The International Journal of Biochemistry & Cell Biology
ReviewEarly signaling pathways activated by c-Kit in hematopoietic cells☆
Introduction
Stem cell factor (SCF) is a growth factor critical in hematopoiesis as well as in the generation of melanocytes and germ cells, reviewed in references [1], [2], [3], [4], [5], [6], [7], [8]. SCF also plays a role in development of the interstitial cells of Cajal in the intestine and in learning functions in the hippocampal region of the brain [9], [10], [11]. Occurring physiologically in either membrane-bound or soluble form, SCF promotes viability as well as proliferation and differentiation of hematopoietic progenitor cells. In addition, SCF is potently synergistic in combination with other growth factors such as Epo, IL-3 and GM–CSF. There are several excellent reviews of SCF biology in relation to hematopoiesis [2], [4], [5], [6]. A comprehensive overview of SCF in relation to its various target tissues can be found in the review by Galli et al. [1]. The purpose of this review is to examine the early signaling pathways activated in response to SCF in hematopoietic cells.
SCF was cloned and characterized in 1990 [12], [13], [14], [15]. The receptor for SCF is the product of the Kit proto-oncogene [16], [17]. The c-Kit gene maps to the White spotting (W) locus in mice, while SCF is encoded by the Steel locus (Sl) [16], [17], [18], [19]. The first descriptions of mutant W and Sl alleles were in 1927 and 1956, respectively [20]. Since that time, multiple alleles of both genes have been discovered and characterized [7], [8], [20]. While the absence of either SCF or c-Kit is lethal in utero, reductions in functional receptor, or ligand, results in aberrations in hematopoiesis, pigmentation and reproduction.
The Kit gene product has been associated with several forms of cancer. The v-Kit oncogene was originally identified as a component of Hardy–Zuckerman strain of feline sarcoma virus [21]. In humans, a series of gain-of-function mutations in the c-Kit juxtamembrane region have been found in gastrointestinal stromal cell tumors [22]. c-Kit is also aberrantly expressed in approximately 70% of all small cell carcinomas of the lung (SCCL), as well as in breast, cervical and ovarian tumors [23], [24], [25], [26], [27]. Coexpression of c-Kit and SCF in SCCL cells generates an autocrine loop that may play a role in the etiology of these cancers. A constitutively active form of human c-Kit (D816 V) has been found with high frequency in patients with mastocytosis and associated hematological disorders [28]. Hematological disorders in these patients range from myelodysplasia to myeloproliferative disease. In addition, these individuals develop leukemia at high frequencies.
The important role of SCF in development of stem cells involved in hematopoiesis, pigmentation, intestinal function and reproduction, as well as its association with some forms of human disease, has lead to interest in understanding its mechanism of action. This review will focus on signaling mechanisms of SCF, specifically in hematopoietic cells.
Section snippets
SCF induces receptor dimerization
c-Kit is a receptor tyrosine kinase (RTK) closely related to the receptors for platelet-derived growth factor and colony-stimulating factor-1. Structurally, the c-Kit extracellular domain can be divided into five immunoglobulin-like regions (Fig. 1). The studies of Yarden and coworkers have shown that the first three immunoglobulin-like regions bind SCF, inducing homodimerization of the receptor [29], [30], [31]. Two models for ligand-induced dimerization of c-Kit have been suggested [32], [33]
c-Kit autophosphorylation
The cDNA sequence of the c-Kit proto-oncogene predicted that the protein was a RTK [41], [42]. Indeed, in vitro kinase assays of c-Kit immunoprecipitates found intrinsic tyrosine kinase activity [41], [43]. The organization of the cytoplasmic domain of c-Kit is similar to that of the receptors for colony-stimulating factor-1 and platelet-derived growth factor. The catalytic domain is divided by a 77 amino acid insert (Fig. 1). The first catalytic domain contains the ATP binding region while the
Phosphatidylinositol-3-kinase and c-Kit signaling
SCF activates multiple signaling components, however, the best characterized of these with regards to structure-function relationships is phosphatidylinositol-3-kinase (PI3 K). PI3 K is a heterodimer composed of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit. The 85 kDa subunit (p85) contains several motifs implicated in protein–protein interactions. These motifs include two SH2 domains, an SH3 domain and a proline-rich domain. Increases in autophosphorylation activity of RTKs
The JAK/STAT pathway and c-Kit signaling
Members of the Janus family of protein tyrosine kinases (JAKs) are activated by ligands interacting with a variety of receptors lacking intrinsic kinase activity. Among these are hematopoietic growth factors that bind receptors in the cytokine receptor superfamily. These include the erythropoietin (Epo) receptor, the granulocyte–macrophage colony-stimulating factor (GM–CSF) receptor, the interleukin 3 (IL-3) receptor as well as numerous others. Reviews relating to activation of Janus kinases by
