Distinct involvement of the Gab1 and Grb2 adaptor proteins in signal transduction by the related receptor tyrosine kinases RON and MET

Although the signal transduction mechanisms of the receptor tyrosine kinase MET are well defined, less is known about its close relative RON. MET initiates intracellular signaling by autophosphorylation on specific cytoplasmic tyrosines that form docking sites for the adaptor proteins Grb2 and Gab1. Grb2 binds directly and is essential for all of the biological activities of MET. Gab1 docks either directly or indirectly via Grb2 and controls only a subset of MET functions. Because MET and RON possess similar adaptor binding sites, it was anticipated that their adaptor interactions would be conserved. Here we show that in contrast to MET, RON relies primarily on Gab1 for signal transmission. Surprisingly, disruption of the Grb2 docking site of RON or Grb2 depletion augments activity, whereas enhancement of Grb2 binding attenuates Gab1 recruitment and signaling. Hence, RON and MET differ in their adaptor interactions; furthermore, Grb2 performs a novel antagonistic role in the context of RON signaling.     

Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.     

Differential expression of receptor tyrosine kinases (RTKs) and IGF-I pathway activation in human uterine leiomyomas

Uterine leiomyomas (fibroids) are benign tumors that are prevalent in women of reproductive age. Research suggests that activated receptor tyrosine kinases (RTKs) play an important role in the enhanced proliferation observed in fibroids. In this study, a phospho-RTK array technique was used to detect RTK activity in leiomyomas compared with myometrial tissue. We found that fifteen out of seventeen RTKs evaluated in this study were highly expressed (P < 0.02-0.03) in the leiomyomas, and included the IGF-I/IGF-IR, EGF/EGFR, FGF/FGF-R, HGF/HGF-R, and PDGF/PDGF-R gene families. Due to the higher protein levels of IGF-IR observed in leiomyomas by us in earlier studies, we decided to focus on the activation of the IGF-IR, its downstream effectors, and MAPKp44/42 to confirm our earlier findings; and validate the significance of the increased IGF-IR phosphorylation observed by RTK array analysis in this study. We used immunolocalization, western blot, or immunoprecipitation studies and confirmed that leiomyomas overexpressed IGF-IRbeta and phosphorylated IGF-IRbeta. Additionally, we showed that the downstream effectors, Shc, Grb2, and MAPKp44/42 (P < 0.02-0.001) were also overexpressed and involved in IGF-IR signaling in these tumors, while IRS-I, PI3K, and AKT were not. In vitro studies showed that IGF-I (100 ng/mL) increased the proliferation of uterine leiomyoma cells (UtLM) (P < 0.0001), and that phosphorylated IGF-IRbeta, Shc, and MAPKp44/42 were also overexpressed in IGF-I-treated UtLM cells (P < 0.05), similar to the tissue findings. A neutralizing antibody against the IGF-IRbeta blocked these effects. These data indicate that overexpression of RTKs and, in particular, activation of the IGF-IR signaling pathway through Shc/Grb2/MAPK are important in mediating uterine leiomyoma growth. These data may provide new anti-tumor targets for noninvasive treatment of fibroids.     

ShcB and ShcC activation by the Trk family of receptor tyrosine kinases

Activation of the neurotrophin Trk receptors is a key process in the survival and development of the nervous system. The signaling adapters ShcB and ShcC, but not ShcA, are thought to be the primary Shc adaptor proteins in neurons as both are highly expressed in both the developing and adult nervous system. Although a previous study suggested that ShcB and ShcC do not strongly interact with the Trk receptors (1), we find that ShcB and ShcC bind the Trk receptors in a phosphotyrosine-dependent manner via their N-terminal phosphotyrosine binding domain at Tyr(499) (TrkA) and Tyr(515) (TrkB), they are tyrosine-phosphorylated in response to neurotrophin stimulation, and they enhance the activation of mitogen-activated protein kinase in Trk-expressing cells. Moreover, neurotrophin treatment of primary cortical neurons stimulates ShcB/ShcC-Trk interaction and the tyrosine phosphorylation of ShcB/ShcC, indicating that they are bona fide targets of the Trk receptors in vivo. Interestingly, two proteins (pp60 and pp75) co-immunoprecipitate with ShcB and ShcC in response to neurotrophin stimulation in primary cortical neurons, suggesting a potential role of these unknown targets in neurotrophin signaling. Collectively, these results demonstrate that ShcB and ShcC, and their co-immunoprecipitating proteins, are activated by the Trk receptors in primary neurons.     

