Regulation of c-Kit signaling

The balance between switching-on and -off intracellular signaling pathways is essential for the cell homeostasis. This balance is ensured by negative feedback loops which guarantee that the activation by itself triggers the shutdown mechanisms. The general mechanisms of TKRs downregulation comprise (i) binding of antagonist ligands, (ii) inhibition of the kinase activity and (iii) dephosphorylation of tyrosine residues by tyrosine phosphatases. Signaling extinction is related to the degradation of the receptor following its irreversible endocytosis (Schlessinger, 2000). In this section, we described the mechanisms of KitL/c-Kit signaling downregulation.

IV.3.i. Protein Kinase C (PKC)

The PKC family comprises serine/threonine kinases which activities are controlled by second messengers. Based on the second messenger they require, there are four groups of proteins.

Conventional (a, bI, bII, and g) PKCs are activated by Ca²⁺, DAG, and phospholipid; while novel (d, e, h, and q) are stimulated by DAG alone. Atypical (i and l) PKCs are activated neither by DAG nor by Ca²⁺. As seen in section IV.2.ii, both second messengers Ca²⁺ and DAG are released by the enzymatic activity of the PLCs after TKRs activation. Reviewed in (Cullen, 2003)

PKCs have been implicated in the negative regulation of several RTK (Blume-Jensen et al., 1993; Cochet et al., 1984) but the mechanism of TKRs signal attenuation is less well clear.

PKCs are capable of phosphorylating c-Kit in the kinase insert region (S741 and S746) which inhibits the kinase activity by an unknown mechanism (Jensen et al., 1993; Blume-Jensen et al., 1995). On the other hand, PKCs induce the proteolytic cleavage of the extracellular domain of c-Kit (Yee, Hsiau, et al., 1994). A more recent study showed that PKCd activation induces the recycling of c-Kit in colon cancer cells. Unfortunately, most of the data about the regulation of c-Kit kinase activity by PKCs are relatively old and have not been repeated.

IV.3.ii. Protein tyrosine phosphatases (PTPs)

Proteins tyrosine phosphatase (PTPs) are enzymes that specifically hydrolyze phosphate groups bound to tyrosine residues. The superfamily of PTPs comprises two classes, classical tyrosine phosphates and dual specificity phosphatases (tyrosine and serine/threonine).

Reviewed in (Tonks, 2006)

Tyrosine phosphatases negatively regulate TKRs activated or not by their respective ligands.

In the absence of ligand, PTPs maintain the TKRs at a low level of activation. In fact, any TKR can be activated at the plasma membrane by a tyrosine phosphatase inhibitor such as pervanadate (Jallal et al., 1992). Similarly, the phosphatase PTP-1B dephosphorylates the receptor during its biosynthesis. (Schmidt-Arras et al., 2005). Phosphatases can also act on phospho-tyrosine residues involved in the recruitment of signaling proteins and the activation of intracellular pathways (Kozlowski et al., 1998; Paulson et al., 1996), but also in the phospho-tyrosine of the A-loop (Huse & Kuriyan, 2002). The role of PTPs in the regulation of TKRs was demonstrated by using PTP deficient mice. For example, PTP-1B-deficient mice have an increased sensitivity to insulin, suggesting a role of such phosphatase in the negative control of the insulin receptor (Elchebly et al., 1999; Klaman et al., 2000).

The involvement of the phosphatase SHP-1 in c-Kit signal regulation arose from studies of intergenic complementation by crossing "me/+” and “W^v/+” mice. Mice “motheaten” (me) carry natural mutations on the PTP1C gene which encode a tyrosine phosphatase SHP-1. The

“me" mice manifest a high expansion of several hematopoietic cell populations that leads to a lethal systemic auto-immunity. (Bignon & Siminovitch, 1994). W^v mice carry a c-Kit mutation that induces a partial loss of function which is not lethal but is associated with lack of coat pigment and anemia (Nocka, Tan, et al., 1990). Improvement of both "W^v" and "me"

phenotypes demonstrated the role of SHP-1 in the in vivo regulation of c-Kit in the hematopoietic compartment. (Lorenz et al., 1996; Paulson et al., 1996)

