IV. KitL/c-Kit signaling
IV.2. Signaling pathways activated downstream of c-Kit
The intracellular signaling pathways activated by c-Kit is one of the main subjects of this work.
Here we discuss some of these pathways and their functional implication in KitL/c-Kit axis in this section. It should be noted that much of the data obtained in vitro on the signaling pathways were done under soluble KitL stimulation. Functional results from transgenic animals are in a physiological context in which KitL can be soluble or membranous-bound depending on the tissue.
IV.2.i. The PI3K/AKT pathway
The phosphatidylinositol-3-kinases (PI3Ks) constitute a family of lipid kinases which phosphorylate phosphoinositide on the 3' hydroxyl group of the inositol ring (Morgan et al., 1990). Their lipid substrates are phosphatidylinositol (PtdIns), PtdIns-4-biphosphate (PtdIns4P) and PtdIns(4,5)P2 (Morgan et al., 1990; Okkenhaug, 2013). There are different classes of PI3Ks, but the Class I are preferentially activated by the TKRs, G protein-coupled receptors and Ras (Leevers et al., 1999). Class I PI3Ks catalyzes the conversion of PtdIns(4,5)P2
to PtdIns(3,4,5)P3. They are heterodimers composed of a p85 regulatory subunit and a p110 catalytic subunit (Carpenter et al., 1990). There are four p110 (a, b, d, g) and five p85 or p85-like (p85a, p55a, p50a, p85b, p55g) isoforms. In mammals, p110a and b are ubiquitously expressed while p110d and g are highly enriched in leukocytes (Vanhaesebroeck et al., 2010).
Below we refer to the activation of the class I PI3Ks, and more precisely to the p85a/p110 heterodimer.
The p85a subunit can interact via its SH2 domain with a phosphorylated tyrosine (pY) of an activated receptor. This tyrosine residue is part of the consensus binding motif (Y-X-X-M) for the SH2 domain of p85a (Songyang et al., 1993; Yoakim et al., 1994). Once engaged, the p85 subunit brings the p110 catalytic subunit near the membrane where it encounters its lipid substrates and produces PtdIns(3,4,5)P3. The generation of PtdInsP3 allows the recruitment
of effector proteins containing pleckstrin homology (PH) domains such as Akt, GAB2, PDK1, and modulators of the small GTPase activity. Following recruitment to PtdInsP3, PDK1 phosphorylates and activates Akt (Vanhaesebroeck & Alessi, 2000; Vanhaesebroeck et al., 2010).
The role of the PI3K/Akt pathway downstream of c-Kit activation has been studied in both hematopoietic and non-hematopoietic cells. It should be noted that the p85a subunit is the major isoform in hematopoietic cells. In the c-Kit receptor, the p85a subunit can bind directly to the murine pY719 (Serve et al., 1994; Serve et al., 1995) or human pY721 residue (Blume-Jensen et al., 1998) (Figure 7). Until now, p85a is the only known interactor for this c-Kit tyrosine residue. After KitL stimulation, PI3K can also be indirectly recruited to the tyrosine-phosphorylated GAB2 (Nishida et al., 2002). The c-Kit downstream activation of the PI3K is critical for mast cell proliferation and survival as well as mast cell secretion, adhesion, and actin polymerization (Kissel et al., 2000).
The cellular functions of class I PI3K were studied in mast cells derived from PI3K-/- mice lacking the p85a subunit. Mice defective for p85a have reduced number and function of mastocytes in the intestinal cavity and peritoneum (Fukao et al., 2002). Similarly, deficiency of p85a reduced fetal liver-derived mast cell proliferation and function in response to KitL.
Moreover, (Blume-Jensen et al., 1998)showed that PI3K is required for KitL-induced cell proliferation and survival in vitro. In agreement with this, KitL-dependent PI3K activity and Akt activation were blocked in p85a-deficient mast cells (Lu-Kuo et al., 2000). The function of PI3K/Akt pathway was also explored by disrupting the p85a binding site on c-Kit (mouse Y719). Indeed, KitL-induced PI3K signaling and Akt activation were almost completely abolished in c-KitY719F/Y719F mast cells (Blume-Jensen et al., 2000; Kissel et al., 2000). It was further shown that c-Kit could also activate the PI3K/Akt pathway through the indirect association of the scaffolding protein Gab2 (J. Sun et al., 2008).
