Mutation D816V alters the internal structure and dynamics of c-KIT receptor cytoplasmic region: implications for dimerization and activation mechanisms

The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites.     

Allosteric Communication across the Native and Mutated KIT Receptor Tyrosine Kinase

A fundamental goal in cellular signaling is to understand allosteric communication, the process by which signals originated at one site in a protein propagate dependably to affect remote functional sites. Here, we describe the allosteric regulation of the receptor tyrosine kinase KIT. Our analysis evidenced that communication routes established between the activation loop (A-loop) and the distant juxtamembrane region (JMR) in the native protein were disrupted by the oncogenic mutation D816V positioned in the A-loop. In silico mutagenesis provided a plausible way of restoring the protein communication detected in the native KIT by introducing a counter-balancing second mutation D792E. The communication patterns observed in the native and mutated KIT correlate perfectly with the structural and dynamical features of these proteins. Particularly, a long-distance effect of the D816V mutation manifested as an important structural re-organization of the JMR in the oncogenic mutant was completely vanished in the double mutant D816V/D792E. This detailed characterization of the allosteric communication in the different forms of KIT, native and mutants, was performed by using a modular network representation composed of communication pathways and independent dynamic segments. Such representation permits to enrich a purely mechanistic interaction-based model of protein communication by the introduction of concerted local atomic fluctuations. This method, validated on KIT receptor, may guide a rational modulation of the physiopathological activities of other receptor tyrosine kinases.     

Protein tyrosine phosphatase receptor type E (PTPRE) regulates the activation of wild-type KIT and KIT mutants differently

Activation of receptor tyrosine kinases needs tight control by tyrosine phosphatases to keep their normal function. In this study, we investigated the regulation of activation of the type III receptor tyrosine kinase KIT by protein tyrosine phosphatase receptor type E (PTPRE). We found that PTPRE can associate with wild-type KIT and inhibit KIT activation in a dose-dependent manner, although the activation of wild-type KIT is dramatically inhibited even when PTPRE is expressed at low level. The D816V mutation of KIT is the most frequently found oncogenic mutation in mastocytosis, and we found that PTPRE can associate and inhibit the activation of KIT/D816V in a dose dependent manner, but the inhibition is much weaker compared with wild-type KIT. Similar to mastocytosis, KIT mutations are the main oncogenic mutations in gastrointestinal stromal tumors (GISTs) although GISTs carry different types of KIT mutations. We further studied the regulation of the activation of GISTs-type KIT mutants and other mastocytosis-type KIT mutants by PTPRE. Indeed, PTPRE can almost block the activation of GISTs-type KIT mutants, while the activation of mastocytosis-type KIT mutants is more resistant to the inhibition of PTPRE. Taken together, our results suggest that PTPRE can associate with KIT, and inhibit the activation of both wild-type KIT and GISTs-type KIT mutants, while the activation of mastocytosis-type KIT mutants is more resistant to PTPRE.     

Neoplasia driven by mutant c-KIT is mediated by intracellular, not plasma membrane, receptor signaling

Activating mutations in c-KIT are associated with gastrointestinal stromal tumors, mastocytosis, and acute myeloid leukemia. In attempting to establish a murine model of human KIT(D816V) (hKIT(D816V))-mediated leukemia, we uncovered an unexpected relationship between cellular transformation and intracellular trafficking. We found that transport of hKIT(D816V) protein was blocked at the endoplasmic reticulum in a species-specific fashion. We exploited these species-specific trafficking differences and a set of localization domain-tagged KIT mutants to explore the relationship between subcellular localization of mutant KIT and cellular transformation. The protein products of fully transforming KIT mutants localized to the Golgi apparatus and to a lesser extent the plasma membrane. Domain-tagged KIT(D816V) targeted to the Golgi apparatus remained constitutively active and transforming. Chemical inhibition of intracellular transport demonstrated that Golgi localization is sufficient, but plasma membrane localization is dispensable, for downstream signaling mediated by KIT mutation. When expressed in murine bone marrow, endoplasmic reticulum-localized hKIT(D816V) failed to induce disease in mice, while expression of either Golgi-localized HyKIT(D816V) or cytosol-localized, ectodomain-deleted KIT(D816V) uniformly caused fatal myeloproliferative diseases. Taken together, these data demonstrate that intracellular, non-plasma membrane receptor signaling is sufficient to drive neoplasia caused by mutant c-KIT and provide the first animal model of myelomonocytic neoplasia initiated by human KIT(D816V).     

