Structure of a c-kit product complex reveals the basis for kinase transactivation

The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.     

Interspecies molecular chimeras of kit help define the binding site of the stem cell factor.

The extracellular portion of the kit-encoded receptor for the stem cell factor (SCF) comprises five immunoglobulin (Ig)-like domains. To localize the ligand recognition site, we exploited the lack of binding of human SCF to the murine receptor by using human-mouse hybrids of Kit and species-specific monoclonal antibodies (MAbs) that inhibit ligand binding. Replacement of the three N-terminal Ig-like domains of the murine Kit with the corresponding portion of the human receptor conferred upon the chimeric receptor high-affinity binding of the human ligand as well as of human-specific ligand-inhibitory MAbs. By constructing five chimeric murine Kit proteins which individually contain each of these three human Ig-like units or pairs of them, we found that the second human domain confers upon the mouse Kit high-affinity binding of the human ligand and also binding of species-specific SCF-competitive MAbs. Nevertheless, the flanking Ig-like domains also affect high-affinity recognition of SCF. Moreover, it appears that the determinants that define ligand specificity of the murine and the human receptors do not structurally coincide. This observation allowed us to identify a chimeric receptor that displayed a dual specificity; namely, it bound with high affinity either the human or the murine SCF molecules and reacted with mouse- as well as human-specific ligand-inhibitory MAbs. Conversely, another chimera, which included all of the five Ig-like domains, bound neither ligand. In conclusion, interdomain packing involving the second Ig-like domain of human Kit and noncontiguous structural motifs of the receptor are involved in SCF recognition.     

Synergistic growth of stem cell factor and granulocyte macrophage colony-stimulating factor involves kinase-dependent and -independent contributions from c-Kit

Stem cell factor (SCF) binds and activates the receptor tyrosine kinase c-Kit, and this interaction is critical for normal hematopoiesis. SCF also synergizes with a variety of growth factors, including those binding members of the cytokine receptor superfamily. The mechanisms mediating this synergy remain to be defined. The present study investigates both structural and biochemical cross-talk between c-Kit and the receptor for granulocyte macrophage colony-stimulating factor (GM-CSF). We have found that c-Kit forms a complex with the beta-chain of the GM-CSF receptor, and this interaction involves the first part of the c-Kit kinase domain. Although inhibition of c-Kit kinase activity completely blocked SCF-induced proliferation, there was still greater than additive growth induced by SCF in combination with GM-CSF. In contrast, an inhibitory antibody against the extracellular domain of c-Kit (K-27) completely inhibited growth in response to SCF alone or in combination with GM-CSF. These results support a kinase-independent component of the synergistic growth induced by SCF and GM-CSF that may relate to interaction of these receptors. It is also clear that a significant part of the synergistic growth is dependent of c-Kit kinase activity. Although synergistic increases in phosphorylation of c-Kit and the beta-chain of the GM-CSF receptor were not observed, SCF and GM-CSF in combination prolonged the duration of Erk1/2 phosphorylation in a phosphatidylinositol 3-kinase-dependent manner. Consistent with these findings, phosphatidylinositol 3-kinase is synergistically activated by SCF and GM-CSF together. Hence, c-Kit makes both kinase-independent and -dependent contributions to the proliferative synergy induced by SCF in combination with GM-CSF.     

Early signaling pathways activated by c-Kit in hematopoietic cells

c-Kit is a receptor tyrosine kinase that binds stem cell factor (SCF). Structurally, c-Kit contains five immunoglobulin-like domains extracellularly and a catalytic domain divided into two regions by a 77 amino acid insert intracellularly. Studies in white spotting and steel mice have shown that functional SCF and c-Kit are critical in the survival and development of stem cells involved in hematopoiesis, pigmentation and reproduction. Mutations in c-Kit are associated with a variety of human diseases. Interaction of SCF with c-Kit rapidly induces receptor dimerization and increases in autophosphorylation activity. Downstream of c-Kit, multiple signal transduction components are activated, including phosphatidylinositol-3-kinase, Src family members, the JAK/STAT pathway and the Ras-Raf-MAP kinase cascade. Structure-function studies have begun to address the role of these signaling components in SCF-mediated responses. This review will focus on the biochemical mechanism of action of SCF in hematopoietic cells.     

Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket.     