Peroxynitrite signaling: receptor tyrosine kinases and activation of stress-responsive pathways

Peroxynitrite, generated for example in inflammatory processes, is capable of nitrating and oxidizing biomolecules, implying a considerable impact on the integrity of cellular structures. Cells respond to stressful conditions by the activation of signaling pathways, including receptor tyrosine kinase-dependent pathways such as mitogen-activated protein kinases and the phosphoinositide-3-kinase/Akt pathway. Peroxynitrite affects signaling pathways by nitration as well as by oxidation: while nitration of tyrosine residues by peroxynitrite modulates signaling processes relying on tyrosine phosphorylation and dephosphorylation, oxidation of phosphotyrosine phosphatases may lead to an alteration in the tyrosine phosphorylation/dephosphorylation balance. The flavanol (-)-epicatechin is a potent inhibitor of tyrosine nitration and may be employed as a tool to distinguish signaling effects due to tyrosine nitration from those that are due to oxidation reactions.     

E-cadherin-mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases

E-cadherin is an essential adhesion protein as well as a tumor suppressor that is silenced in many cancers. Its adhesion-dependent regulation of signaling has not been elucidated. We report that E-cadherin can negatively regulate, in an adhesion-dependent manner, the ligand-dependent activation of divergent classes of receptor tyrosine kinases (RTKs), by inhibiting their ligand-dependent activation in association with decreases in receptor mobility and in ligand-binding affinity. E-cadherin did not regulate a constitutively active mutant RTK (Neu*) or the ligand-dependent activation of LPA receptors or muscarinic receptors, which are two classes of G protein-coupled receptors. EGFR regulation by E-cadherin was associated with complex formation between EGFR and E-cadherin that depended on the extracellular domain of E-cadherin but was independent of beta-catenin binding or p120-catenin binding. Transfection of E-cadherin conferred negative RTK regulation to human melanoma and breast cancer lines with downregulated endogenous E-cadherin. Abrogation of E-cadherin regulation may contribute to the frequent ligand-dependent activation of RTK in tumors.     

Transglutaminases and receptor tyrosine kinases

Transglutaminases are confounding enzymes which are known to play key roles in various cellular processes. In this paper, we aim to bring together several pieces of evidence from published research and literature that suggest a potentially vital role for transglutaminases in receptor tyrosine kinases (RTK) signalling. We cite literature that confirms and suggests the formation of integrin:RTK:transglutaminase complexes and explores the occurrence and functionality of these complexes in a large fraction of the RTK family.     

Overexpression and activation of the RON receptor tyrosine kinase in a panel of human colorectal carcinoma cell lines

RON is a receptor tyrosine kinase belonging to the MET proto-oncogene family. The purposes of this study are to determine the expression and activation of RON in a panel of human colon carcinoma cell lines. Western blotting showed that RON is barely detectable in normal and SV-40-transformed colon epithelial cells, but highly expressed and constitutively activated in several colon carcinoma cell lines including Colo201, HT-29, HCT116, and SW837. Moreover, a novel RON variant with a molecular mass of 160 kDa (RONDelta160) was identified from HT-29 cells. The cDNA encoding RONDelta160 has an in-frame deletion of 109 amino acids in the extracellular domain of the RON beta chain, which is caused by splicing out of two exons in the RON mRNA. No mutations were found in the kinase domain of the RON gene in five carcinoma cell lines screened. By expressing RON in colon epithelial cells, we found that RON activation increases cell motile-invasive activities and protects cells against apoptotic death. These data suggest that RON expression and activation are deregulated in colon carcinoma cell lines. By abnormal activation of RON, this receptor and its variant may regulate motile-invasive phenotypes of certain colon carcinoma cells in vivo.     

