Oncogenic signaling by activated c-Kit

The gain-of-function mutations responsible for the constitutive activation of TKRs can have quantitative and qualitative consequences of the kinase activity as well as the activated downstream signaling pathways. The quantitative changes can be the result of continuous signaling or modifications in the activation threshold to signal. Qualitative changes may result from variations in the catalytic properties of oncogenic receptors or their unusual location.

This section aims to illustrate specific features of oncogenic signaling and the molecular mechanisms that account for its specificity.

As exposed in section 0, the extent of receptor signaling depends on the downregulation mechanism including the action of phosphatases or receptor degradation. In many oncogenic situations, signaling does not cease and become persistent because TKRs escape the negative regulation. Escape to negative regulation mediated by the action of phosphatases has been

WT D814Y

Piao et al. observed that SHP-1 is not phosphorylated and its expression is reduced probably due to protein degradation in mutated cells (Piao et al., 1996).

Oncogenic receptors also escape the mechanism of endocytosis and degradation and often involves recruitment of Cbl. The most potent oncogenes that avoid the action of Cbl are the viral oncogenes. Some TKRs have a viral oncogenic counterpart. For instance, Fms and c-Kit have v-Fms and v-Kit, respectively. Compared to Fms, v-Fms lacks 50 amino acids in the c-terminus that causes the loss of Y969 which recruitment Cbl. Thus v-Fms is refractory to ubiquitination mediated by Cbl (Mancini et al., 2002). Similarly, v-Kit lack the two tyrosines (Y568 and Y936) that recruit Cbl to c-Kit. In conclusion, the viral oncogenic forms lack the tyrosine residues which are essential for the recruitment of Cbl to activated receptors (Reviewed in (Peschard & Park, 2003). Reciprocally, v-cbl lacks the RING domain and consequently its E3-ubiquitin ligase activity. Mutations in Cbl that disrupt the ligase activity are also oncogenic, probably indirectly via a failure in the negative regulation of TKRs (Dikic &

Schmidt, 2007; Sanada et al., 2009)

It exists a correlation between the tumorigenic capacity and the level of activation of oncogenic TKRs. In the case of c-Kit, the duplication A502/Y503 and the substitution K509I cause a GOF effect when overexpressed in COS cells (Bodemer et al., 2010). However, in Ba/F3 cells the level of c-Kit activation is lower and is also poorer than that of the activating mutant D816V (Yang et al., 2010). These types of mutations cause an excessive cell proliferation of mast cells but with a different degree of severity. In pediatric mastocytosis, 45% of patients have mild regulatory-type mutations. In contrast, in adult mastocytosis, which is an incurable disease, catalytic mutations are much more frequent (Bodemer et al., 2010; B. J. Longley, Jr.

et al., 1999).

Because the first substrate of activated TKRs is the receptor itself, several studies have compared the auto-phosphorylation pattern of wild-type concerning mutated TKRs.

Comparison of murine c-Kit^WT and c-Kit^D814V reveals a difference in the auto-phosphorylation array of the receptor (Piao et al., 1996). In using phospho-specific antibodies, Ronnstrand et al. observed qualitative and quantitative differences in tyrosine phosphorylation of wild-type and mutated 3 (Razumovskaya et al., 2009). However, the main limitation of these studies is the lack of specificity of antibodies. Therefore, to evaluate the phosphorylation state, one needs first to immunoprecipitate the protein of interest, a step that reduces the utility of such

a phopsho-antibodies. This limitation we encountered while assessing the phosphorylation status of c-Kit.

A quantitative phosphoproteomics study was carried out on the oncogenic 3-ITD (Internal Tandem Duplication) receptor (Choudhary et al., 2009). This report showed that the Y842 of A-loop is not phosphorylated when 3-ITD is retained in the endoplasmic reticulum (ER). In contrast, Y591 is phosphorylated to the same levels either the receptor is in the ER or the cell membrane. Regarding c-Kit, phosphorylation of the A-loop in Y823 is not required for the kinase activity although it can increase the stability of the active conformation (Mol et al., 2004). It is therefore plausible that the activated receptor in the ER is unable to reach the maximum level of activity. If the tyrosine phosphorylation of the A-loop is carried out in trans, it is possible that the receptor does not dimerize in the ER. Thus, the kinase activity of the receptor might differ between the ER and the membrane, and this might correspond to the activation of different signaling pathways.

If the receptors itself acquire new catalytic properties, this may also be the case of the substrates downstream to TKRs. The study conducted by Piao et al. (1996) showed that c-Kit^D814Y phosphorylates EGFR-peptides but is more effective than c-Kit^WT in phosphorylating peptides of ABL-type (Piao et al., 1996). Another comparative study showed that deletion of the JMD does not differ from c-Kit^WT in the substrate specificity. (Chan et al., 2003).

