addition to the di-tyrosine motif, we think that the W556 is another critical residue of this region. Substitutions or deletions encompassing the W556 (human W557) residue of c-Kit have been found in GISTs and malignant melanoma in humans (cancer.sanger.ac.uk) and in canine mast cell tumors as a kinase-activating mutation (Ma et al., 1999). In humans, mastocytosis have been associated with the D816V activating mutation in the c-Kit kinase domain (Fritsche-Polanz et al., 2001; B. J. Longley et al., 1996; Nagata et al., 1995). It has been previously shown that mutations affecting the c-Kit kinase domain could stabilize the A-loop therefore favoring the active conformation of the receptor (Mol et al., 2004).
To analyze the involvement of the JMD and the TKD, c-Kit oncogenic mutations (c-KitW556A or c-KitD814V/H) were analyzed for their ability to spread on immobilized-KitL. Compared with the c-KitWT, both c-Kit mutants W556A and D814V/H blocked COS cells spreading. The spreading test revealed a spreading defect of around 16% for the JMD mutant W556A ( Figure 22-A)
and 6-10% for the kinase domain mutants D814V and D814H ( Figure 22-B). We also assessed the function of S819 and Y821 located in the A-loop of the kinase domain. The residue S819 is phosphorylated in a KitL-stimulated PKC-dependent manner ((Blume-Jensen et al., 1995) that in turns seems to inhibit the c-Kit kinase activity. The amino acid Y821 is one of the major phosphorylation sites within the kinase domain. When overexpressed in COS cells, c-KitS819A and c-KitS819D mutants did not change the spreading response ( Figure 22-B). However, both c-KitY821D and c-KitY821F induced a half reduction of the c-KitWT spreading response ( Figure 22-C). We pursued the investigation with the JMD activating mutation W556A and the kinase domain D816V.
Figure 22. c-Kit activating mutations block cell spreading on fibronectin. Transfected COS cells were plated on surfaces coated with low fibronectin concentrations in the presence of immobilized-KitL (FN2.2/PA17.8/immobilized-KitL-GFP-IgG) and allowed to spread for 15 minutes. A-C) Percentage of spread cells expressing mutants of the A) juxtamembrane domain (KitW556A); B) the kinase domain c-KitD814H/V and c-KitS819A/D; and C) the A-loop within the kinase domain c-KitY821D/F all fused with tagRFP. Results represent means ± SEM from 3 independent experiments. Significant differences versus the WT are expressed by: (*, p<0.01); (**, p<0.001); (***, p<0.0001).
V.1.Function of the di-tyrosine motif in the context of the activated receptor c-KitW556A and c-KitD814V
To ruled-out a delayed response, we evaluated the spreading kinetics over 60 minutes. As shown in ( Figure 23-A), around 40% of mutant cells spread after 60 minutes. However, this response still almost half of that showed by cells expressing a normal receptor (∼80%). This assay revealed that c-Kit activating mutations cannot spread on low fibronectin concentrations towards immobilized-KitL in compare to the WT. Failed spreading could be related to a lower cell surface expression in compare to the WT. Moreover, both mutants did
not differ in their response suggesting that a constitutively active kinase, while being associated with more proliferative phenotype, might interfere with cell spreading.
In view of the results, we wonder whether W556A and D814V blocked cell interfere with cell spreading by the same mechanism. Is the c-Kit kinase activity of W556A and D816V dependent on the di-tyrosine motif? To answer these questions, we modified the mouse c-Kit sequence to generate the double mutants: c-c-KitW556A/YYFF and c-KitD814V/YYFF both fused to a tagRFP at the c-terminus. Like previous experiments, COS cells were transfected with these mutants and their spreading capacity on fibronectin and i-KitL was compared to the WT receptor.
Figure 23. Function of the di-tyrosine motif (Y567/Y569) in cell spreading mediated by c-Kit activating mutants. Transfected COS cells were plated on surfaces coated with fibronectin 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.
Percentage of spread cells as a function of time is represented in A) c-KitWT versus c-KitW556A and c-KitW556A/YYFF. In B) c-KitWT versusc-KitD814V and c-KitD814V/YYFF all fused with tagRFP. Results represent means ± SEM from 3 independent experiments. Significant differences versus the WT are expressed by (*, p<0.01); (**, p<0.001); (***, p<0.0001). No significant differences (ns).
As shown in Figure 23, by combining the c-Kit activating and YYFF mutations COS cells managed to spread more than those cells expressing mutants W556A ( Figure 23-A) or D814V ( Figure 23-B) alone. Although not significant, these differences were kept over time with the double mutants attaining spreading levels close to the WT receptor. These responses were no so strong but indicate that both mutations behave equally in response to immobilized-KitL.
What is the exact function of the JMD in this context still unclear.
V.2.Spreading sensitivity of c-KitW556A and c-KitD814V to imatinib and dasatinib
As already mentioned in the introduction, regulatory and catalytic mutations differ in their sensitivity to kinase inhibitors. Inhibitors like imatinib bind an inactive kinase stabilizing this conformation (Mol et al., 2004; Mol et al., 2003). These inhibitors are effective in inhibiting the regulatory but not the catalytic mutants which form is constitutively active. We therefore wondered whether the spreading in the context of W556A and S814V can be recovered with imatinib or dasatinib? To this end, COS cells overexpressing the c-Kit mutations W556A and D816V were treated with different concentrations of imatinib or dasatinib for 1 hour and then plated on low fibronectin and i-KitL coated surfaces.
In contrast to MC9 cells (Figure 16), spreading of COS cells overexpressing a c-Kit wild-type receptor was efficiently blocked with 10µM of imatinib (Figure 24-A). On the other hand, dasatinib significantly reduced COS-c-KitWT cell spreading to less than 20% compared to non-treated cells (Figure 24-D). Treatment with 10nM of dasatinib only showed an effect after 15 minutes. Surprisingly, despite no significant results, imatinib tend to recover spreading of COS cells overexpressing the active c-KitW556A receptor (Figure 24-B). However, c-KitW556A expressing cells did not improve cell spreading when treated with dasatinib (Figure 24-E).
Instead, these cells spread even less than the non-treated cells. As expected, treatment of COS cells overexpressing the activating c-KitD814V mutation were insensitive to imatinib.
Figure 24. Dose-response of imatinib and dasatinib inhibition of spreading of c-Kit activating mutants. Transfected COS cells were treated 1h with imatinib and dasatinib at the indicated concentrations (DMSO=control). Cell spreading on FN and immobilized-KitL was follow for 1h and the percentage of spread cells determined at the indicated time points for 12 fields per condition.
Percentage of spread cells as a function of time is represented for imatinib (A-C) and dasatinib (D-E). Results represent means ± SEM from 3 independent experiments. Significant differences versus the WT are expressed by (**, p<0.001); (***, p<0.0001). No significant differences (ns).
Discussion