introduced mutations in the c-Kit receptor. The c-Kit wild-type or mutated variants were transiently expressed in COS cells. The effects of such mutants in immobilized-KitL mediated spreading. In contrast to MC/9 cells, COS cells growth in adherence. Therefore, the following tests were done on lower fibronectin concentrations (FN2.2/PA17.8) that that used for MC/9 cells. Moreover, the percentage of spread cells was quantified 15 minutes after cell seeding.
IV.1. PI3K recruitment is required for immobilized-KitL-induced spreading on low fibronectin concentrations
As previously reported by our group and others, recruitment of PI3K is required for cell spreading. We confirm once again this observation and validated further experiences with COS cells expressing the PI3K recruitment mutant c-KitY719F. As shown in Figure 19, COS cells expressing c-KitY719F mutant showed significantly lower spreading compared to c-KitWT– expressing cells. This reduced response corroborates that activation of c-Kit by i-KitL requires the phosphorylation of Y719 and the recruitment of PI3K to this site.
Figure 19. Reduced spreading of c-KitY719F transfected cells. Percentage of COS_c-KitY719F expressing cells that spread on low fibronectin in the presence of immobilized-KitL (FN2.2/PA17.8/KitL-GFP-IgG). On the right, representative immuno-stained images showing the phenotype of c-KitWT (spread) and c-KitY719F (non-spread) expressing cells. The c-Kit-tagRFP is in red and the actin (phalloidin staining) in green. Non-transfected (NT) cells were employed as a negative control.
Results represent means ± SEM from 3 independent experiments. Significant differences versus the WT are expressed by **(p<0.01), ***(p<0.001).
IV.2. Function of the c-Kit c-terminal tail in cell spreading
Our data suggest that c-Kit can be mechanically activated. Auto inhibition of the PDGFRb involves the C-terminal tail of the receptor (Chiara et al., 2004). Even though the C-terminal tail of PDGFRb and c-Kit are quite different we inferred the same in the case of c-Kit.
Therefore, we postulated that upon immobilized-KitL stimulation c-Kit is stretched inducing
the release of the c-tail inhibition. The cytoplasmic tail of the mouse c-Kit protein spans from leucine 921 to alanine 975. This region contains a tyrosine in position 934 (Y934, Table 1) which is phosphorylated upon receptor activation and recruit signaling proteins such GRB2 and Cbl. Therefore, we wonder what the function of this region in the mechanical activation of c-Kit is? To answer this question, we created deletions and point substitutions of tyrosine Y934 and assessed their effects in cell spreading towards immobilized-KitL presentation on low fibronectin.
Using the panel of c-Kit deletion mutants, we observed that in the absence of the last 25-37 amino acids did not change the spreading capacity. COS cells expressing either the deletions d949-975 and d938-975 or the full-length WT receptor spread to comparable levels ( Figure 20-A, B). By contrast, the spreading response significantly decreased to almost 40% with a truncation encompassing the last 46 amino acids (d929-975) ( Figure 20-A, B). These data suggest that residues 930-937 make some contribution to cell spreading.
To detect whether the spreading defect of the deletion d929-975 is due to the absence of the Y934, we replaced the tyrosine 934 by alanine, aspartate or phenylalanine and tested their effect on cell spreading. As shown in Figure 20-C, none of the Y934 substitutions interfered with cell spreading on low fibronectin an immobilized-KitL. These suggest that other not yet identified c-terminal motifs might be required for cell spreading. Therefore, we design another mutation in which added back the last 25 c-terminal amino acids but residues 929-948 still absent. Interestingly, COS cells transfected with this c-Kit variant (d929-929-948) recovered cell spreading to levels comparable to that of the full-length protein. However, when we reintroduced the fragments 929-948 and 949-975 (d941-948 mutant) spreading of COS cells was blocked. Unfortunately, in both cases the c-Kit was not well expressed at the cell surface (data not shown) invalidating the use of theses mutants for further analysis.
Figure 20. Influence of c-Kit c-terminal deletions in KitL-induced cell spreading. A) Representation of the deletion mutants spanning the mouse c-Kit c-terminal tail from leucine 921 to alanine 975.