Therefore, it seems that mutations of the kinase domain produce a global change in kinase selectivity. These mutations cause key conformational modifications in the catalytic loop which are evidenced by their resistance to inhibitors targeting the inactive conformations (Schittenhelm et al., 2006; Zermati et al., 2003). It is, therefore, possible that tyrosine residues, normally hidden in the structure, become accessible to phosphorylation and thus recruit new substrates. It is possible that they can modify the catalytic selectivity of the enzyme.

V.2.i. c-Kit signaling from a nonstandard compartment

Maturation defects that prevent the correct localization of membrane proteins have already been implicated in disease (C. R. Sanders & Myers, 2004). Several studies have shown a reduction of the surface expression of constitutively active TKRs in respect of their wild-type counterpart. This reduced expression was ascribed to a more rapid degradation of the mutated receptor variants, either c-Kit (Moriyama et al., 1996) or CSF-1R (Li & Stanley, 1991).

However, more recently the lower surface expression has been attributed to an early maturation defect in the ER. This phenomenon was examined in the case of the FGFR3 (Lievens & Liboi, 2003), 3 (Lievens et al., 2004; Schmidt-Arras et al., 2005) and c-Kit (Bougherara et al., 2009). In the example of c-Kit, the activating mutations V560G and D816V lead to a quasi-total disappearance of the mutated receptor from the surface, which is found in the ER in an immature glycosylated but not phosphorylated form (Bougherara et al., 2009).

As for 3-ITD, the inhibition of the c-Kit kinase activity is accompanied by an increase in their surface expression (Bougherara et al., 2009). This effect can be due to an increase in the export of the inhibited forms or to a stabilization of the membrane-anchored forms which are not degraded when in the inactive state. However, endocytosis of c-Kit^WT depends on the integrity of the lipid RAFT and is affected by treatment with methyl-β-cyclodextrin (Jahn et al., 2002) which does not impact on the membrane expression of c-Kit activating mutants of the JMD (S. Tabone-Eglinger et al., 2008). This finding suggests that the reduced cell surface expression observed for oncogenic receptors is not only due to a higher rate of degradation.

Are the expression levels of oncogenic receptors high enough to induce cell transformation?

Can oncogenic signaling occur in an intracellular compartment?

The c-Kit activating mutations found related to GISTs are sufficient to induce cell transformation in vivo (S. Tabone-Eglinger et al., 2008) and in vitro (Xiang et al., 2007). By targeting the c-Kit^D816V mutated receptor in different cell compartments (membrane, Golgi, cytoplasm), it was shown that all the retained forms could induce cytokine-independent proliferation of Ba/F3 cells. Moreover, an intracellular form of c-Kit^D816V induced a lethal myeloproliferative disorder (Xiang et al., 2007). Similarly, an ER-restricted 3-ITD is effective in inducing cell transformation in vitro and MPD in vivo (Choudhary et al., 2009). Accordingly, intracellular forms of oncogenic RTKs would be sufficient to support cell transformation.

Differences between normal and oncogenic TKRs signaling have been evidenced in various studies. In the case of c-Kit, for example, the PI3K/AKT pathway is used by both the c-Kit^WT and the mutated c-Kit^D816 receptors ((Chian et al., 2001; Hashimoto et al., 2003). The specificities of c-Kit^D816V oncogenic signaling have also been investigated for SFKs. For example, FES kinase is the major effector c-Kit^D816V-mediated cell proliferation, but it does not intervene in the c-Kit^WT-induced proliferation (Voisset et al., 2007). In contrast to c-Kit^WT, neither c-Kit^D814V phosphorylation nor ubiquitination depends on SFK. The requirement of SFK is also bypassed in D816V-induced Erk activation as well as cell survival, proliferation and

colony formation (J. Sun et al., 2009). These findings can be explained by the differential localization of the WT and mutated forms which might recruit distinct adaptors proteins.

Serve et al. compared the phosphorylation profile of proteins activated by membrane- and retained forms of 3. While membrane-3 activated the MAPK and AKT pathway, the ER-retained 3-ITD activate the STAT5-PIM pathway (Choudhary et al., 2009).

To summarize this section, oncogenic signaling from activated RTKs differs from physiological signaling at multiple levels. Quantitative changes of an oncogenic signal are related to the capacities of oncogenes to escape the negative regulatory mechanisms. Direct escape concern mutations that affect kinase self-control mechanisms, and the recruitment of negative regulators such as phosphatases or elements of the endocytic machinery. Indirect mechanisms are mostly linked to an abnormal location of the oncogenic forms with respect to their normal counterpart. Qualitative changes are also features of oncogenic signaling.

Mutations that result in conformational changes of TKRs can alter the catalytic specificity of the enzymes. Indirectly, the subcellular localization may influence the precise nature of signaling by activating alternative pathways. This activation can occur via the recruitment of specific proteins or by changing the qualitative nature of receptor phosphorylation. All these mechanisms are not exclusive and likely account for the differences between oncogenic and normal signaling. This concept is well illustrated by viral oncogenes. Lastly, it is not excluded that some mutants may use several mechanisms at the same time, but it seems that each oncogene may use a unique combination of such mechanism.