DEFINITION
Stem cell factor, also termed Kit ligand, steel factor or mast cell growth factor is the ligand of the c-kit protooncogene product. It is a glycoprotein existing in both soluble and membrane bound forms, after alternative splicing and proteolytic cleavage. Therefore, SCF has been first described as a pluripotent growth factor involved in the early stages of haematopoiesis, as well as in the development and function of germ cells and melanocytes. In addition, SCF may be implicated in inflammatory processes
CHEMICAL STRUCTURE AND IMAGES
Structural basis for activation of the tyrosine kinase KIT by stem cell factor. Cell 2007
Crystal Structure of the KIT Ectodomain
KIT ectodomain shows an elongated serpentine shape with approximate dimensions of 170 X 60 X 50 A°.D1, D2, D3, D4, and D5 domains of KIT exhibit a typical immunoglobulin super family (IgSF) fold, composed of eight b strands, designated ABCC0DEFG, assembled into a b sandwich consisting of two antiparallel b sheets. D1, D2, D3, and D5 each contain a conserved disulfide bond connecting cysteine residues at B5 and F5 (Fifth amino acids of strand B and F, respectively), positions that bridge the two b sheets to form the center of the hydrophobic core of the Ig-like fold. D2 and D5 contain two disulfide bonds and D4 does not contain any cysteine residue; nevertheless,
the integrity of the Ig-like fold of D4 is maintained even though the conserved cysteine residues at B5 and F5 are replaced by a valine and phenylalanine residues, respectively. The angle between D1 and D2 along the axis of the two domains is 76°, resembling the orientation between the first and second Ig-like domains of the interleukin-1b receptor. In contrast, the angle between D2 and D3 is 150°, between D3 and D4 is 119°, and between D4 and D5 is 162°. The orientation between the ABED and A0GFC b sheets for the different Ig-like domains are~180° for D1-D2, ~180° for D2-D3, ~90° for D3-D4, and ~180° for D4-D5. The superposition of all five Ig-like domains of KIT ectodomain with telokin used as a standard for Ig-folds reveals root-mean-square deviations
(rmsd) of 1.5–2.9 A° for equivalent Ca atoms. D2 is the most divergent among the five KIT Ig-like domains as revealed by its higher rmsd values when superimposed with telokin. Based on structural conservation of key amino acids in Ig-like domains and their secondary structural topology D1, D2, D3, and D4 belong to the I-subset and D5 is related to the C2 and IgCAM subsets of IgSF . Furthermore, among the structurally conserved 20 finger-print residues of IgSF , 10–14 residues are conserved in the five Ig-like domains of KIT.
Overall Structure of the SCF-KIT Complex
Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor
Cell, Volume 130
The structure of SCF-KIT complex shows 2:2 stoichiometry,in which two sets of 1:1 complexes in the asymmetric unit are related by a noncrystallographic 2-fold symmetry. The observed SCF-KIT 2:2 complex in the crystal lattice is consistent with experiments demonstrating that KIT dimerization is driven by the dimeric SCF ligand. The two sets of KIT ectodomains and SCF molecules resemble an upside down ‘‘A’’ letter with approximate dimensions of 170 X
130 X 70 A°. The overall structure of SCF bound to KIT is similar to the previously described structures of free SCF. The structure of SCF-KIT 2:2 complex shows that an individual SCF protomer binds directly to D1, D2, and D3 of an individual KIT protomer. Consequently, a single-receptor protomer forms a symmetric complex with a similar 2-fold related surface on an SCF protomer. Dimerization of KIT is also mediated by homotypic interactions between the two membrane-proximal Ig-like domains of KIT, namely by D4-D4 and D5-D5 interactions. This results in dramatically altered configurations of D4 and D5 relative to the rest of the molecule, and these configurations bring the C termini within 15 A° of each other close to the place where they connect to the transmembrane domain. The structure is also characterized by the existence of a large cavity at the center of the complex with dimensions of ~50 3 503 15A°. The crystal structure demonstrates that each protomer of SCF binds exclusively to a single KIT molecule and that receptor dimerization is driven by SCF dimers that facilitate additional receptor-receptor interactions.
Protein Aminoacids Percentage
SYNTHESIS AND TURNOVER
Stem cell factor and its receptor c-Kit as targets for inflammatory diseases 2005
The SCF gene is encoded at the steel (Sl) locus on human chromosome 12q22-q24 and murine chromosome 10.