Systematic search for putative new domain families in Mycoplasma gallisepticum genome

Background

Stem cell factor (SCF) receptor c-Kit is recognized as a key signaling molecule, which transduces signals for the proliferation, differentiation and survival of stem cells. Binding of SCF to its receptor triggers transactivation, leading to the recruitment of kinases and phosphatases to the docking platforms of c-Kit catalytic domain. Tyrosine phosphatase-1 (Shp-1) deactivates/attenuates 'Kit' kinase activity. Whereas, Asp816Val mutation in the Kit activation loop transforms kinase domain to a constitutively activated state (switch off-to-on state), in a ligand-independent manner. This phenomenon completely abrogates negative regulation of Shp-1. To predict the possible molecular basis of interaction between c-Kit and Shp-1, we have performed an in silico protein-protein docking study between crystal structure of activated c-Kit (phosphorylated c-Kit) and full length crystal structure of Shp-2, a close structural counterpart of Shp-1.

Findings

Study revealed a stretch of conserved amino acids (Lys818 to Ser821) in the Kit activation domain, which makes decisive H-bonds with N-sh2 and phosphotyrosine binding pocket residues of the phosphatase. These H-bonds may impose an inhibitory steric hindrance to the catalytic domain of c-Kit, there by blocking further interaction of the activation loop molecules with incoming kinases. We have also predicted a phosphotyrosine binding pocket in SH2 domains of Shp-1, which is found to be predominantly closer to a catalytic groove like structure in c-Kit kinase domain.

Conclusions

This study predicts that crucial hydrogen bonding between N-sh2 domain of Shp-1 and Kit activation loop can modulate the negative regulation of c-Kit kinase by Shp-1. Thus, this finding is expected to play a significant role in designing suitable gain-of-function c-Kit mutants for inducing conditional proliferation of hematopoietic stem cells.     

Nociceptive tuning by stem cell factor/c-Kit signaling

The molecular mechanisms regulating the sensitivity of sensory circuits to environmental stimuli are poorly understood. We demonstrate here a central role for stem cell factor (SCF) and its receptor, c-Kit, in tuning the responsiveness of sensory neurons to natural stimuli. Mice lacking SCF/c-Kit signaling displayed profound thermal hypoalgesia, attributable to a marked elevation in the thermal threshold and reduction in spiking rate of heat-sensitive nociceptors. Acute activation of c-Kit by its ligand, SCF, resulted in a reduced thermal threshold and potentiation of heat-activated currents in isolated small-diameter neurons and thermal hyperalgesia in mice. SCF-induced thermal hyperalgesia required the TRP family cation channel TRPV1. Lack of c-Kit signaling during development resulted in hypersensitivity of discrete mechanoreceptive neuronal subtypes. Thus, c-Kit can now be grouped with a small family of receptor tyrosine kinases, including c-Ret and TrkA, that control the transduction properties of sensory neurons.     

Expression of c-Kit receptor tyrosine kinase and effect on beta-cell development in the human fetal pancreas

The receptor, c-Kit, and its ligand, stem cell factor (SCF), are critical for hematopoietic stem cell differentiation and have been implicated in the development, function, and survival of rodent islets. Previously, we reported that exogenous SCF treatments of cultured human fetal (14-16 wk fetal age) islet-epithelial clusters enhanced islet cell differentiation and proliferation (Li J, Goodyer CG, Fellows F, Wang R. Int J Biochem Cell Biol 38: 961-972, 2006). In the present study, we examined the expression pattern of c-Kit in early to midgestation human fetal pancreata and the relevance of c-Kit receptor tyrosine kinase for insulin gene expression and beta-cell survival. c-Kit is expressed in the intact pancreas in a cell-specific manner, with a significant decrease in immunoreactivity in the duct regions from 8 to 21 wk fetal age, paralleled by a significant increase in expression within endocrine regions. These c-Kit-positive cells are highly proliferative and show frequent coexpression with insulin and glucagon. Treatment of islet-epithelial clusters with anti-ACK45 antibody stimulates c-Kit phosphorylation paralleled by a significant increase in PDX-1 and insulin expression, increased cell proliferation, and reduced beta-cell death. In contrast, transient transfection with c-Kit siRNA results in a three- to fourfold decrease in c-Kit, PDX-1, and insulin expression and decreased cell proliferation. This study describes important changes in the distribution and dynamics of c-Kit-expressing cells during human fetal pancreatic neogenesis, suggesting that c-Kit may be a marker for human pancreatic islet progenitor cells. Functional analysis of the c-Kit receptor tyrosine kinase provides evidence that phosphorylation of c-Kit receptor may be involved in mediating early beta-cell differentiation and survival.     

The alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor interacts with c-Kit and inhibits c-Kit signaling

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates hematopoiesis and the function of mature host defense cells through the GM-CSF receptor (GMR), which is composed of alpha (alphaGMR) and beta (betaGMR) subunits. Stem cell factor is another important hematopoietic cytokine that signals through c-Kit, a receptor tyrosine kinase, and regulates hematopoietic stem cell maintenance and erythroid development. Like other cytokine receptors, GMR and c-Kit are generally deemed as independent adaptor molecules capable of transducing cytokine-specific signals. We found that the alphaGMR directly interacts with c-Kit and that the interaction is mediated by the cytoplasmic domains. Furthermore, alphaGMR inhibited c-Kit auto-phosphorylation induced by the ligand stem cell factor. Consistent with the inhibitory effect, the expression of alphaGMR was suppressed in cells whose viability was dependent on c-Kit signaling. In contrast, the alternatively spliced alpha2 isoform of the alphaGMR could not inhibit c-Kit signaling, providing a rationale for the existence of the alpha2 isoform. Our results suggest that in addition to having the commonly appreciated roles in cytokine signal transduction, the receptors alphaGMR and c-Kit could interact to coordinate their signal initiation.     

Structure of the active core of human stem cell factor and analysis of binding to its receptor kit

Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding portions composed of immunoglobulin-like repeats. We have determined the crystal structure of selenomethionyl soluble human SCF at 2.2 A resolution by multiwavelength anomalous diffraction phasing. SCF has the characteristic helical cytokine topology, but the structure is unique apart from core portions. The SCF dimer has a symmetric 'head-to-head' association. Using various prior observations, we have located potential Kit-binding sites on the SCF dimer. A superimposition of this dimer onto VEGF in its complex with the receptor Flt-1 places the binding sites on SCF in positions of topographical and electrostatic complementarity with the Kit counterparts of Flt-1, and a similar model can be made for the complex of PDGF with its receptor.     

Changes in Conformation and Stability upon SCF/sKit Complex Formation

Stem cell factor (SCF) is thought to be a member of the four-helical bundle cytokine superfamily, and exists in solution as a noncovalent homodimer. It is the ligand for Kit, a tyrosine kinase type III receptor. The interaction of SCF and Kit affects early hematopoietic progenitors, as well as gametocytes, melanocytes, and mast cells. Upon binding of SCF the Kit undergoes dimerization and transphosphorylation. Circular dichroism (CD), intrinsic fluorescence, and Fourier transform infrared (FTIR) spectroscopy were used for conformational analyses of free SCF, soluble Kit (sKit), and the complex. The sKit consisted of the extracellular domain of Kit, contained five Ig-like domains, and was prepared from the conditioned media of transfected Chinese hamster ovary cells. With these techniques, a reproducible conformational change was seen upon ligand/receptor binding. The far-UV CD and FTIR spectroscopy indicated a slight increase in the -helical content. The near-UV CD and fluorescence spectra showed changes in the environments of the aromatic amino acids. The thermal denaturation of SCF was not affected by complex formation, while the melting temperature of sKit increased only a few degrees when binding SCF. This indicates that binding is temperature dependent, consistent with titration calorimetry results published previously which demonstrated that there is a large enthalpy of binding. The conformational changes which accompany SCF/sKit binding could play a role in the receptor dimerization and signal transduction which follow.     

Stem cell factor/c-Kit interactions regulate human islet-epithelial cluster proliferation and differentiation

Stem cell factor (SCF), a progenitor cell growth factor, binds to and activates the c-Kit receptor tyrosine kinase, which is critical for early stem cell differentiation in haematopoiesis and gametogenesis. Nothing is known regarding these interactions during islet development in the human fetal pancreas. The present study was to investigate whether an increase in c-Kit receptor activity in isolated human fetal islet-epithelial clusters, by giving exogenous SCF, would promote beta-cell development. In the intact fetal pancreas, SCF and c-Kit were observed co-localizing with cytokeratin 19 in both ductal and newly forming islet cells. Islet cells isolated from 14 to 16 weeks fetal pancreata were cultured with SCF (50 ng/ml) or vehicle for 48 h. We observed an increase in the number of c-Kit-, pancreatic and duodenal homeobox gene 1- (PDX-1-), insulin- and glucagon-expressing cells in the SCF-treated group (PDX-1 and insulin, p < 0.05). PDX-1 and c-Kit mRNA levels were also up-regulated in the SCF group (PDX-1, p < 0.05), with no change in preproinsulin or proglucagon gene expression. Co-localization of insulin with PDX-1 or c-Kit was observed frequently in SCF-treated cultures. A significantly (p < 0.05) greater proliferative capacity of islet-epithelial clusters was found in the SCF group in parallel with increased (p < 0.02) phosphorylation of Akt in a phosphatidylinositol-3 kinase (PI3K)-dependent manner. Our results demonstrate that SCF/c-Kit interactions are likely to be involved in mediating islet cell differentiation and proliferation during human fetal pancreatic development, and that phosphorylated Akt may have a role downstream of SCF/c-Kit signaling.     