Cross-talk between RON receptor tyrosine kinase and other transmembrane receptors

RON is a transmembrane receptor tyrosine kinase that mediates biological activities of Macrophage Stimulating Protein (MSP). MSP is a multifunctional factor regulating cell adhesion, motility, growth and survival. MSP binding to RON causes receptor tyrosine phosphorylation leading to up-regulation of RON catalytic activity and subsequent activation of downstream signaling molecules. Recent studies show that RON is spatially and functionally associated with other transmembrane molecules including adhesion receptors integrins and cadherins, and cytokine and growth factor receptors IL-3 betac, EPOR and MET. For example, MSP-induced cell shape change is mediated via RON-activated IL-3 betac receptor. Activation of integrins causes MSP-independent RON phosphorylation, and the integrin/RON collaboration regulates cell survival. Thus, RON can be activated without MSP by ligand stimulation of RON-associated receptors, and MSP-activated RON can cause ligand-independent activation of RON-associated receptors. As a result of the receptor cross-activation RON-specific pathways become a part of a signal transduction network of other receptors, and conversely signaling pathways activated by other receptors can be used by RON. This receptor collaboration extends the spectrum of cellular responses generated by MSP and by putative ligands of RON-associated receptors. However signaling pathways involved in the receptor cross-talk and underlying activation mechanisms remain to be investigated. The purpose of this review is to summarize data and to discuss a role of cross-talk between RON and other transmembrane receptors.     

A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases

Eph receptor tyrosine kinases (RTKs) mediate numerous developmental processes. Their activity is regulated by auto-phosphorylation on two tyrosines within the juxtamembrane segment (JMS) immediately N-terminal to the kinase domain (KD). Here, we probe the molecular details of Eph kinase activation through mutational analysis, X-ray crystallography and NMR spectroscopy on auto-inhibited and active EphB2 and EphA4 fragments. We show that a Tyr750Ala gain-of-function mutation in the KD and JMS phosphorylation independently induce disorder of the JMS and its dissociation from the KD. Our X-ray analyses demonstrate that this occurs without major conformational changes to the KD and with only partial ordering of the KD activation segment. However, conformational exchange for helix alphaC in the N-terminal KD lobe and for the activation segment, coupled with increased inter-lobe dynamics, is observed upon kinase activation in our NMR analyses. Overall, our results suggest that a change in inter-lobe dynamics and the sampling of catalytically competent conformations for helix alphaC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation.     

Induction of antagonistic soluble decoy receptor tyrosine kinases by intronic polyA activation

Alternative intronic polyadenylation (IPA) can generate truncated protein isoforms with significantly altered functions. Here, we describe 31 dominant-negative, secreted variant isoforms of receptor tyrosine kinases (RTKs) that are produced by activation of intronic poly(A) sites. We show that blocking U1-snRNP can activate IPA, indicating a larger role for U1-snRNP in RNA surveillance. Moreover, we report the development of an antisense-based method to effectively and specifically activate expression of individual soluble decoy RTKs (sdRTKs) to alter signaling, with potential therapeutic implications. In particular, a quantitative switch from signal transducing full-length vascular endothelial growth factor receptor-2 (VEGFR2/KDR) to a dominant-negative sKDR results in a strong antiangiogenic effect both on directly targeted cells and on naive cells exposed to conditioned media, suggesting a role for this approach in interfering with angiogenic paracrine and autocrine loops.     

Structural analysis of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γ-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.     