Exploring the cause of drug resistance by the detrimental missense mutations in KIT receptor: computational approach

In this work, we computationally identified the most detrimental missense mutations of KIT receptor causing gastrointestinal stromal tumors and analyzed the drug resistance of these missense mutations. Out of 31 missense mutations, 19 variants were commonly found less stable, deleterious and damaging by I-Mutant 2.0, SIFT and PolyPhen programs, respectively. Subsequently, we performed modeling of these 19 variants to understand their change in conformations with respect to native KIT receptor by computing their RMSD. Further, the native and 19 mutants were docked with the drug ‘Imatinib’ to explain the drug resistance of these detrimental missense mutations. Among the 19 mutants, we found by docking studies that 12 mutants, namely, F584C, F584L, V654A, L656P, T670I, R804W, D816F, D816V, D816Y, N822K, Y823D and E839K had less binding affinity with Imatinib than the native type. Finally, we analyzed that the loss of binding affinity of these 12 mutants, was due to altered flexibility in their binding amino acids with Imatinib as compared with native type by normal mode analysis. In our work, we found the novel data that the majority of the drug-binding amino acids in those 12 mutants had encountered loss of flexibility, which could be the theoretical basis for the cause of drug insensitivity.     

Role of ELA region in auto-activation of mutant KIT receptor: a molecular dynamics simulation insight

KIT receptor is the prime target in gastrointestinal stromal tumor (GISTs) therapy. Second generation inhibitor, Sunitinib, binds to an inactivated conformation of KIT receptor and stabilizes it in order to prevent tumor formation. Here, we investigated the dynamic behavior of wild type and mutant D816H KIT receptor, and emphasized the extended A-loop (EAL) region (805–850) by conducting molecular dynamics simulation (～100?ns). We analyzed different properties such as root mean square cutoff or deviation, root mean square fluctuation, radius of gyration, solvent-accessible surface area, hydrogen bonding network analysis, and essential dynamics. Apart from this, clustering and cross-correlation matrix approach was used to explore the conformational space of the wild type and mutant EAL region of KIT receptor. Molecular dynamics analysis indicated that mutation (D816H) was able to alter intramolecular hydrogen bonding pattern and affected the structural flexibility of EAL region. Moreover, flexible secondary elements, specially, coil and turns were dominated in EAL region of mutant KIT receptor during simulation. This phenomenon increased the movement of EAL region which in turn helped in shifting the equilibrium towards the active kinase conformation. Our atomic investigation of mutant KIT receptor which emphasized on EAL region provided a better insight into the understanding of Sunitinib resistance mechanism of KIT receptor and would help to discover new therapeutics for KIT-based resistant tumor cells in GIST therapy.     

Drug binding and resistance mechanism of KIT tyrosine kinase revealed by hydrogen/deuterium exchange FTICR mass spectrometry

Mutations of the receptor tyrosine kinase KIT are linked to certain cancers such as gastrointestinal stromal tumors (GISTs). Biophysical, biochemical, and structural studies have provided insight into the molecular basis of resistance to the KIT inhibitors, imatinib and sunitinib. Here, solution‐phase hydrogen/deuterium exchange (HDX) and direct binding mass spectrometry experiments provide a link between static structure models and the dynamic equilibrium of the multiple states of KIT, supporting that sunitinib targets the autoinhibited conformation of WT‐KIT. The D816H mutation shifts the KIT conformational equilibrium toward the activated state. The V560D mutant exhibits two low energy conformations: one is more flexible and resembles the D816H mutant shifted toward the activated conformation, and the other is less flexible and resembles the wild‐type KIT in the autoinhibited conformation. This result correlates with the V560D mutant exhibiting a sensitivity to sunitinib that is less than for WT KIT but greater than for KIT D816H. These findings support the elucidation of the resistance mechanism for the KIT mutants.     

