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.     

Integration of signals from receptor tyrosine kinases and g protein-coupled receptors

Activation of G protein-coupled receptors (GPCRs) leads to stimulation of classical G protein signaling pathways. In addition, GPCRs can activate the mitogen-activated protein kinases (MAPKs) such as the extracellular signal-regulated kinases, c-Jun NH(2)-terminal kinases (JNKs), and p38 MAPKs, and thereby influence cell proliferation, cell differentiation and mitogenesis. Cross talk between GPCRs and receptor tyrosine kinases (RTKs) is an incredibly complex process, and the exact signaling molecules involved are largely dependent on the cell type and the type of receptor that is activated. In this review we investigate recent advances that have been made in understanding the mechanisms of cross talk between GPCRs and RTKs, with a focus on GPCR-mediated activation of the Ras/MAPK pathway, GPCR-induced transactivation of RTKs, GPCR-mediated activation of JNK, and p38 MAPK, integration of signals by RhoGTPases, and activation of G protein signaling pathways by RTKs.     

Receptor tyrosine kinase activation: From the ligand perspective

Receptor tyrosine kinases (RTKs) are single-span transmembrane receptors in which relatively conserved intracellular kinase domains are coupled to divergent extracellular modules. The extracellular domains initiate receptor signaling upon binding to either soluble or membrane-embedded ligands. The diversity of extracellular domain structures allows for coupling of many unique signaling inputs to intracellular tyrosine phosphorylation. The combinatorial power of this receptor system is further increased by the fact that multiple ligands can typically interact with the same receptor. Such ligands often act as biased agonists and initiate distinct signaling responses via activation of the same receptor. Mechanisms behind such biased agonism are largely unknown for RTKs, especially at the level of receptor–ligand complex structure. Using recent progress in understanding the structures of active RTK signaling units, we discuss selected mechanisms by which ligands couple receptor activation to distinct signaling outputs.     

The Epidermal Growth Factor Receptor (EGFR) Promotes Uptake of Influenza A Viruses (IAV) into Host Cells

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake.     

Growth factor-specific signaling pathway stimulation and gene expression mediated by ErbB receptors

The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1beta acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1beta acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2beta acting through the same heterodimeric receptor species. Surprisingly, NRG1beta and NRG2beta also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes.     

Selection and growth regulation of genetically modified cells with hapten‐specific antibody/receptor tyrosine kinase chimera

Although receptor tyrosine kinases (RTKs) play a pivotal role in the development and maintaining the homeostasis of the body, overexpression or mutation of RTKs often induces tumorigenesis or metastasis. To mimic the function of RTKs, we developed two fusion receptors consisting of anti‐fluorescein antibody single‐chain Fv, extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of epidermal growth factor receptor or c‐fms based on previously constructed antibody/cytokine receptor chimeras. The expression of these chimeric receptors in the hematopoietic cell line Ba/F3 and non‐hematopoietic cell line NIH/3T3 resulted in the activation of receptors themselves, downstream signaling molecules and cell proliferation in response to fluorescein‐conjugated BSA, leading to selective expansion of transduced cells up to almost 100%. These results indicate that the cognate antigen could activate the chimeric receptors even though the wild‐type extracellular domains were switched to the antibody fragment. This is the first study to show that our antigen‐mediated genetically modified cell amplification (AMEGA) system could be applied to non‐hematopoietic cells by utilizing antibody/RTK chimeras. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Activation of receptor protein-tyrosine kinases from the cytoplasmic compartment

It is widely accepted that receptor protein-tyrosine kinases (RTKs) are activated upon dimerization by binding to their extracellular ligands. However, EGF receptor (EGFR) dimerization per se does not require ligand binding. Instead, its cytoplasmic kinase domains have to form characteristic head-to-tail asymmetric dimers to become active, where one 'activator' domain activates the other 'receiver' domain. The non-catalytic, cytoplasmic regions of RTKs, namely the juxtamembrane and carboxy terminal portions, also regulate kinase activity. For instance, the juxtamembrane region of the RTK MuSK inhibits the kinase domain probably together with a cellular factor(s). These findings suggest that RTKs could be activated by cytoplasmic proteins. Indeed, Dok-7 and cytohesin have recently been identified as such activators of MuSK and EGFR, respectively. Given that failure of Dok-7 signaling causes myasthenia, and inhibition of cytohesin signaling reduces the proliferation of EGFR-dependent cancer cells, cytoplasmic activators of RTKs may provide new therapeutic targets.     