The shadow of death on the MET tyrosine kinase receptor

The MET tyrosine kinase receptor is a high-affinity receptor for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF-MET system is necessary for embryonic development, and aberrant MET signalling favours tumorigenesis and metastasis. MET is a prototype of tyrosine kinase receptor, which is able to counteract apoptosis through the initiation of a survival signal involving notably the PI3K-Akt pathway. Paradoxically, the MET receptor is also able to promote apoptosis when activated by HGF/SF or independently of ligand stimulation. The molecular mechanisms underlying this uncommon response have been recently investigated and revealed dual antiapoptotic or proapoptotic property of MET according to the cell type or stress conditions. Although the involvement of MET in the regulation of integrated biological responses mostly took into account its efficient antiapoptotic function, its proapoptotic responses could also be important for regulation of the survival/apoptosis balance and play a role during the development or tumour progression.     

Hyaluronan constitutively regulates activation of multiple receptor tyrosine kinases in epithelial and carcinoma cells

Hyaluronan (HA) is enriched in the pericellular matrices of many malignant human tumors, and manipulations of HA interactions have strong effects on tumor progression in animal models. Increased HA production stimulates ERBB2 activation, leading to increased cell survival activities and several malignant cell properties. On the other hand, inhibition of constitutive HA-tumor cell interactions in malignant cells inhibits these properties. We have now investigated the role of HA in activation of several additional receptor tyrosine kinases (RTKs), i.e. IGF1R-beta, PDGFR-beta, EGFR and c-MET, in colon, prostate, and breast carcinoma cells. In each case we show that antagonists of endogenous HA interactions inhibit their tyrosine phosphorylation, i.e. activation. On the other hand, we show that these RTKs are activated in phenotypically normal or relatively benign tumor cells by experimentally increasing HA production. We also investigated the role of HA in constitutive versus ligand-induced activation of RTKs. In HCA7 colon and C4-2 prostate carcinoma cells, ERBB2 is constitutively activated in a ligand-independent manner, whereas IGF1R-beta and PDGFR-beta require ligand interaction for activation. We show that both constitutive activation of ERBB2 and ligand-mediated activation of IGF1R-beta and PDGFR-beta are reversed by co-treatment of the cells with a HA antagonist. We conclude that HA serves a general function in RTK activation.     

VEGF stimulates MAPK through a pathway that is unique for receptor tyrosine kinases

We demonstrate that stimulation of primary cultures of endothelial cells with vascular endothelial cell growth factor (VEGF) results in a rapid increase in labeled guanine nucleotide bound to p21ras. Surprisingly, although VEGF stimulates ras activity, adenoviral-mediated gene transfer of a dominant negative form of ras (N17ras) had no effect on VEGF-stimulated mitogen-activated protein kinase (MAPK) activity. In contrast, treatment of endothelial cells with two structurally unrelated inhibitors of protein kinase C (PKC) abrogated VEGF-stimulated MAPK activity. In addition, inhibition of ras-Raf interactions by expression of a truncated form of Raf containing only the ras binding domain blocked VEGF-stimulated MAPK activation. These results suggest that VEGF stimulation of MAPK in endothelial cells differs from the pathway used by other members of the receptor tyrosine kinase family. In contrast, analogous to certain G-coupled receptors, VEGF appears to activate MAPK through a PKC-dependent pathway that requires a stable ras-Raf interaction but is not inhibited by N17ras expression.     

Spatio‐temporally precise activation of engineered receptor tyrosine kinases by light

Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low‐intensity blue light. We screened light‐oxygen‐voltage (LOV)‐sensing domains for their ability to activate RTKs by light‐activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto‐RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.     

Trafficking of receptor tyrosine kinases to the nucleus

It has been known for at least 20 years that growth factors induce the internalization of cognate receptor tyrosine kinases (RTKs). The internalized receptors are then sorted to lysosomes or recycled to the cell surface. More recently, data have been published to indicate other intracellular destinations for the internalized RTKs. These include the nucleus, mitochondria, and cytoplasm. Also, it is recognized that trafficking to these novel destinations involves new biochemical mechanisms, such as proteolytic processing or interaction with translocons, and that these trafficking events have a function in signal transduction, implicating the receptor itself as a signaling element between the cell surface and the nucleus.