The D816V Mutation of c-Kit Circumvents a Requirement for Src Family Kinases in c-Kit Signal Transduction

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis, and gain-of-function mutations of the receptor are frequently seen in several malignancies, including acute myeloid leukemia, gastrointestinal stromal tumors, and testicular carcinoma. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V), leading to constitutive activation of the receptor. In this study, we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as of activation of downstream signaling pathways including receptor ubiquitination and the Ras/Mek/Erk pathway. Our data demonstrate that, unlike wild-type c-Kit, the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition, we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. We further present evidence that, in contrast to wild-type c-Kit, Src family kinases are dispensable for c-Kit/D816V cell survival, proliferation, and colony formation. Taken together, we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival, thereby contributing to the transforming potential of c-Kit/D816V.The receptor for stem cell factor (SCF),² c-Kit, is a type III receptor tyrosine kinase that belongs to the same subfamily as the platelet-derived growth factor receptors, the Flt3 receptor, and the macrophage colony-stimulating factor receptor (1). The c-Kit gene is identical to the white spotting locus (W) in the mouse. c-Kit is expressed in the hematopoietic system, in the gastrointestinal system, in melanocytes, and in germ cells, and therefore loss-of-function mutations in c-Kit lead to defects in hematopoiesis, melanogenesis, and gametogenesis. Stimulation of the c-Kit receptor with its ligand, SCF, leads to receptor dimerization and activation of its intrinsic tyrosine kinase activity. Specific tyrosine residues are autophosphorylated, which results in the activation of downstream signaling pathways, including the Ras/Erk pathway and the PI3-kinase pathway (for review, see Ref. 2).One of the crucial steps in oncogenic transformation of cells is the gain of independence of external growth stimuli. This can be achieved in several different ways, including mutations that render receptor tyrosine kinases constitutively active in the absence of ligand stimulation. In the case of c-Kit, these mutations most commonly occur either in exon 11 (encoding the juxtamembrane region) and are found predominantly in gastrointestinal stromal tumors or in exon 17 (encoding the activation loop of the kinase domain). A frequently occurring type of mutation in exon 17 in c-Kit is at codon 816. This type of mutation has been found in several human malignancies including acute myeloid leukemia, mastocytosis, germ cell tumors of the seminoma or dysgerminoma types, sinonasal natural killer/T-cell lymphomas, and in intracranial teratomas (3–10). These mutations at codon 816 lead to conversion of an aspartic acid residue to a valine, a tyrosine, a phenylalanine, an asparagine, or a histidine residue. The recently elucidated crystal structure of the c-Kit kinase domain has helped define the mechanism of activation by this type of mutation (11). In the unstimulated wild-type c-Kit, the juxtamembrane region inserts directly into the clefts between the amino- and carboxyl-terminal lobes of the kinase domain, disrupting the c-Kit control helix, and physically blocking the conserved kinase DFG motif from attaining a productive conformation. The activation loop folds back over the substrate binding groove and interacts with the active center of the kinase as a pseudosubstrate. It is not fully known how mutation of aspartic acid 816 leads to activation of c-Kit. It has been suggested either that the mutation inverts the conformation of the protein backbone so that the side chain of arginine 815 is being flipped from its position in the autoinhibited or that the effect of aspartic acid 816 mutations may be derived from its ability to stabilize the small positively charge α-helical dipole through its negative charge of its side chain. Asp-816 mutations in c-Kit promote receptor autophosphorylation and thereby constitutively activate downstream signaling pathways independent of SCF binding and therefore contribute to cell transformation (12). Imatinib (Gleevec) is a well known inhibitor of c-Kit juxtamembrane mutations and has been used in the treatment of gastrointestinal stromal tumors with activating mutations in the juxtamembrane region of c-Kit. In contrast, cells expressing c-Kit/D816V are resistant to Imatinib, whereas the Abl/Src dual inhibitor Dasatinib also inhibits the D816V mutant of c-Kit (13).Negative regulation of c-Kit signaling has been shown to occur mainly through ubiquitin-mediated internalization and degradation of the receptor (14, 15). Ubiquitination is mediated by ubiquitin E3 ligases that attach ubiquitin to their target proteins, resulting in either monoubiquitination or polyubiquitination. Key components in this machinery are the Cbl family of ubiquitin E3 ligases, represented by Cbl, Cbl-b, and Cbl-c (16). Signaling through receptor tyrosine kinases must be tightly regulated, and inhibition of Cbl activation and receptor ubiquitination can lead to cell transformation (17). It has been shown that both direct and indirect binding of Cbl to wild-type c-Kit can induce Cbl activation and receptor ubiquitination followed by receptor internalization and degradation (15, 18). In contrast, the mechanisms behind negative regulation of the oncogenic c-Kit/D816V are so far unknown.Extracellular signal-regulated kinase (Erk) proteins are the evolutionary conserved products of the two genes, Erk1 and Erk2, and are central proteins in the Ras/Erk signaling pathway. Erk1 and Erk2 are activated by dual phosphorylation on their regulatory tyrosine and threonine residues (19). The serine/threonine kinase Akt, also known as protein kinase B, is activated downstream of PI3-kinase and plays a central role in signaling induced by growth factors, cytokines, and other cellular stimuli (20). The Ras/Erk pathway and the PI3-kinase pathway are key signaling pathways involved in the regulation of cell proliferation, survival, and differentiation induced by c-Kit. A key player in the relay of signals from c-Kit into the cells is Src. Binding of Src to Tyr-568 in c-Kit leads to its activation and subsequent phosphorylation of Shc and activation of the Ras/Erk pathway (21). PI3-kinase is activated by c-Kit through two alternate routes, either through direct binding to Tyr-721 in c-Kit (22) or through indirect binding to the scaffolding protein Gab2, which associates to c-Kit via the adapter protein Grb2. Activation of PI3-kinase is dependent on Src-mediated phosphorylation of Gab2 (23). Other investigators have shown that neither Erk nor Akt is constitutively activated in cells expressing c-Kit/D816V (24).In this report, we demonstrate that in c-Kit/D816V-expressing Ba/F3 cells, a low constitutive activation of both Erk and Akt exists and that this activation can be further augmented by SCF stimulation. We also present data showing that c-Kit/D816V evades the need of Src family kinases for receptor ubiquitination and Erk activation by having an altered substrate specificity resembling Src family kinases. We conclude that Src family kinases play different roles in wild-type c-Kit and c-Kit/D816V-induced cell survival and growth.     