The biology of stem cell factor and its receptor C-kit

The receptor tyrosine kinase c-Kit and its ligand Stem Cell Factor (SCF) are essential for haemopoiesis, melanogenesis and fertility. SCF acts at multiple levels of the haemopoietic hierarchy to promote cell survival, proliferation, differentiation, adhesion and functional activation. It is of particular importance in the mast cell and erythroid lineages, but also acts on multipotential stem and progenitor cells, megakaryocytes, and a subset of lymphoid progenitors. SCF exists in soluble or transmembrane forms which appear to differ in function. Multiple isoforms of c-Kit also exist as a result of alternate mRNA splicing, proteolytic cleavage and the use of cryptic internal promoters in certain cell types. This review focuses on what is known about the regulation of c-Kit expression, the functions of SCF and c-Kit isoforms, and the nature of the biological responses elicited by this receptor-ligand pair with emphasis on the haemopoietic system.     

The c-Kit/D816V mutation eliminates the differences in signal transduction and biological responses between two isoforms of c-Kit

Activating mutations of codon 816 of the Kit gene have been implicated in malignant cell growth of acute myeloid leukemia (AML), systemic mastocytosis and germ cell tumors. Substitution of aspartic acid with valine (D816V) renders the receptor independent of ligand for activation and signaling. Wild-type c-Kit is a tyrosine kinase receptor that requires its ligand, stem cell factor (SCF), for activation. Several isoforms of c-Kit exist as a result of alternative mRNA splicing, of which two are characterized by the presence or absence of four amino acids (GNNK? and GNNK+, respectively) in the extracellular domain. The two isoforms show differences in signal transduction and biological activities and the shorter isoform seems to be highly expressed than the longer isoform in human malignancies. In this study we analysed the signal transduction downstream of the oncogenic c-Kit mutant D816V in an isoform specific context, using the hematopoietic cell line Ba/F3 stably transfected with the different versions of isoform and mutant receptor. Our data show that in contrast to the differences shown in the activation of wild-type c-Kit isoforms, both isoforms of c-Kit/D816V are constitutively phosphorylated to the same extent. By the use of Western blot analysis we investigated the activation of different signaling proteins and found that both D816V/GNNK? and D816V/GNNK+ constitutively phosphorylated Gab2, Shc, SHP-2 and Cbl to almost the same extent as c-Kit/GNNK?. In addition, both isoforms of c-Kit/D816V induced SCF-independent cell survival and proliferation equally well. This is in contrast to wild-type c-Kit, where c-Kit/GNNK? induced better cell survival and stronger proliferation than c-Kit/GNNK+, and both required stimulation with SCF. Taken together, these findings reveal that the differences in downstream signal transduction and biological responses between the two GNNK isoforms are eliminated by the D816V mutant.     

Signaling conformations of the tall cytokine receptor gp130 when in complex with IL-6 and IL-6 receptor

gp130 is a shared cytokine signaling receptor and the founding member of the 'tall' class of cytokine receptors. A crystal structure of the ligand-binding domains of gp130 in complex with human interleukin-6 (IL-6) and its a-receptor (IL-6Ralpha) revealed a hexameric architecture in which the gp130 membrane-distal regions were approximately 100 A apart, in contrast to the close apposition seen between short cytokine receptor complexes. Here we used single-particle EM to visualize the entire extracellular hexameric IL-6-IL-6Ralpha-gp130 complex, containing all six gp130 domains. The structure reveals that gp130 is bent such that the membrane-proximal domains of gp130 are close together at the cell surface, enabling activation of intracellular signaling. Variation in the receptor bend angles suggests a possible conformational transition from open to closed states upon ligand binding; this transition is probably representative of the other tall cytokine receptors.     