Regulation of EphA 4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function

Signaling by receptor tyrosine kinases (RTKs) is mediated by their intrinsic kinase activity. Typically, kinase-activating mutations result in ligand-independent signaling and gain-of-function phenotypes. Like other RTKs, Ephs require kinase activity to signal, but signaling by Ephs in vitro also requires clustering by their membrane bound ephrin ligands. The relative importance of Eph kinase activity and clustering for in vivo functions is unknown. We find that knockin mice expressing a mutant form of EphA4 (EphA4(EE)), whose kinase is constitutively activated in the absence of ephrinB ligands, are deficient in the development of thalamocortical projections and some aspects of central pattern generator rhythmicity. Surprisingly, other functions of EphA4 were regulated normally by EphA4(EE), including midline axon guidance, hindlimb locomotion, in vitro growth cone collapse, and phosphorylation of ephexin1. These results suggest that signaling of Eph RTKs follows a multistep process of induced kinase activity and higher-order clustering different from RTKs responding to soluble ligands.     

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.     

The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) transduce signals via cytoplasmic adaptor proteins to downstream signaling components. We have identified loss-of-function mutations in dshc, the Drosophila homolog of the mammalian adaptor protein SHC. A point mutation in the phosphotyrosine binding (PTB) domain completely abolishes DSHC function and provides in vivo evidence for the function of PTB domains. Unlike other adaptor proteins, DSHC is involved in signaling by only a subset of RTKs: dshc mutants show defects in Torso and DER but not Sevenless signaling, which is confirmed by epistasis experiments. We show by double-mutant analysis that the adaptors DOS, DRK, and DSHC act in parallel to transduce the Torso signal. Our results suggest that DSHC confers specificity to receptor signaling.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatase-1B

Receptor tyrosine kinases (RTKs) are key regulators of cellular homeostasis. Based on in vitro and ex vivo studies, protein tyrosine phosphatase-1B (PTP1B) was implicated in the regulation of several RTKs, yet mice lacking PTP1B show defects mainly in insulin and leptin receptor signaling. To address this apparent paradox, we studied RTK signaling in primary and immortalized fibroblasts from PTP1B(-/-) mice. After growth factor treatment, cells lacking PTP1B exhibit increased and sustained phosphorylation of the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR). However, Erk activation is enhanced only slightly, and there is no increase in Akt activation in PTP1B-deficient cells. Our results show that PTP1B does play a role in regulating EGFR and PDGFR phosphorylation but that other signaling mechanisms can largely compensate for PTP1B deficiency. In-gel phosphatase experiments suggest that other PTPs may help to regulate the EGFR and PDGFR in PTP1B(-/-) fibroblasts. This and other compensatory mechanisms prevent widespread, uncontrolled activation of RTKs in the absence of PTP1B and probably explain the relatively mild effects of PTP1B deletion in mice.     

Should I stay or should I go?: Shedding of RPTPs in cancer cells switches signals from stabilizing cell-cell adhesion to driving cell migration

Dissolution of cell-cell adhesive contacts and increased cell-extracellular matrix adhesion are hallmarks of the migratory and invasive phenotype of cancer cells. These changes are facilitated by growth factor binding to receptor protein tyrosine kinases (RTKs). In normal cells, cell-cell adhesion molecules (CAMs), including some receptor protein tyrosine phosphatases (RPTPs), antagonize RTK signaling by promoting adhesion over migration. In cancer, RTK signaling is constitutive due to mutated or amplified RTKs, which leads to growth factor independence or autonomy. An alternative route for a tumor cell to achieve autonomy is to inactivate cell-cell CAMs such as RPTPs. RPTPs directly mediate cell adhesion and regulate both cadherin-dependent adhesion and signaling. In addition, RPTPs antagonize RTK signaling by dephosphorylating molecules activated following ligand binding. Both RPTPs and cadherins are downregulated in tumor cells by cleavage at the cell surface. This results in shedding of the extracellular, adhesive segment and displacement of the intracellular segment, altering its subcellular localization and access to substrates or binding partners. In this commentary we discuss the signals that are altered following RPTP and cadherin cleavage to promote cell migration. Tumor cells both step on the gas (RTKs) and disconnect the brakes (RPTPs and cadherins) during their invasive and metastatic journey.Key words: receptor protein tyrosine kinase, receptor-like protein tyrosine phosphatase, cadherins, cell adhesion, signal transduction, phospholipase C gamma, protein kinase C, catenins, IQGAP1 protein, regulated intramembrane proteolysis     

Directing traffic in neural cells: determinants of receptor tyrosine kinase localization and cellular responses

The trafficking of receptor tyrosine kinases (RTKs) to distinct subcellular locations is essential for the specificity and fidelity of signal transduction and biological responses. This is particularly important in the PNS and CNS in which RTKs mediate key events in the development and maintenance of neurons and glia through a wide range of neural processes, including survival, proliferation, differentiation, neurite outgrowth, and synaptogenesis. The mechanisms that regulate the targeting of RTKs to their subcellular destinations for appropriate signal transduction, however, are still elusive. In this review, we discuss evidence for the spatial organization of signaling machinery into distinct subcellular compartments, as well as the role for ligand specificity, receptor sorting signals, and lipid raft microdomains in RTK targeting and the resultant cellular responses in neural cells.     

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.