Functional deregulation of KIT: link to mast cell proliferative diseases and other neoplasms

Abstract

In this review, the authors discuss common gain-of-function mutations in the stem cell factor receptor KIT found in mast cell proliferation disorders and summarize the current understanding of the molecular mechanisms by which these transforming mutations may affect KIT structure and function leading to altered downstream signaling and cellular transformation. Drugs targeting KIT have shown mixed success in the treatment of mastocytosis and other hyperproliferative diseases. A brief overview of the most common KIT inhibitors currently used, the reasons for the varied clinical results of such inhibitors and a discussion of potential new strategies are provided.

Keywords: KIT inhibitors; KIT mutations; KIT signaling; KIT trafficking; Mastocytosis.

Figure 1

Structure of KIT. KIT comprises five extracellular immunoglobulin-like domains, a membrane spanning domain, and two catalytic kinase domains. The first three immunoglobulin-like domains are responsible for binding to the KIT ligand, SCF. The two immunoglobulin-like domains proximal to the plasma membrane interact and facilitate dimerization of KIT. A region of four amino acids (GNNK) lies adjacent to the plasma membrane region, and alternative splicing of KIT results in GNNK+ and GNNK− isoforms. The juxtamembrane (JM) domain of KIT contains the Tyr residues Y568 and Y570, which become phosphorylated upon activation releasing its auto-inhibitory function. Point mutations in the JM domain change its conformation and prevent its regulatory function. The V560G mutation is an example of an activating mutation in the JM domain, particularly in association with GISTs. There are two catalytic kinase domains (KD) separated by a kinase insert domain. Several activating mutations have been reported in the KD of KIT. The D816V mutation is a common mutation and is associated with mastocytosis.

Figure 2

KIT signaling pathways. KIT signaling occurs through several pathways. Phosphorylation of the juxta-membrane Tyr residues recruits Src family kinases (SFKs), in particular Lyn, initiating PI3K signaling and the phosphorylation of other signaling and adaptor proteins. These events most likely occur within lipid rafts where the PI3K substrate, PI(4,5)P₂, is converted to PI(3,4,5)P₃, a signaling lipid that contributes to the activation of other enzymes. Adaptor proteins (NTAL and Grb2) are phosphorylated by the receptor or by Src kinases and recruit other signaling proteins, forming signaling complexes. PLCγ1, for example is recruited by the adaptor protein NTAL to lipid rafts, activated by phosphorylation and by PI(3,4,5)P₃, cleaving PI(3,4)P₂ to form IP₃, which induces the release of Ca²⁺ from intracellular stores; and DAG, together with Ca²⁺ activates PKC. Tyr residues within the kinase insert domain of KIT interact with PI3K and Grb2, leading to the activation of the Ras/Raf/MAPK pathway. Another pathway critical for KIT mediated proliferation, particularly in gain-of-function KIT mutants, is the activation of the transcription factors STAT1/3/5 (signal transducers and activators of transcription). Janus kinase (JAK) is phosphorylated after KIT activation, and in turn phosphorylates STAT allowing its translocation to the nucleus, where it exerts its function. Inhibitory pathways of KIT also regulate responsiveness to SCF. The juxtamembrane Tyr residues of KIT can recruit the phosphatases, SHP1, SHP2 and SHIP1, which may contribute to negative regulation of KIT signaling. In addition, recruitment of the ubiquitin E3 ligase, c-Cbl, via the dimeric adaptor protein APS, may be a critical determinant for ubiquitination and degradation of KIT. Both Tyr 936 and Tyr 570 appear to regulate the recruitment of c-Cbl and binding of APS. APS exists as a dimer and thus may require two simultaneously phosphorylated KIT residues to recruit c-Cbl. For simplification, only one extracellular domain of KIT is represented and bound ligand is not depicted.

Figure 3

Receptor tyrosine kinase trafficking may affect signal transduction. Trafficking of receptor tyrosine kinases (RTKs), such as KIT, may regulate signal transduction pathways by binding to adaptor proteins specific to endosomal compartments and altering the type of signaling complexes recruited. Initiation of RTK signaling triggers recruitment to lipid rafts where interactions with plasma membrane anchored signaling molecules is facilitated. In particular, lipid rafts may be critical for signaling through the Src family kinases, PI3K and PLCγ1. Internalization of RTKs can induce the activation of additional signaling pathways by recruitment of adaptor proteins such as adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) in very early endosomes, which can bind AKT and direct signaling through GSK3β. As the early endosome matures it becomes positive for PtdIns(3)P which recruits the early early endosomal antigen 1 (EEA1) protein. At this point, the early endosome may sort receptors for recycling back to the plasma membrane. Receptors that are not recycled remain in the endosome as it matures to a late endosome and control switches from Rab5 to Rab7. Signaling still occurs within the limiting membrane of late endosomes where adaptor protein complexes of p14, p18 and MP1, which are specific to the late endosomal compartments, may promote unique signaling to the MAPK pathway. The localization of late endosomes may also promote signal transduction to the nucleus because their close proximity to the centrosome allows weak signals to travel shorter distances and reach the nucleus. Eventually, receptors passage into intraluminal vesicles of multi-vesicular bodies and lysosomes, which results in cessation of signaling and receptor degradation. The schematic is an oversimplification, as multiple signaling pathways may well occur at all stages of receptor trafficking, but enrichment of particular adaptor proteins may promote a particular pathway by facilitating specific interactions. Activating mutations in RTKS favor their intracellular localization and trigger signaling from intracellular compartments in the absence of ligand. Further studies are needed to determine how the constitutively active RTKs traffic and signal in cells. Normal RTKs are depicted in green and mutated RTKs in red.