig. 1d and S1e), interferes with the ability of FAK to bind endogenous paxillin and therefore prevents FA targeting. To confirm this, we performed co-immunoprecipitation experiments to isolate FAK and co-precipitated proteins, from extracts of induced Hela cells that stably express LD2-LD4 (uninduced Hela cells as well as GFP expressing cells were used as controls). As shown in Fig. 8f, the levels of co-precipitated Paxillin in induced cells are markedly reduced compared to control cells. These data clearly show that expression of the polypeptide disrupts the interaction of FAK with endogenous paxillin, thus confirming the molecular mechanism of action.
Dimers of a single LD motif can effectively displace FAK from FAs and reduce tumor cell migration
The polypeptide used throughout this study consists of two separate LD motifs that have different sequences. However, designing and delivering a small molecule inhibitor consisting of two molecules would be quite challenging and complicated. We thus went on to examine the possibility that a single LD motif could bind to both HPs of the FAT domain and displace FAK.
We therefore proceeded to generate two new constructs encoding either LD2 or LD4 fused to GFP, hereunto referred to as GFP LD2 and GFP LD4 respectively (Fig. 9a) and examined the efficiency of the stable polypeptides expressed (Fig. 9b), to displace FAK from FAs. LD2 has been shown to bind to both HPs with equally high affinity, whereas LD4 only binds HP1 with high affinity, thus we expected that GFP LD2 would be more efficient in displacing FAK compared to GFP LD4 [43, 58, 59]. However, both LD2 and LD4 as monomers failed to displace FAK from FAs (Fig. 9 c and d). These results, are in agreement with previous studies showing that peptides containing both LD2 and LD4 display higher affinity for FAT compared to single LDs [31, 43, 58, 60], as well as the results discussed earlier, showing that LD2 and LD4 have to be connected through a flexible linker of the proper size in order to displace FAK from FAs. Taken together, these data provide strong evidence that an LD motif dimer is required for a high affinity interaction with FAK to take place.
To test this possibility and at the same time determine if a single LD motif could in fact target both HPs, we generated two new constructs encoding LD2-LD2 or LD4-LD4, separated by the optimized 30 amino acid linker described earlier (Fig. 9a and b). As shown (Fig. 9c-e) both LD2-LD2 and LD4-LD4 effectively displace FAK from FAs, with a similar efficiency as LD2-LD4 suggesting that both LD2 and LD4 can bind both HPs. Importantly, expression of either polypeptide reduced the migratory capacity of MDA MB-231 cells, as efficiently as LD2-LD4 (Fig. 9f).
These results lead to the conclusion that the function of FAK, specifically at FAs, can be efficiently targeted using the above described strategy, as long as the peptide topology is maintained (two LD motifs connected with an appropriate linker). More importantly, it suggests that a single small molecule mimic could potentially bind both HPs eliminating the need for two individual molecules.