Src family members and c-Kit signaling
Src family members are involved in a wide range of cellular functions including cell adhesion, cell motility, cell cycle progression, survival, differentiation, protein trafficking and cellular architecture, reviewed in [102], [103], [104]. They interact with one or more components of most known signaling pathways. With regard to signaling through RTKs, Src family members are activated in response to numerous RTK ligands including platelet-derived growth factor (PDGF), epidermal growth factor
The Ras–Raf–MAP kinase cascade and c-Kit signaling
One signaling pathway activated in response to many growth factors is the Ras–Raf–MAP kinase cascade, reviewed in [116], [117], [118]. In brief, phosphorylated tyrosine residues on ligand-activated receptors recruit multiple SH2-containing proteins to the receptor complex and these proteins couple RTKs to activation of Ras. Included among these are Grb2, Shc, SHP2 and Grap. A Grb family member is constitutively associated with Sos, a guanine nucleotide exchange factor. Recruitment of Grb2 to
Negative regulators of SCF signaling
Several lines of evidence suggest that SHP1, an SH2-containing protein tyrosine phosphatase specific for hematopoietic cells, is a negative regulator of c-Kit signaling. SCF induces association of the SHP1 SH2 domain with phosphorylated tyrosine 569 of murine c-Kit [89], [90]. Transfection of Y569F c-Kit into BaF3 cells resulted in increases in SCF-induced proliferation [90]. Genetic evidence also suggests that SHP1 is a negative regulator of c-Kit. Motheaten mice (me) and motheaten viable mice
Other signaling components activated by SCF
A variety of other signaling components are activated in response to SCF, although the signaling pathways these proteins are involved with, and their role in SCF-mediated responses, remain to be defined. For example, PLCγ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). SCF induces weak association between the SH2 domain of PLCγ and tyrosine 936 of human c-Kit as well as small increases in tyrosine phosphorylation of PLCγ
C-Kit signaling and cellular matrix components
Hematopoietic progenitor cells interact with both stromal cells and components of the extracellular matrix. These events, in conjunction with signals mediated by soluble and membrane-bound growth factors, are important in the regulation of hematopoiesis. Interaction of differentiated progeny, such as mast cells, with components of the extracellular matrix is also important in their functional responses. SCF-induced adhesion occurs through multiple mechanisms. One mechanism involves interaction
Integrated signaling mechanisms
Although delineation of linear signaling pathways is conceptually attractive, many of the signaling components activated by SCF play roles in multiple pathways. In most cases, SCF-mediated responses result from the input of multiple, interconnected signaling pathways (summarized in Fig. 3). For example, SHP2 is likely involved in activation of the Ras–Raf–MAP kinase cascade but may also negatively regulate other signaling pathways [90], [123]. Phosphorylation of c-Kit by PKC isoforms decreases
C-Kit structure-function summary
SCF activates multiple signal transduction components including PI3 K, Src family members, the Ras–Raf–MAP kinase cascade and the JAK/STAT pathway. Activation of many of these pathways is dependent on interaction of one or more upstream signaling components with specific sites on c-Kit. Table 1 is a summary of tyrosine residues on c-Kit that interact with components of these pathways. In addition, if known, the biological consequence of mutation of each site is also shown. Fig. 4 summarizes
SCF-mediated synergy
A remarkable feature of SCF is its capacity to synergize with other hematopoietic growth factors. This is a critical because stem cells and multipotential progenitor cells respond optimally to growth factors in combination. The mechanisms mediating synergy are poorly understood. Studies from the laboratory of Keller and coworkers [160] demonstrated that synergistic responses of hematopoietic progenitor cells to combinations of ligands binding members of the cytokine receptor superfamily
Conclusions
In the nine years since SCF was identified as the c-Kit ligand, a remarkable amount has been learned about its mechanism of action. SCF activates multiple signaling pathways and these pathways lead to a variety of biological responses. While many of the early studies focused on signaling pathways activated by SCF in fibroblasts transfected with c-Kit, subsequent work in hematopoietic cell lines generally supports these findings. Understanding the role of these signaling pathways in SCF-mediated
Acknowledgements
The author would like to thank Dr Doug Lowy, Dr Virginia Broudy and Ms Bridget O’Laughlin for their critical review of this manuscript. The efforts of Ms Karen Canon in preparation of the references were also greatly appreciated.
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