The interaction of c-Kit and SHP-1 depends on c-Kit activation by KitL (Yi & Ihle, 1993) and occurs at Y569 of the JMD (Kozlowski et al., 1998). SHP-1 is also involved in the negative regulation of signals initiated by EGFR (Keilhack et al., 1998) and Fms (Umeda et al., 1999). In general, there is no absolute selectivity of the PTPs towards the TKRs, the same PTP being able to act on several TKRs and the other way around. The cell/tissue context might also be responsible for the PTPs/TKRs specificity.

IV.3.iii. Suppressor Of Cytokine Signaling (SOCS)

The SOCS proteins are well-known for their role in the negative regulation of the JAK/STAT pathway. All the members of the family contain an SH2 domain and a SOCS box, as well as a Kinase Inhibitory Region (KIR) domain (Yoshimura et al., 2007). Briefly, SOCS1 and SOCS3 can inhibit the kinase activity of JAKs either by acting as a pseudosubstrate or by interacting via

their SH2 domain with a catalytic tyrosine. Reviewed in (Croker et al., 2008). SOCS1 binds to c-Kit as well as GRB2 and VAV. In hematopoietic cells, SOCS1 suppressed KitL-mediated proliferation but maintained cell survival signals. However, it did not block the c-Kit kinase activity (De Sepulveda et al., 1999). The authors suggested that SOCS might behave like a competitor and thus block the recruitment and phosphorylation of other interactors involved in cell growth.

IV.3.iv. Endocytosis and degradation

The primary function of endocytosis of activated receptors is to remove them from the cell surface so that the cell can respond to a new stimulation.

Ubiquitination is a cascade of reactions catalyzed by three classes of ligases enzymes (E1, E2, E3) leading to the covalent attachment of a ubiquitin moiety to a lysine residue of a substrate protein. Substrates can be mono- or poly-ubiquitinated. While poly-ubiquitination on defined lysine residues is linked to protein degradation by the proteasome, mono-ubiquitination is more related to endocytic trafficking, DNA repair, and inflammation. Reviewed in (Miranda &

Sorkin, 2007). Many RTKs are ubiquitinated in a ligand-dependent manner by Cbl (for Casitas B-lineage Lymphoma) proteins. Cbl proteins are E3-ubiquitin ligase enzymes, but they can also function as adapters protein. They interact directly with phospho-tyrosine residues of TKRs via their Tyrosine Kinase Binding (TKB) domain or are recruited indirectly by adapters such as GRB2. Reviewed in (Thien & Langdon, 2005)). Cbl is activated by phosphorylation.

Active Cbl transfers ubiquitin from an E2-ligase to an active TKR. In the case of EGFR, it appears that mono-ubiquitination is sufficient for their endocytosis and that polyubiquitination can accelerate the process (Haglund et al., 2003).

Following their activation, the TKRs are phosphorylated and recruit Cbl. Once ubiquitinated the TKRs are recruited in coated pits which bud in the cytoplasm to become clathrin-coated vesicles. These vesicles are further excised, lose their coat and then fuse with the early endosomes, forming the sorting endosomes (sorting endosomes). At this stage, the TKRs may be recycled to the membrane or continue in the endocytic pathway. Ubiquitinated receptors can be recruited to sorting endosomes, internalized and incorporated into the Multi Vesicular Bodies (MVBs). The MVBs then fuse with lysosomes, resulting in the degradation of receptors.

Reviewed in (Katzmann et al., 2002; Wiley & Burke, 2001).

In KitL-stimulated mastocytes, c-Kit is rapidly removed from the cell surface. After approximately 30 minutes of stimulation, the percentage of receptors remaining on the surface is reduced by at least 50% (Yee, Hsiau, et al., 1994). This loss is related to the internalization of the receptor which remains coupled to KitL (Yee, Hsiau, et al., 1994) and which appears in punctate intracellular structures (Broudy et al., 1998; Jahn et al., 2002).

