A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.     

Functional specificity of cytoplasmic and transmembrane tyrosine kinases: identification of 130- and 75-kilodalton substrates of c-fps/fes tyrosine kinase in macrophages.

c-fps/fes encodes a 92-kDa protein-tyrosine kinase (NCP92) that is expressed at the highest levels in macrophages. To determine if c-fps/fes can mediate the action of the colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) and to identify potential targets of c-fps/fes in macrophages, we have overexpressed c-fps/fes in a CSF-1-dependent macrophage cell line. A 30- to 50-fold overexpression of c-fps/fes partially released these cells from their factor dependence by a nonautocrine mechanism, and this correlated with the tyrosine phosphorylation of two proteins of 130 and 75 kDa (P130 and P75). c-fps/fes did not cause tyrosine phosphorylation or activation of CSF-1 dependent targets, including CSF-1R, Shc, and phosphatidylinositol 3-kinase, and conversely, CSF-1 did not induce tyrosine phosphorylation of P130 and P75. P75 appears to be a novel phosphotyrosyl protein, whereas P130 cross-reacts with a known substrate of v-src. P130 and P75 may be direct substrates of c-fps/fes: P130 was tightly associated with NCP92, and the src homology 2 domain of NCP92 specifically bound phosphorylated P130 and P75 but not the CSF-1-induced phosphotyrosyl proteins, consistent with the possibility that P130 and P75 are physiological targets of c-fps/fes. We conclude that although c-fps/fes can functionally substitute for CSF-1R to a certain extent, these tyrosine kinases act largely independently of each other and that P130 and P75 are novel targets whose mechanisms of action may be unrelated to the signalling pathways utilized by receptor tyrosine kinases.     

The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase

Unlike prototypical receptor tyrosine kinases (RTKs), which are single-chain polypeptides, the insulin receptor (InsR) is a preformed, covalently linked tetramer with two extracellular α subunits and two membrane-spanning, tyrosine kinase-containing β subunits. A single molecule of insulin binds asymmetrically to the ectodomain, triggering a conformational change that is transmitted to the cytoplasmic kinase domains, which facilitates their trans-phosphorylation. As in prototypical RTKs, tyrosine phosphorylation in the juxtamembrane region of InsR creates recruitment sites for downstream signaling proteins (IRS [InsR substrate] proteins, Shc) containing a phosphotyrosine-binding (PTB) domain, and tyrosine phosphorylation in the kinase activation loop stimulates InsR’s catalytic activity. For InsR, phosphorylation of the activation loop, which contains three tyrosine residues, also creates docking sites for adaptor proteins (Grb10/14, SH2B2) that possess specialized Src homology-2 (SH2) domains, which are dimeric and engage two phosphotyrosines in the activation loop.Insulin is a highly potent anabolic hormone that is critical for tissue development and for glucose homeostasis (Taniguchi et al. 2006). Released from the β cells of the pancreas, insulin regulates glucose output from the liver and glucose uptake into (primarily) skeletal muscle and adipose tissue. In addition, insulin promotes the synthesis and storage of carbohydrates, lipids, and protein. Insulin’s actions are mediated by the insulin receptor (InsR), a plasma membrane-resident glycoprotein and member of the receptor tyrosine kinase (RTK) family. Other members of the InsR subfamily of RTKs include the insulinlike growth factor-1 receptor (IGF1R) and insulin receptor-related receptor, the latter of which has no known ligand. As an RTK, InsR is ligand-activated through mechanisms that are both prototypical and atypical of RTKs. These mechanisms will be the focus of this article.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.     