The reconstitution of BMMC deficient for c-Kit with the Y719F mutant receptor demonstrated the role of the pathway in SCF-dependent mast cell-effector functions such as adhesion and degranulation. BMMCs from animals deficient for p85a have a chemotaxis defect to KitL (Tan, Yazicioglu, et al., 2003) whereas pure IgE-dependent degranulation is unaffected. The PI3K activity, therefore, appears to be involved in the KitL-mast cell-dependent effector functions in vitro and in vivo. Y719F knock-in mice revealed discrete phenotypes in hematopoietic
compartments. However, they also demonstrated a non-redundant fundamental role of c-Kit activation of the PI3K pathway in the reproductive function. Indeed, both males and females have a lack of fertility. Males are sterile due to blockage of spermatogenesis and females blocked during follicular development (Blume-Jensen et al., 2000; Kissel et al., 2000).
IV.2.ii. The gamma phospholipase (PLCg)
Phospholipases C (PLCs) are enzymes that hydrolyze the ester bond between the glycerol and phosphate of phospholipids, releasing a diglyceride (DAG) and a phospho-alcohol. In vivo, PLC hydrolyzes PtdIns(4,5)P2 to DAG and inositol-1-4-5-triphosphate (IP3). The second messenger DAG activates PKC while IP3 triggers the release of Ca2+ from the endoplasmic reticulum.
Gamma-type PLC (PLCg) are activated by RTKs. There are two isoforms of PLCg with different expression patterns. While PLCg1 is ubiquitously expressed, PLCg2 is restricted to the hematopoietic compartment (Wilde & Watson, 2001). Consequently, deficiency of PLCg2, while viable, induces growth delay and internal hemorrhage due to defective platelet aggregation (D. Wang et al., 2000).
After membrane-KitL stimulation, PLCg interacts with pY730 of c-Kit via its SH2 domain (Rottapel et al., 1991) (Figure 7). This interaction leads to PLCg activation which is required for cell proliferation (Gommerman et al., 2000; Herbst et al., 1991; Trieselmann et al., 2003).
It has been suggested that PLCg is activated only by membrane-KitL. However, soluble-KitL can indirectly activate PLCg by activating PLD (Koike et al., 1993) in a PI3K-dependent way (Kozawa et al., 1997). KitL protects cells that express c-Kit from apoptosis (Neta et al., 1993).
The radioprotective effect of KitL against apoptosis implicates PLCg1 as a major player. By mutating Y730 or using a PLCg inhibitor (U-73122), c-Kit was unable to protect cells from radiations (Maddens et al., 2002; Plo et al., 2001).
IV.2.iii. The MAPK pathway
The MAPK cascade is a module made up of three enzymes activated in series. The first kinase to be activated is the MAP kinase kinase kinase (MAP3K). Then it activates the MAP kinase kinase (MAP2K) by phosphorylation on serine and threonine residues. In turn, MAP2K activates a MAPK (e.g., ERK) by phosphorylation on a tyrosine residue and then on a serine.
The three most studied MAPK modules are: (i) Raf1/MAP2K1,2/MAPK 1,2; (ii) the p38 MAPKs;
and (iii) the JNK1, 2, 3.
All TKRs activate the ERK pathway by activating the small G protein Ras. Ras proteins as other members of the family are active when bound to GTP and inactive when bound to GDP. The hydrolysis of GTP is facilitated by the action of GTPase activating proteins (GAPs), while Guanine nucleotide Exchange Factors (GEFs) catalyze the conversion of GDP to GTP. The best-characterized pathway that activates Ras involves the Ras-GEF named SOS.
SOS is linked to GRB2 which can interact directly with c-Kit Y703 and Y936 (Thommes et al., 1999) (Figure 7). GRB2 can also be indirectly recruited to c-Kit via APS (Wollberg et al., 2003), SHP-2 (Tauchi et al., 1994) or SHC (D. J. Price et al., 1997), all described to interact directly with c-Kit on Y568/Y570. “Add-back" experiments revealed that the restitution of Y568/Y570 was sufficient to restore activation of the MAPK pathway (L. Hong et al., 2004). In agreement with this result, BMMCs derived from the YY568/570FF knock-in mice exhibit a MAPK pathway activation defect (Kimura et al., 2004). Besides, GRB2 can also be recruited on tyrosine phosphorylated GAB2 following activation of c-Kit at least in mast cells (Nishida et al., 2002).