The name of each mutant is given before the sequence. In red, the mutation that block cell spreading and in green the tyrosine 934. B and C) Percentage of COS-c-Kit-tagRFP cells that spread on low fibronectin concentrations in the presence of immobilized-KitL. D) Representative immuno-stained images showing the phenotype of c-KitWT, c-Kitd929-975 (mostly non-spread) and c-KitY934F expressing cells. The c-Kit-tagRFP is in red and the actin in green (phalloidin staining). Results represent means
± SEM from 3 independent experiments. Significant differences versus the WT are expressed by
**(p<0.01), ***(p<0.001).
The pathways activated by an immobilized or a membrane-bound ligand still not well known.
To progress in the study of the molecular mechanism involved in the mechanical activation of c-Kit by immobilized-KitL. Therefore, we extended our analysis to other regions of the protein including the juxtamembrane and the kinase domain.
IV.3. Function of the di-tyrosine motif Y567/Y569 in cell spreading
As described in the introduction, the JMD of c-Kit function as conformational inhibitor of the receptor kinase activity. Following KitL binding, receptor dimerization leads to the
phosphorylation of Y567 and Y569. This phosphorylation release the JMD that in turns opens the access to the catalytic site shifting the receptor to the active conformation (Yuzawa et al., 2007). Once phosphorylated, Y567/569 can interact with proteins containing SH2 domains such as SFKs, SHP1/2 and APS (Table 1). Mutations in the JMD lead to a constitutive activation of c-Kit and have been found in GISTs. In addition to conformational destabilization, such mutations could lead to a loss of a negative regulation due to defective interactions with regulators such as SHP1/2. The di-tyrosine motif has been implicated in KitL-dependent cell proliferation as well as cell migration. Here, we assessed the function of Y567/569 in cell spreading towards immobilized KitL.
As with the other mutants, COS cells were transfected with mouse c-Kit cDNAs carrying either the single mutants Y567F, Y569F or the double mutant Y567F/Y569F (hereafter YYFF), all fused with a tagRFP. Transfected cells were incubated in suspension for 1 hour in starvation medium then plated on surfaces coated with low fibronectin and immobilized-KitL (FN2.2/PA17.8/KitL-GFP-IgG). Cell spreading was followed for 60 minutes by taking pictures of 16 fields per condition every 15 minutes (see Material and Methods).
As shown in Figure 21-A, the c-Kit mutants Y567F and Y569F and YYFF spread less than the WT but these differences were not statistically significant. Moreover, the spreading response was delayed and doubled at 30 minutes reaching the wild-type levels (75%). Compared to the c-KitWT, cells expressing the double mutant YYFF showed half reduction of cell spreading mainly between 15 and 30 minutes. Like for the single mutants, the YYFF variant showed a delayed spreading. Although no significant differences exist, the spreading response after 60 minutes never reach the WT levels.
These results suggest that cell spreading towards immobilized-KitL do not require a functional di-tyrosine motif. The question is whether the YYFF mutation lower cell spreading because a loss of interactions with the adapters or regulatory proteins. To answer this question, we transiently transfected COS cells with the c-KitYYFF-tagRFP alone or in combination with a mouse cDNA encoding the phosphatase SHP2 fused to the GFP. Less than 20% of SHP2-expressing cells spread from 15-60 minutes (Figure 21-B). However, overexpression of SHP2 together with c-KitWT induced spreading comparable to that observed with c-KitWT alone (Figure 21-C). In the case of the c-KitYYFF mutant, introduction of SHP2 did not improved spreading (Figure 21-C). These results indicate that signals coming from the di-tyrosine
Y567/569 motif are required for early spreading. However, recruitment of SHP2 to the Y567/569 motif do not seems to be implicated in cell spreading.
Figure 21. Function of the di-tyrosine motif Y567/Y569 in cell spreading. A and B) Kinetics of spreading of COS cells expressing: in A) c-KitWT, c-KitY567F, c-KitY569F or c-KitYYFF all fused with tagRFP and in B) SHP2-GFP, c-KitWT-SHP2, c-KitYYFF-SHP2. Transfected COS cells were plated on surfaces coated with low fibronectin concentrations in the presence of immobilized-KitL (FN2.2/PA17.8/KitL-GFP-IgG). Cell spreading was follow for 1h and the percentage of spread cells was determined at the indicated time points for 12 fields per condition. C) Comparison of cell spreading between different mutants 15 minutes after seeding. Results represent means ± SEM from 3 independent experiments. Significant differences are represented by asterisks (*) versus the c-KitWT(A) and the SHP2WT (B). Statistical differences: *(p<0.01), ** (p<0.01), *** (p<0.001), **** (p<0.0001).
V. Effect of oncogenic c-Kit mutations in the JMD and the TKD in cell spreading on fibronectin