SCF is expressed as two isoforms after alternative splicing of the sixth exon. The first SCF isoform is a 248 aa (45 kD) glycoprotein (SCF248) expressed at the cell membrane which is cleaved by proteases to generate a 165 aa (31 kD) soluble protein (sSCF or SCF165). The cleavage site is encoded by the sixth exon (Val-Ala-Ala-Ser, aa 163–166). The second SCF isoform is a 220 aa (32 kD) glycoprotein also expressed at the cell membrane. This form, lacking the sixth exon, remains membrane bound (mSCF or SCF220) but may also be shed by proteases to generate a soluble form. The secondary cleavage site used to generate the soluble form has been reported in the mouse, and is located at exon 7 (mutagenesis studies located it at or near Lys-Ala-Ala-Ser, aa 178–181). It seems to be used in the absence of the exon 6 primary cleavage site. The primary and secondary proteolytic cleavage sites of murine SCF have been mutated enabling the generation of cell lines producing only membrane-bound SCF, suggesting the absence of other major cleavage sites.
The c-Kit tyrosine kinase receptor is encoded at the white spotting (W) locus on human chromosome 4q11–q12 and murine chromosome 5.
Four different isoforms of c-Kit have been reported as a result of alternative splicing events. Alternative 5′ splice donor sites at the exon/intron junction of exon 9 lead to the presence or absence of a four amino acid sequence
GNNK (glycine-asparagine-asparagine-lysine, codons 510–513) in the juxtamembrane domain. Splice acceptor site, in human but not mouse, resulting in the presence or absence of a serine residue in the cytoplasmic interkinase domain. As described for
SCF, the c-Kit receptor can also be proteolytically cleaved, allowing shedding from the surface of hematopoietic cells, mast cells, and endothelial cells. This shedding occurs in the fifth immunoglobulin-like domain of c-Kit, but the proteolycic cleavage site is unknown.
SCF gene is composed of 8 exons. Exon 1 encodes a 5′ untranslated sequence and the first 5 aa of a 25 aa signal peptide. Exons 2–7 encode the extracellular domain. Exon 7 also encodes a 23 aa transmembrane domain, and exon 8 a short (36 aa) intracellular domain. sSCF is found as a noncovalently linked homodimer , which spontaneously dissociates and re-associates in solution.
The c-Kit receptor belongs to the subclass III of the tyrosine kinase receptor family. The c-Kit gene is composed of 21 exons. Exon 1 encodes the 5′-untranslated region and the signal peptide. Exons 2–9 encode the extracellular domain, exon 10 the transmembrane domain and exons 11–20 the intracellular domain. The extracellular domain (520 aa) consists of five immunoglobulin (Ig)-like domains. The c-Kit intracellular domain is composed of a tyrosine kinase domain split in two by an insert region. Dimerization of the receptor leads to autophosphorylation on several tyrosines in the cytoplasmic domain.
Downregulation of c-Kit signalling
Stem cell factor and its receptor c-Kit as targets for inflammatory diseases 2005
The protein kinase C (PKC) interacts with c-Kit, and phosphorylates on S741 and S746 of the kinase insert. These phosphorylations lead to inhibition of the c-Kit kinase activity. PKC also mediates downregulation of c-Kit by activating a pathway leading to shedding of the extracellular domain of c-Kit . The protein phosphatase SHP-1 (SH2 domain-containing tyrosine phosphatase-1) negatively regulates c-Kit signalling through binding to the phosphorylated Y570 in the juxtamembrane domain. The suppressors of cytokine signalling (SOCS)-1 and 6 also downregulate c-Kit signalling. SOCS-1 binds Grb2 (Growth factor Receptor-Bound protein 2) and the protein vav (human vaccinia virus oncogene homolog), leading to inhibition of SCF-induced proliferation. SOCS-6 interacts with the phosphorylated Y568 in the juxtamembrane domain of c-Kit. This leads to downregulation of SCF-induced p38 and ERK1/2 phosphorylations, and proliferation. Cbl (Casitas B-lineage) family members are newly established as components of the ubiquitin ligation machinery involved in the degradation of phosphorylated proteins. Upon SCF stimulation, recruitment of Src kinases or maybe other proteins to phosphorylated Y568/Y570 leads to recruitment of Cbl proteins. Once phosphorylated, Cbl proteins mediate c-Kit ubiquitination and degradation through the proteasome and/or lysosome pathways, leading to c-Kit downregulation
CELLULAR FUNCTION
Stem cell factor and its receptor c-Kit as targets for inflammatory diseases 2005
SCF is expressed in vitro by various cells from the airways, including the bronchial epithelial cells, bronchial subepithelial myofibroblasts, lung fibroblasts , bronchial smooth muscle cells, endothelial cells, peripheral blood eosinophils and isolated human lung mast cells.
c-Kit is principally expressed on hematopoietic stem cells, and on human lung mast cells in the airways . It has also been described on peripheral blood eosinophils and circulating basophils. Expression of c-Kit has been reported on some structural cells, like human vascular smooth muscle cells, epithelial cells, and human umbilical vein endothelial cells. These various c-Kit expressing cells also produce SCF, which may therefore act through an autocrine pathway.