The ERK pathway is involved in the differentiation of hematopoietic cells, and the activation kinetics of the pathway seems to influence the cell fate (Uchida et al., 2001). Engagement of ERK following the activation of c-Kit must have a role during the development of the different c-Kit dependent cell lineages, but this function had not been addressed directly. The phenotypic defects of YY568/570FF knock-in mice suggest that other signaling pathways are probably affected.
IV.2.iv. The Src Family of Kinases (SFKs)
In mammals, the family of Src kinases (SFK) comprises two classes. Class A is composed of Src, Yes, Fgr, Yrk, and Fyn. The kinases Lyn, Hck, Lck, and Blk, belong to the class B. While Src, Fyn and Yes are ubiquitously expressed, the other members are more restricted to specific tissues such as the hematopoietic compartment. For a review see (Thomas & Brugge, 1997)
The SFKs share a common structure and activation mode. They are composed of an SH3 domain, an SH2 domain, and a catalytic domain. In the inactive state, the SFKs have a compact closed structure maintained by intramolecular protein-protein interactions. When phosphorylated, a C-terminal tyrosine residue (Y527 for Src) interacts with the SH2 domain.
This interaction facilitates the contact of the SH3 domain with a polyproline motif located in a linker region. Thus, SFKs exhibit a self-inhibited conformational structure which can be
released by (i) dephosphorylation of Y527 residue (ii) competitive binding of the SH2 domain to another phosphotyrosine, (iii) competitive binding of the SH3 domain (Reviewed in (Ingley, 2008)).
The most used SFK inhibitors are SU6656, and the Protein Phosphatase (PP) 1 and 2 inhibitors (Bain et al., 2007). Results obtained with PP1 and PP2 under c-Kit stimulation should be taken with caution since PP1 and PP2 are also inhibitors of c-Kit (Tatton et al., 2003). Dasatinib is also an excellent SFK inhibitor, but it also blocks c-Kit in its kinase active state (Lombardo et al., 2004).
Activation of SFKs is presented here without specifying the member of the family concerned.
In BMMCs, the main SFK member is Lyn; Fyn and Hck being present ten to twenty times less (H. Hong et al., 2007). In the hematopoietic system, the activation of c-Kit by KitL leads to SFK association with c-Kit and activation (Linnekin et al., 1997)). This interaction depends on the association of the SH2 domain of SFK with pY568 and the di-phosphorylated unit pY568/pY570 of c-Kit (Lennartsson et al., 1999; D. J. Price et al., 1997; Timokhina et al., 1998) (Figure 7). Most of the studies investigating the biochemical and functional consequences of SFK activation by c-Kit come from the analysis of pY568/pY570 mutation to phenylalanine (YY568/570FF) which block SFK binding and activation. However, because other interactors with these phosphosites exist (Table 1), it seems too reductive to analyze these mutants with the sole prospect of SFK activation.
The contribution of SFKs in the cellular responses mediated by KitL/c-Kit have been studied outside the context of the mutant YY568/570FF. Indeed, BMMCs from mice deficient in Lyn, Fyn and Hck revealed the role of Lyn kinase in KitL/c-Kit-dependent proliferation (Linnekin et al., 1997). Also, Fyn-deficient BMMCs uncover a function of the kinase in the reorganization of the actin cytoskeleton during migration of mast cells towards KitL (Samayawardhena et al., 2007). BMMCs deficient in Hck have a KitL-dependent proliferation defect in culture even though the Hck-/- mice do not present any visible defect in the mast cell compartment. The results from studies on BMMCs Lyn-/- are contradictory. A study showed proliferation and migration defects of Lyn-/- BMMCs (O'Laughlin-Bunner et al., 2001). However, another study suggested that Lyn contributes to the negative regulation of KitL-dependent mast cell proliferation. Moreover, the authors demonstrated an increase in the number of tissue mast cells in Lyn-/- mice (Hernandez-Hansen et al., 2004). Besides cell proliferation, Lyn also participates in the negative regulation of KitL-induced degranulation (Odom et al., 2004).