Dimerization and activation of the kit receptor by monovalent and bivalent binding of the stem cell factor.

The protooncogene c-kit encodes a tyrosine kinase receptor for the stem cell factor (SCF). Mutants of c-kit were shown to confer a pleiotropic defective phenotype and often display negative dominance in heterozygous mice. To explore the involvement of receptor dimerization in this genetic phenomenon, we employed both a human ligand, which does not recognize the murine receptor, and a rodent SCF, which binds to the human receptor with 100-fold reduced affinity as compared with human SCF. SCF binding to living cells was found to induce rapid and complete receptor dimerization that involved activation of the catalytic tyrosine kinase function. Although receptor dimerization can be attributed to the dimeric nature of the ligand, no dissociation of Kit dimers occurred at high excess of SCF, suggesting that receptor-receptor interactions are also involved in dimer stabilization. This was supported by in vitro formation of heterodimers between the human and murine Kit proteins through monovalent binding of species-specific human SCF. By coexpression of human and mouse Kit in murine fibroblasts, we found that receptor heterodimerization in living cells involved an increase in the affinity of human Kit for rat SCF and also an accelerated rate of receptor down-regulation. When a human Kit mutant lacking the kinase insert domain was coexpressed with the murine wild-type receptor, we observed a significant decrease in both the activation of the intact tyrosine kinase and its coupling to an effector protein, namely phosphatidylinositol 3'-kinase. Our results favor a receptor activation model that assumes an initial step of monovalent ligand binding, followed by an intermediate receptor dimer bound by one arm of the ligand molecule. This model predicts the existence of an intrinsic receptor dimerization site and provides a structural basis for genetic dominance of mutant SCF receptors.     

Blocking SCF/c-Kit signal did not inhibit the proliferation of cultured liver progenitor cells

Oval cells are the putative liver stem cells that proliferate during hepatocarcinogenesis and chemically-induced severe liver injury. Antigens traditionally associated with haematopoietic cells, such as c-Kit, have been reported to be expressed by oval cells. Previous studies suggested that stem cell factor (SCF) and c-Kit were critical to oval cell development. However, the role of SCF/c-Kit signals in oval cell proliferation still remains unclear. Recently, we reported the establishment of oval cell-derived liver epithelial progenitor cells (LEPCs). In this work, we showed LEPCs co-expressed c-Kit and its ligand SCF. The involvement of SCF/c-Kit signals in LEPCs proliferation was investigated either by exposing LEPCs to c-Kit inhibitors (STI571 and AG1296), SCF, anti-SCF neutralized antibody or by using small interfering RNA to knock-down c-Kit expression. Our data demonstrate that blocking SCF/c-Kit signal did not inhibit the proliferation of LEPCs, which suggest SCF/c-Kit is not necessary for the proliferation of oval cells, at least for the cultured oval cell counterpart LEPCs.     

The unique ligand-binding pocket for the human prostacyclin receptor. Site-directed mutagenesis and molecular modeling

The human prostacyclin receptor is a seven-transmembrane alpha-helical G-protein coupled receptor, which plays important roles in both vascular smooth muscle relaxation as well as prevention of blood coagulation. The position of the native ligand-binding pocket for prostacyclin as well as other derivatives of the 20-carbon eicosanoid, arachidonic acid, has yet to be determined. Through the use of prostanoid receptor sequence alignments, site-directed mutagenesis, and the 2.8-A x-ray crystallographic structure of bovine rhodopsin, we have developed a three-dimensional model of the agonist-binding pocket within the seven-transmembrane (TM) domains of the human prostacyclin receptor. Upon mutation to alanine, 11 of 29 candidate residues within TM domains II, III, IV, V, and VII exhibited a marked decrease in agonist binding. Of this group, four amino acids, Arg-279 (TMVII), Phe-278 (TMVII), Tyr-75 (TMII), and Phe-95 (TMIII), were identified (via receptor amino acid sequence alignment, ligand structural comparison, and computer-assisted homology modeling) as having direct molecular interactions with ligand side-chain constituents. This binding pocket is distinct from that of the biogenic amine receptors and rhodopsin where the native ligands (also composed of a carbon ring and a carbon chain) are accommodated in an opposing direction. These findings should assist in the development of novel and highly specific ligands including selective antagonists for further molecular pharmacogenetic studies of the human prostacyclin receptor.