Internalization is accompanied by degradation of the receptor which is no longer detectable at all after one hour of stimulation (Shimizu et al., 1996). In the absence of KitL, the half-life of c-Kit is one to two hours, while this time is reduced to 30 minutes after ligand stimulation.

Recovery of the receptor to the cell surface follows a much slower kinetics. It takes more than 48 hours to regain the basal expression level (Shimizu et al., 1996). Furthermore, re-expression of c-Kit at the cell surface seems to be entirely dependent on the de novo synthesis and translation of new messengers (Broudy et al., 1998; Jahn et al., 2002; Shimizu et al., 1996).

The c-Kit kinase activity is required for internalization (Jahn et al., 2002; Yee, Hsiau, et al., 1994). Study of mutants Y719F and YY568/570FF suggested that Src, but not PI3K, is involved in c-Kit internalization (Gommerman et al., 1997; Jahn et al., 2002; Yee, Hsiau, et al., 1994).

For instance, Lyn-deficient cells which exhibit reduced c-Kit internalization (Broudy et al., 1999). Similarly, inhibition of Src kinase activity with the kinase inhibitor SU6656 reduced c-Kit degradation (Masson et al., 2006; Zeng et al., 2005).

Cbl is involved in the process of endocytosis and degradation of c-Kit. It has been shown that Cbl-b and c-Cbl interact with c-Kit and are phosphorylated following its activation (Zeng et al., 2005). Members of the Cbl family could be recruited on c-Kit indirectly via APS (Wollberg et al., 2003; Yokouchi et al., 1999) or GRB2 (Brizzi et al., 1996; J. Sun et al., 2007). A direct interaction dependent on the phosphorylation of c-Kit is described on pY568 and pY936 (see Table 1). The recruitment of Cbl on c-Kit and its phosphorylation are required for c-Kit ubiquitination and internalization which leads to lysosomal degradation. The specificity of this interaction is dictated by a hydrophobic residue isoleucine/leucine in +3-position of the phospho-tyrosine residues: I571 and L939 (Masson et al., 2006).

It has been shown that c-Kit is poly-ubiquitinated following its activation (Miyazawa et al., 1994; Yee, Hsiau, et al., 1994; Zeng et al., 2005). However, Masson et al. showed that c-Kit is rather mono-ubiquitinated (Masson et al., 2006). Co-immunoprecipitation experiments (Gommerman et al., 1997) and confocal imaging of a c-Kit chimera construct suggest that

upon stimulation c-Kit localize to clathrin-coated pits. Moreover, the integrity and functionality of the lipid RAFTs is a prerequisite for its internalization (Jahn et al., 2002).

SOCS proteins can form a complex which has E3-ubiquitin ligase activity. Indeed, the partners of SOCS can thus be ubiquitinated and directed towards the 26S proteasome. Reviewed in (Kile et al., 2002; Piessevaux et al., 2008). SOCS5 and EGFR interact directly via the SH2 domain of SOCS5. This interaction decreases the mitogenic capacity of EGFR and regulates its half-life of EGFR (Kario et al., 2005; Nicholson et al., 2005). Similarly, SOCS6 interacts directly with the JMD of c-Kit (pY568) and reduce KitL-dependent proliferation and signaling (Bayle et al., 2004). Thanks to its SOCS box, SOCS6 can act as a ubiquitin ligase and target c-Kit to degradation by the proteasome (Zadjali et al., 2011).

In summary, the control of c-Kit signaling, (intensity and duration) is critical for cell homeostasis. Downregulation of c-Kit activity is carried out mainly by three feedback mechanisms: PKC activity, dephosphorylation by tyrosine phosphatases and receptor degradation. Any disturbance leads to an aberrant signaling resulting in pathological disorders and malignancies. In the following section, we consider the involvement of KitL/c-Kit in malignant transformation.