Detailed conformational dynamics of juxtamembrane region and activation loop in c-Kit kinase activation process

The stem cell factor receptor (c-Kit) plays critical roles in initiating cell growth and proliferation. Its kinase functional abnormality has been thought to associate with several human cancers. The regulation of c-Kit kinase activity is achieved by phosphorylation on the residues Tyr568 and Tyr570 within juxtamembrane region (JMR) and subsequent structural transition of JMR and activation loop (A-loop). However, the detailed conformational dynamics of JMR and A-loop are far from clear, especially whether their conformational changes are coupled or not during the kinase activation transition. In this investigation, the complete conformational transition pathway was determined using a series of nanosecond conventional molecular dynamics (MD) and targeted molecular dynamics (TMD) simulations in explicit water systems. The results of the MD simulations show that the phosphorylation of residues Tyr568 and Tyr570 within JMR induces the detachment of JMR from the kinase C-lobe and increases the fluctuation in the structure of JMR, thus appearing to initiate the kinase activation process. During the course of the TMD simulation, which characterizes the conformational transition of c-Kit from autoinhibitory to activated state, the JMR undergoes a rapid departure from the allosteric binding site and drifts into solvent, followed by the conformational flip of A-loop from inactive (fold) state to active (extended) state. A change in the orientation of helix alphaC in response to the motion of JMR and A-loop has also been observed. The computational results presented here indicate that the dissociation of JMR from the kinase domain is prerequisite to c-Kit activation, which is consistent with previous experiments.     

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.     

Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.     

Profiling Y561-dependent and -independent substrates of CSF-1R in epithelial cells

Receptor tyrosine kinases (RTKs) activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs), which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC) analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK.     