The involvement of SFKs in c-Kit-mediated proliferation (L. Hong et al., 2004; Lennartsson et al., 1999; Tan, Hong, et al., 2003; Timokhina et al., 1998) and migration (L. Hong et al., 2004;
Ueda et al., 2002) was also analyzed by using the c-Kit YY568-570FF mutant. Finally, the engagement of SFKs in c-Kit internalization (Broudy et al., 1999) is discussed in section 0.
IV.2.v. Janus Kinases and Signal Transducer And Activator of Transcription (JAK/STAT) The mammalian family of Janus Kinase (JAK) includes four cytoplasmic tyrosine kinases JAK-1, -2, -3 and TYK-2. JAKs are intracellular signaling effectors of cytokine receptors and provide them with a kinase activity. JAK-1 is involved in signaling downstream of IL-2 and IL-4 receptors, gp130 family receptors and class II cytokine receptors. JAK-2 signals are downstream of single chain receptors such as the Epo receptor (EpoR), the IL-3 receptor family, the gp130 family and the type II cytokine receptors (e.g., IFN-g). JAK-3 is predominantly expressed in the hematopoietic compartment and acts in the transduction of signals from cytokines receptors which use the gamma chain. Reviewed in (Babon et al., 2014).
Signal Transducers and Activators of Transcription (STATs) are transcription factors that transduce signals from many cytokines. Upon activation by the JAKs, STATs are recruited to cytokine receptors via their SH2 domain and phosphorylated by JAKs proteins. Afterwards, they dimerize and move to the nucleus where they then bind to specific promoters inducing the transcription of target genes. In mammals, there are seven STAT proteins which share a common structural arrangement of functional domains. Reviewed in (Mitchell & John, 2005).
Little is known about the link between KitL/Kit and the JAK/STAT pathway. Stimulation of Kit by KitL is accompanied by JAK-2 phosphorylation which is constitutively associated with c-Kit (Brizzi, Zini, et al., 1994; Linnekin et al., 1996; Weiler et al., 1996). Experiments using antisense oligonucleotides (Weiler et al., 1996) or fetal liver progenitors from JAK-2 deficient mice show that JAK-2 activation Is essential for KitL/c-Kit-mediated proliferation (Radosevic et al., 2004). Following activation of c-Kit, STAT1, STAT5A, and STAT5B are recruited to the receptor and tyrosine phosphorylated (Brizzi et al., 1999). This modification is accompanied by an increase in their binding to DNA (Deberry et al., 1997; Ryan et al., 1997). In KitL stimulated-BMMCs STAT1/2/3 activation is rapid and transient. This response depends on the c-Kit D816V kinase activity (Chaix et al., 2011). However, additional observations concerning the activation of the JAK-STAT pathway downstream of c-Kit are contradictory. For example, in erythroid cell lines, stimulation by KitL alone does not activate the JAK-2/STAT5 pathway
which is specifically activated by EpoR stimulation (Jacobs-Helber et al., 1997; Joneja et al., 1997). Similarly, in a murine mast cell line, activation of the JAK-2/STAT5 pathway was observed under IL-3 stimulation but not by KitL (O'Farrell et al., 1996). These inconsistencies are probably due to the cell context as well as the stimuli used in the cited studies.
Here we have presented the conventionally signaling pathways downstream of c-Kit. Other pathways exist and are probably activated in certain cellular and tissue contexts.
Figure 7. C-Kit signaling and regulation. KitL binding to c-Kit induces the receptor dimerization and autophosphorylation of cytoplasmic tyrosine residues and the recruitment of diverse proteins. The proteins recruited include Grb2 (growth-factor-receptor-bound protein 2); SRC-family kinases (SFKs); signalling enzymes such as the Phospholipase Cγ (PLCγ) and the Phosphatidylinositol 3-kinase (PI3K). The subsequent activation of these proteins, as well as the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) and the RAS/MAPK pathway leads to cell survival, proliferation and differentiation; cell adhesion and spreading; and maste cell chemotaxis and secretory functions. The red box highlights the proteins involved in the downregulation of c-Kit such as the SH2-domain-containing protein tyrosine phosphatases (SHP1/2); Supressor Of Cytokine Signaling (SOCS); the E3-ubiquitin ligase Cbl; and the Protein Kinase C (PKC). SOS: Son Of Sevenless. See text for more details.