Effect of SCF on inflammatory cells
Both mast cells and eosinophils express SCFand its c-Kit receptor at the cell membrane . Because SCF is upregulated in inflammatory conditions both in vitro and in vivo, it may affect inflammatory cell function, and therefore be a potential therapeutic target for inflammatory diseases.
SCF-induced mast cell development. In human , SCF is the main mast cell growth factor, promoting by itself their development from bone marrow, cord blood, or peripheral blood progenitor cells in vitro. SCF-induced development of mast cells from CD34+ cells is regulated by other co-factors including IL-9, thrombopoietin, nerve growth factor (NGF), and IL-3 . In the presence of IL-6, cultured SCF-dependent mast cells have a decreased proliferation and expression of c-Kit receptor, together with increased intracellular histamine levels. The addition of IL-6 to cultures containing mast cells resulted in a substantial reduction of the number of progenies grown by SCF in the liquid culture. This IL-6-mediated inhibition of mast cell growth may be due in part to the suppression at the precursor level. The stimulation of the 10-week cultured mast cells with SCF+IL-6 for 24 hours did not influence the cell number, but increased the histamine concentrations of both the cell lysate and the supernatant compared with the values obtained with SCF alone. Thus, the increased level of intracellular histamine may reflect an upregulation of histamine production rather than an increment in histamine storage. Also, SCF-induced mast cell development is shown to be inhibited by GM-CSF , and by IL-4.
The SCF/c-Kit pathway has also been involved in mast cell survival in human. SCF acts by itself to induce mast cell survival, or in synergism with other growth factors such as IL-3 or NGF.
SCF is a chemotactic factor for mast cells in vitro. The transduction pathways involved in the SCF-induced mast cell migration include activation of the p38 MAP kinase , activation of PI3-kinase since genetic or pharmacological inactivation of the p110δ isoform of PI3-kinase in mast cells leads to defective SCF-mediated migration in vitro , and activation of the Src family kinase Lyn, since Lyn-deficient mast cells show impairment of SCF-induced chemotaxis as compared to wild-type mast cells .SCF-induced mast cell adhesion. Involvement of the c-Kit receptor in SCF-induced mast cell adhesion was demonstrated in mast cells derived from W/W mice, which do not express the extracellular domain of c-Kit, and cannot bind to fibroblasts. SCF-induced mast cell adhesion requires c-Kit tyrosine kinase activity. Mast cells derived from W/Wv mice, which have a missense mutation in the kinase insert of c-Kit, do not adhere to fibronectin upon SCF stimulation . Adhesion of mast cell induced by SCF occurs through an integrin receptor, as it is calcium-dependent and can be blocked by an RGD (Arginine–Glycine–aspartic acid)-containing peptide.
SCF by itself stimulates mast cell degranulation. Cord-blood-derived human mast cells produce IL-13 in the presence of SCF. SCF can also induce the release of IL-6 from murine mast cells in vitro. Small amounts of TNF-α and IL-4 are released in response to SCF stimulation. SCF can also potentiate mast cell degranulation during IgE-dependent activation and release of histamine and arachidonic acid metabolites, or serotonin from peritoneal mast cells.
Direct effects of SCF on eosinophils include development of eosinophils by murine marrow cells, an effect which is enhanced by granulocyte colony stimulating factor G-CSF. SCF induces murine eosinophils to degranulate and release eosinophil peroxidase (EPO) and leukotriene C4 (LTC4) in a dose-dependent manner. SCF also induces eosinophils to produce CC chemokines, including RANTES (regulated on activation, normal T expressed and secreted), macrophage-derived chemokine (MDC), macrophage inflammatory protein-1β (MIP-1β), and CCL6 (C10).
REGULATION
SCF promoter activity is increased by cyclic AMP (cAMP). SCF production is increased by IL-1β, tumor necrosis factor-α (TNF-α) or phorbol 12-myristate 13-acetate (PMA) in vascular endothelial cells, and lung fibroblasts .
SCF is upregulated in vitro by pro-inflammatory stimuli. On the other hand, glucocorticoids downregulate SCF production in human lung fibroblasts, suggesting that SCF might be a good target for anti-inflammatory treatments. c-Kit expression is downregulated by various pro-inflammatory signals, such as IL-1β, TNF-α, and PMA, in human endothelial cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4 and IL-10 downregulate c-Kit expression in the human mast cell line HMC-1.
Internalization of the receptor may be a mechanism to attenuate cellular response to SCF. c-Kit is rapidly internalized after SCF binding. c-Kit internalization requires kinase activity, PI3-kinase activation and Ca 2+ influx.
DIAGNOSTIC USE
The SCF/C-KIT COMPLEX as a target for inflammatory disease
Skin inflammation
Rheumatoid arthritis
Allergic rhinitis
Asthma