A juxtamembrane tyrosine in the colony stimulating factor-1 receptor regulates ligand-induced Src association, receptor kinase function, and down-regulation

Recent literature implicates a regulatory function of the juxtamembrane domain (JMD) in receptor tyrosine kinases. Mutations in the JMD of c-Kit and Flt3 are associated with gastrointestinal stromal tumors and acute myeloid leukemias, respectively. Additionally, autophosphorylated Tyr559 in the JMD of the colony stimulating factor-1 (CSF-1) receptor (CSF-1R) binds to Src family kinases (SFKs). To investigate SFK function in CSF-1 signaling we established stable 32D myeloid cell lines expressing CSF-1Rs with mutated SFK binding sites (Tyr559-TFI). Whereas binding to I562S was not significantly perturbed, Y559F and Y559D exhibited markedly decreased CSF-1-dependent SFK association. All JMD mutants retained intrinsic kinase activity, but Y559F, and less so Y559D, showed dramatically reduced CSF-1-induced autophosphorylation. CSF-1-mediated wild-type (WT)-CSF-1R phosphorylation was not markedly affected by SFK inhibition, indicating that lack of SFK binding is not responsible for diminished Y559F phosphorylation. Unexpectedly, cells expressing Y559F were hyperproliferative in response to CSF-1. Hyperproliferation correlated with prolonged activation of Akt, ERK, and Stat5 in the Y559F mutant. Consistent with a defect in receptor negative regulation, c-Cbl tyrosine phosphorylation and CSF-1R/c-Cbl co-association were almost undetectable in the Y559F mutant. Furthermore, Y559F underwent reduced multiubiquitination and delayed receptor internalization and degradation. In conclusion, we propose that Tyr559 is a switch residue that functions in kinase regulation, signal transduction and, indirectly, receptor down-regulation. These findings may have implications for the oncogenic conversion of c-Kit and Flt3 with JMD mutations.     

Vitamin D(3) and analogues modulate the expression of CSF-1 and its receptor in human dendritic cells

The active vitamin D(3)-metabolite 1,25(OH)(2)D(3) inhibits the interleukin 4/granulocyte-macrophage colony-stimulating factor (IL-4/GM-CSF)-induced differentiation of human monocytes into dendritic cells without altering survival. Colony-stimulating factor 1 (CSF-1) is an important survival factor for cells of the monocytic lineage. We therefore investigated whether the inhibitory activity of 1,25(OH)(2)D(3) is paralleled by a regulation of CSF-1 and its receptor. Purified human monocytes were cultured together with IL-4/GM-CSF in the presence of 1,25(OH)(2)D(3), its analogue tacalcitol, the low-affinity vitamin D receptor ligand 24,25(OH)(2)D(3), or the solvent ethanol for up to 5 days. Expression of CSF-1, CSF-1R, and GM-CSF mRNA was measured by RT-PCR. Protein secretion for CSF-1 was measured by ELISA, expression of CSF-1R by flow cytometry. The results showed that 1,25(OH)(2)D(3) and tacalcitol significantly up-regulated CSF-1 mRNA-expression and protein secretion in a dose-dependent manner. The effect of 1,25(OH)(2)D(3) occurred already after 1h of pre-treatment. In contrast, CSF-1R mRNA- and cell surface-expression was down-regulated simultaneously. The solvent ethanol and 24,25(OH)(2)D(3) were without effect. GM-CSF mRNA expression was not modulated in 1,25(OH)(2)D(3)-treated cells. These data point towards a distinct and specific regulation of CSF-1 and its receptor by 1,25(OH)(2)D(3) and its analogue tacalcitol in human monocytes which parallels the inhibition of differentiation into dendritic cells without altering survival.     

Differences in signaling pathways and expression level of the phosphoinositide phosphatase SHIP1 between two oncogenic mutants of the receptor tyrosine kinase KIT

Oncogenic mutations of the receptor tyrosine kinase KIT are encountered in myeloid leukemia and various solid tumors, including gastrointestinal stromal tumors. We previously identified the human oncogenic germ line mutant KIT(K642E), a substitution in the tyrosine kinase 1 domain (TK1D) in a familial form of gastrointestinal stromal tumors. The effects of oncogenic KIT mutants on cell signaling and regulation are complex. Cellular models are valuable basic tools to tailor novel strategies on specific cellular and molecular bases for tumors expressing KIT oncogenic mutants. Murine KIT(WT) and the murine homologues of human KIT oncogenic mutants, further referred to as KIT(K641E) and KIT(del559), a point deletion in the juxtamembrane domain (JMD), were stably expressed in IL-3-dependent Ba/F3 cells. Major differences in the constitutively activation of Akt/PKB, MAP kinases and STATs pathways were observed between KIT(K641E) and KIT(del559), whereas KIT ligand elicited responses in both mutants. Noteworthy, the protein level of the phosphoinositide phosphatase SHIP1, but not SHIP2 and PTEN, was reduced in KIT(K641E) only while inhibition of KIT phosphorylation reversibly raised SHIP1 level in both JMD and TK1D oncogenic mutants, unraveling the control of SHIP protein level by KIT phosphorylation.     