Activation Status of Receptor Tyrosine Kinases as an Early Predictive Marker of Response to Chemotherapy in Osteosarcoma

Receptor tyrosine kinases (RTKs) are membrane receptors that play a vital role in various biological processes, in particular, cell survival, cell proliferation, and cell differentiation. These cellular processes are composed of multitiered signaling cascades of kinases starting from ligand binding to extracellular domains of RTKs that activate the entire pathways through tyrosine phosphorylation of the receptors and downstream effectors. A previous study reported that, based on proteomics data, RTKs were a major candidate target for osteosarcoma. In this study, activation profiles of six candidate RTKs, including c-Met, c-Kit, VEGFR2, HER2, FGFR1, and PDGFRα, were directly examined from chemonaive fresh frozen tissues of 32 osteosarcoma patients using a multiplex immunoassay. That examination revealed distinct patterns of tyrosine phosphorylation of RTKs in osteosarcoma cases. Unsupervised hierarchical clustering was calculated using Pearson uncentered correlation coefficient to classify RTKs into two groups—Group A (c-Met, c-Kit, VEGFR2, and HER2) and Group B (FGFR1 and PDGFRα)—based on tyrosine phosphorylation patterns. Nonactivation of all Group A RTKs was associated with shorter overall survival in stage IIB osteosarcoma patients. Percentages of tumor necrosis in patients with inactive Group A RTKs were significantly lower than those in patients with at least one active Group A RTK. Paired primary osteosarcoma cells with fresh osteosarcoma tissue were extracted and cultured for cytotoxicity testing. Primary cells with active Group A RTKs tended to be sensitive to doxorubicin and cisplatin. We also found that osteosarcoma cells with active Group A RTKs were more proliferative than cells with inactive Group A RTKs. These findings indicate that the activation pattern of Group A RTKs is a potential risk stratification and chemoresponse predictor and might be used to guide the optimum chemotherapy regimen for osteosarcoma patients.     

Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway

Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and β1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.     

Quantitative Phosphoproteomic Analysis Identifies Activation of the RET and IGF-1R/IR Signaling Pathways in Neuroblastoma

Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.     

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.     

p120 Catenin is required for growth factor-dependent cell motility and scattering in epithelial cells

Cadherin-mediated cell-cell adhesion is dynamically modulated during epithelial-mesenchymal transition triggered by activation of receptor tyrosine kinases (RTK) in epithelial cells. Several cadherin-binding proteins have been identified that control cell-cell adhesion. However, the mechanisms by which intercellular adhesion and cell motility are coregulated are still unknown. Here, we delineate a hitherto uncharted cooperation between RTKs, RhoA GTPase, and p120 catenin in instructing a motile behavior to epithelial cells. We found that expression of an N-terminus-deleted p120 catenin in a variety of epithelial cell types, including primary keratinocytes, effectively competes for endogenous p120 at cadherin binding sites and abrogates EGF-stimulated cell motility as well as HGF-induced cell scattering. The deleted mutant also inhibits the PI3K-dependent RhoA activation ensuing receptor activation. Conversely, we also show that the ectopic expression of full-length p120 in epithelial cells promotes cytoskeletal changes, stimulates cell motility, and activates RhoA. Both motogenic response to p120 and RhoA activation require coactivation of signaling downstream of RTKs as they are suppressed by ablation of the Ras/PI3K pathway. These studies demonstrate that p120 catenin is a necessary target of RTKs in regulating cell motility and help define a novel pathway leading to RhoA activation, which may contribute to the early steps of metastatic invasion.     

Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.Receptor tyrosine kinase (RTK) signaling is initiated on binding of soluble growth factors to growth factor receptors such as the insulin receptor (IR) or epidermal growth factor receptor (EGFR), or on binding of membrane-bound ephrins, as is the case for Eph receptors. Intracellular signaling is then propagated through PTMs, which commonly serve to regulate protein function by acting as docking sites for recruitment of modular protein interaction domains. Phosphorylation is the best studied PTM, and is a principle mechanism regulating intracellular signaling.A common element in RTK signaling involves autophosphorylation of the intracellular portion of the receptor (Fig. 1). RTKs become activated as a result of ligand-stabilized dimerization or oligomerization. For instance, in the EGFR subfamily (which includes ErbB and EGF receptors), the formation of homo- or heterodimers is initiated by ligand binding and subsequent exposure of a dimerization domain (Hynes and Lane 2005). Dimerization of the RTKs allows autophosphorylation of the RTKs; EGFR is exceptional in that an allosteric interaction between the kinase domains of adjacent monomers is responsible for the receptor activation (Zhang et al. 2006). However, in the majority of cases dimerization enhances RTK catalytic activity through phosphorylation of the kinase activation loop, and in some instances the juxtamembrane region, and recruits signaling effectors through the creation of pTyr docking sites. The specific interaction of signaling proteins with these pTyr-binding motifs activates signaling pathways, such as canonical signaling through the Ras-mitogen activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K)-Akt, and phospholipase C-gamma (PLC-γ) pathways. These RTK pathways can result in a variety of cellular processes, including differentiation, proliferation, survival, and migration (Fig. 1). The cellular context of signaling can dictate the biological outcome, and how each RTK initiates a given cellular process remains an area of active research.Open in a separate windowFigure 1.Receptor tyrosine kinases activate downstream pathways through recruitment of proteins containing pTyr-binding domains. Receptor tyrosine kinases are activated on growth factor binding to the extracellular domain of the receptor, leading to receptor dimerization and tyrosine phosphorylation (yellow circles labeled with a P) of their cytoplasmic tails, which act as docking sites for recruitment of PTB and SH2 domains. Various RTKs can mediate a diverse set of cellular processes (colored boxes) determined by the recruitment of specific SH2- and PTB-domain-containing proteins. The gray box displays how the adaptor Grb2 is recruited to an RTK through recognition of the pY-x-N (pY = pTyr, x = any natural amino acid) and activates cell growth and survival pathways such as MAPK and AKT, respectively, through complex formation via its SH3 domains.Tyrosine phosphorylation mediates RTK signaling through the recruitment and activation of proteins involved in downstream signaling pathways, mediated through pTyr binding of the SH2 and PTB domains of signaling effectors. SH2 and PTB domains are found in an otherwise diverse set of proteins containing a range of distinct catalytic and interaction domains, and provide a degree of specificity through their recognition of both a pTyr residue and surrounding amino acids. Here we will discuss the properties of proteins that contain SH2 and PTB domains and their roles in signaling downstream of RTKs, as well as the mechanisms by which they regulate the activity of these signaling effectors.     

Robust co-regulation of tyrosine phosphorylation sites on proteins reveals novel protein interactions

Cell signaling networks propagate information from extracellular cues via dynamic modulation of protein-protein interactions in a context-dependent manner. Networks based on receptor tyrosine kinases (RTKs), for example, phosphorylate intracellular proteins in response to extracellular ligands, resulting in dynamic protein-protein interactions that drive phenotypic changes. Most commonly used methods for discovering these protein-protein interactions, however, are optimized for detecting stable, longer-lived complexes, rather than the type of transient interactions that are essential components of dynamic signaling networks such as those mediated by RTKs. Substrate phosphorylation downstream of RTK activation modifies substrate activity and induces phospho-specific binding interactions, resulting in the formation of large transient macromolecular signaling complexes. Since protein complex formation should follow the trajectory of events that drive it, we reasoned that mining phosphoproteomic datasets for highly similar dynamic behavior of measured phosphorylation sites on different proteins could be used to predict novel, transient protein-protein interactions that had not been previously identified. We applied this method to explore signaling events downstream of EGFR stimulation. Our computational analysis of robustly co-regulated phosphorylation sites, based on multiple clustering analysis of quantitative time-resolved mass-spectrometry phosphoproteomic data, not only identified known sitewise-specific recruitment of proteins to EGFR, but also predicted novel, a priori interactions. A particularly intriguing prediction of EGFR interaction with the cytoskeleton-associated protein PDLIM1 was verified within cells using co-immunoprecipitation and in situ proximity ligation assays. Our approach thus offers a new way to discover protein-protein interactions in a dynamic context- and phosphorylation site-specific manner.     

PTP1B Targets the Endosomal Sorting Machinery: DEPHOSPHORYLATION OF REGULATORY SITES ON THE ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT COMPONENT STAM2*

Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated STAM2 specifically suppressed Akt activation, and a phosphorylation-deficient STAM2 mutant displayed prolonged localization on endosomes following EGF stimulation. These results reveal a novel link between the dephosphorylation and endocytic machinery and suggest that PTP1B can affect RTK signaling in a previously unrecognized manner.     

Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2

Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.     

Structural basis for UBA-mediated dimerization of c-Cbl ubiquitin ligase

Ligand-induced down-regulation by the ubiquitin-protein ligases, c-Cbl and Cbl-b, controls signaling downstream from many receptor-tyrosine kinases (RTK). Cbl proteins bind to phosphotyrosine residues on activated RTKs to affect ligand-dependent ubiquitylation of these receptors targeting them for degradation in the lysosome. Both c-Cbl and Cbl-b contain a ubiquitin-associated (UBA) domain, which is important for Cbl dimerization and tyrosine phosphorylation; however, the mechanism of UBA-mediated dimerization and its requirement for Cbl biological activity is unclear. Here, we report the crystal structure of the UBA domain of c-Cbl refined to 2.1-A resolution. The structure reveals the protein is a symmetric dimer tightly packed along a large hydrophobic surface formed by helices 2 and 3. NMR chemical shift mapping reveals heterodimerization can occur with the related Cbl-b UBA domain via the same surface employed for homodimerization. Disruption of c-Cbl dimerization by site-directed mutagenesis impairs c-Cbl phosphorylation following activation of the Met/hepatocyte growth factor RTK and c-Cbl-dependent ubiquitination of Met. This provides direct evidence for a role of Cbl dimerization in terminating signaling following activation of RTKs.     

Receptor tyrosine kinases mediate cell-cell interactions during Drosophila development

In vertebrates, receptor tyrosine kinases (RTKs) have been identified as growth factor receptors and proto-oncogenes. Many of these RTKs appear to play a key role in the regulation of cell growth. Recent analyses of several Drosophila genes encoding putative RTKs indicate that this class of proteins also serves an important role in cell fate decisions which depend on cellular interactions during development. The sevenless RTK mediates the position-dependent specification of a particular photoreceptor cell type (R7) in the eye. The local specification of R7 cells requires a functional tyrosine kinase domain of the sevenless protein but does not depend on the spatially restricted expression of the sevenless gene. The Drosophila EGF receptor homolog serves multiple functions during development, some of which are clearly unrelated to regulation of cell growth. Finally, the torso gene encodes an RTK required for the specification of the terminal regions of the Drosophila larva. A number of other genes have been genetically identified that appear to function in the same developmental processes upstream or downstream of these three RTKs. These loci are excellent candidates for genes encoding other components of the signalling pathways such as ligands or substrates of the RTKs.     

Physical and functional interactome atlas of human receptor tyrosine kinases

Much cell‐to‐cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP‐MS) to characterize stable binding partners and RTK–protein complexes, proximity‐dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase‐deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well‐studied RTKs, offers insights into the functions of less well‐studied RTKs, and highlights RTK‐RTK interactions and shared signaling pathways.     

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.     

Inactivation of Receptor Tyrosine Kinases Overcomes Resistance to Targeted <Emphasis Type="Italic">B-RAF</Emphasis> Inhibitors in Melanoma Cell Lines

The discovery of B-RAF activating mutations in malignant melanoma cells has led to the development of a number of targeted drugs, which block exclusively the mutant B-RAF protein. Tumor cells often acquire resistance to B-RAF inhibitors via activation of alternative signaling pathways. One of the resistance mechanisms is activation of PDGF, VEGF, c-KIT, and certain other tyrosine kinases. The possibility of overcoming the resistance to the B-RAF inhibitor Vemurafenib by inactivating receptor tyrosine kinases (RTKs) was studied in metastatic melanoma cell lines differing in B-RAF mutations and RTK activity. It was found that RTK inactivation may help to overcome resistance to B-RAF inhibitors via inhibition of tyrosine kinase phosphorylation and a subsequent blocking of the PI3K-AKT-mTOR and MEK-ERK1/2 downstream signaling pathways. The changes eventually mitigated the cell growth and enhanced the Vemurafenibdependent cell cycle arrest.