Colony-stimulating factor-1 modulates alpha 2-macroglobulin receptor expression in murine bone marrow macrophages.

Primary cultures of murine bone marrow macrophages (BMMs) were prepared from marrow cell suspensions. These cells expressed specific receptors that recognized the transformed conformation of human alpha 2-macroglobulin (alpha 2M) generated by reaction with CH3NH2. alpha 2M receptor expression was regulated by colony-stimulating factor-1 (CSF-1). The BMMs were deprived of CSF-1 for 6 h and then treated with different concentrations of the purified cytokine. After 18 h, binding of 125I-alpha 2M-CH3NH2 was examined at 4 degrees C. Analysis of the saturation isotherms and Scatchard transformations indicated that the KD was not affected by CSF-1 (1.9-2.4 nM), whereas the maximum specific radioligand binding capacity (Bmax) was increased from 5.6 x 10(4) receptors/cell in the absence of CSF-1 to 2.2 x 10(5) and 2.6 x 10(5) receptors/cell for BMMs treated with 1,000 and 10,000 units/ml CSF-1, respectively. The difference in total cellular protein after exposure to different levels of CSF-1 for 18 h was small (1.50-1.92 ng/cell) and not statistically significant. A 6-12-h lag phase was identified between the time of CSF-1 exposure and increased alpha 2M receptor expression. Cycloheximide completely blocked the increase in alpha 2M receptor expression when added simultaneously with the CSF-1; greater than 50% inhibition was observed when the cycloheximide was added up to 8 h later. The RNA synthesis inhibitors, actinomycin D and daunomycin, prevented increased alpha 2M receptor expression when added up to 4 h after the CSF-1, but had no effect at 8 h. At 37 degrees C, uptake and digestion of 125I-alpha 2M-CH3NH2 was increased in BMMs treated with 1,000 units/ml CSF-1 for 18 h compared with untreated cells. These studies demonstrate that CSF-1 increases the expression of alpha 2M receptors in BMMs through a pathway that requires new RNA and protein synthesis. We hypothesize that increased alpha 2M receptor expression may play an important role in cellular growth and differentiation.     

Functional dissection of structural domains in the receptor for colony-stimulating factor-1.

Receptor tyrosine kinases (RTKs) transduce external signals to the interior of the cell via a cytoplasmic kinase domain. We demonstrated previously that ligand-induced kinase activation of the colony-stimulating factor-1 receptor (CSF-1R) occurs via receptor oligomerization without propagation of conformational changes through the transmembrane (TM) domain (Lee, A. W., and Nienhuis, A. W. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7270-7274). We have now examined the role of the different subdomains in the metabolic and signaling properties of CSF-1R. Two types of chimeric receptors have been utilized, Glyfms A, with the extracellular and TM domains of glycophorin A (GpA) and the cytoplasmic domain of CSF-1R, and Glyfms B, where only the extracellular domain originates from GpA. Glyfms A was found to exhibit a higher basal level of in vitro kinase activity, an increased associated phosphatidylinositol (PtdIns) 3-kinase activity and to support enhanced cellular mitogenesis, compared with wild-type CSF-1R or to Glyfms B. The constitutive activation of Glyfms A is consistent with the hypothesis that the TM domain may play a role in receptor oligomerization. Cross-linking with anti-GpA antibodies activated the kinase function of Glyfms B leading to an increase in PtdIns 3-kinase association and to the transmission of a mitogenic signal. Our results indicate that an activated kinase domain contains the major determinant for coupling with PtdIns 3-kinase, independent of extracellular and TM sequences and of ligand binding. Both chimeric receptors underwent internalization in the presence of anti-GpA antibodies but were not degraded, including the tyrosine-phosphorylated and kinase-active population. These results suggest that structural determinants in the extracellular domain must be important for targeting internalized receptors for lysosomal degradation.