The Caenorhabditis elegans EGL-15 Signaling Pathway Implicates a DOS-Like Multisubstrate Adaptor Protein in Fibroblast Growth Factor Signal Transduction

EGL-15 is a fibroblast growth factor receptor in the nematode Caenorhabditis elegans. Components that mediate EGL-15 signaling have been identified via mutations that confer a Clear (Clr) phenotype, indicative of hyperactivity of this pathway, or a suppressor-of-Clr (Soc) phenotype, indicative of reduced pathway activity. We have isolated a gain-of-function allele of let-60 ras that confers a Clr phenotype and implicated both let-60 ras and components of a mitogen-activated protein kinase cascade in EGL-15 signaling by their Soc phenotype. Epistasis analysis indicates that the gene soc-1 functions in EGL-15 signaling by acting either upstream of or independently of LET-60 RAS. soc-1 encodes a multisubstrate adaptor protein with an amino-terminal pleckstrin homology domain that is structurally similar to the DOS protein in Drosophila and mammalian GAB1. DOS is known to act with the cytoplasmic tyrosine phosphatase Corkscrew (CSW) in signaling pathways in Drosophila. Similarly, the C. elegans CSW ortholog PTP-2 was found to be involved in EGL-15 signaling. Structure-function analysis of SOC-1 and phenotypic analysis of single and double mutants are consistent with a model in which SOC-1 and PTP-2 act together in a pathway downstream of EGL-15 and the Src homology domain 2 (SH2)/SH3-adaptor protein SEM-5/GRB2 contributes to SOC-1-independent activities of EGL-15.     

Caenorhabditis elegans Fibroblast Growth Factor Receptor Signaling Can Occur Independently of the Multi-Substrate Adaptor FRS2

The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y¹⁰⁰⁹ and Y¹⁰⁸⁷), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.FIBROBLAST growth factors (FGFs) play important roles in many developmental and physiological processes, including cell migration, angiogenesis, proliferation, differentiation, and survival (Ornitz and Itoh 2001; Polanska et al. 2009). Mammals have a battery of both FGF ligands and high-affinity receptors to carry out this diverse set of important functions. These ligands and their receptors are generated from a set of 18 genes encoding FGFs and 4 genes encoding their receptors (Eswarakumar et al. 2005). Upon ligand binding, fibroblast growth factor receptors (FGFRs) dimerize, activating their intrinsic tyrosine kinase activity, which causes both autophosphorylation on intracellular tyrosine residues and phosphorylation of additional substrates (Eswarakumar et al. 2005). These phosphorylation events lead to the assembly of a signaling complex around the activated receptor, ultimately promoting various downstream signaling pathways (Eswarakumar et al. 2005).A large portion of mammalian FGFR signaling is mediated by the multi-substrate adaptor protein FRS2/snt-1 (Kouhara et al. 1997; Hadari et al. 1998, 2001; Lax et al. 2002; Gotoh et al. 2005). FRS2 constitutively associates with the juxtamembrane region of the FGFR via its amino-terminal PTB domain (Xu et al. 1998; Ong et al. 2000). Upon FGFR activation, FRS2 becomes heavily phosphorylated, allowing it to recruit Grb2 and Shp2 via their SH2 domains (Kouhara et al. 1997; Eswarakumar et al. 2005). Since these components cannot associate with the receptor in the absence of FRS2 (Hadari et al. 2001), FRS2 serves as an essential link between the activated receptor and many downstream signal transduction pathways.The understanding of FGF-stimulated signal transduction pathways has been aided by the study of FGF signaling in model organisms. Powerful genetic screens and the reduced complexity of the set of FGFs and their receptors in both Drosophila melanogaster and Caenorhabditis elegans have helped promote an understanding of the conserved aspects of FGF signaling pathways (Huang and Stern 2005; Polanska et al. 2009). In C. elegans, FGF signaling is mediated by two FGF ligands, EGL-17 and LET-756, and a single FGF receptor, EGL-15 (DeVore et al. 1995; Burdine et al. 1997; Roubin et al. 1999). The EGL-15 FGFR is structurally very similar to mammalian FGF receptors, with the highest level of sequence conservation found within the intracellular tyrosine kinase domain and the three extracellular immunoglobulin (IG) domains.Similar to mammalian FGFRs, alternative splicing also generates functionally distinct EGL-15 isoforms. A major structural difference between EGL-15 and other FGFRs lies in an additional domain located between the first IG domain and the acid box of EGL-15. This EGL-15-specific insert is encoded by a pair of mutually exclusive fifth exons, generating two EGL-15 isoforms, 5A and 5B, with different functions (Goodman et al. 2003). Alternative splicing also affects the sequence at the very end of the carboxy-terminal domain (CTD) of EGL-15 (Goodman et al. 2003), giving rise to four distinct C-terminal isoform types, referred to as types I–IV (see Figure 1A).Open in a separate windowFigure 1.—The SEM-5∷EGL-15 interaction is dependent upon the SEM-5 SH2 binding sites in the CTD of EGL-15. (A) Important residues in the EGL-15 CTD isoforms. The C-terminal portion of the kinase domain and the CTD are shown. The gray box is common to all C-terminal isoforms. Sequences of the CTD isoforms can be found in Figure S5. (B) SEM-5 binds directly to EGL-15 Y1009 and Y1087. A full-length SEM-5 prey was mated to eight EGL-15 baits: empty vector control (pBTM116) and derivatives containing variants of either the type I or the type IV EGL-15(Intra). Growth is an indication of an interaction between SEM-5 and the EGL-15 bait. Dilutions of the culture mixture are indicated above. hFGFR1, a bait containing the corresponding portion of the intracellular domain of the human FGFR1. Similar results were obtained using the human SEM-5 ortholog, Grb2.EGL-15, like its mammalian orthologs, is also involved in a large variety of functions, including cell migration guidance affecting the sex myoblast (SM) (Stern and Horvitz 1991; DeVore et al. 1995; Goodman et al. 2003) and CAN cells (Fleming et al. 2005), muscle arm extension (Dixon et al. 2006), a number of processes controlling terminal axon morphology (Bulow et al. 2004), muscle protein degradation (Szewczyk and Jacobson 2003), and fluid homeostasis (Huang and Stern 2004). A conserved FGFR signaling pathway in C. elegans was established by identifying the genes necessary for the role of EGL-15 in fluid homeostasis, and much of this same pathway is utilized in other functions of EGL-15 (DeVore et al. 1995; Borland et al. 2001; Huang and Stern 2004, 2005). The identification of these genes was facilitated by a temperature-sensitive mutation affecting a receptor tyrosine phosphatase, CLR-1 (CLeaR), which functions to negatively regulate EGL-15 signaling (Kokel et al. 1998). Mutations in clr-1 abolish this regulatory constraint on EGL-15, resulting in fluid accumulation in the pseudocoelomic cavity due to hyperactive EGL-15 signaling. The buildup of this clear fluid, resulting in the Clr phenotype, is easily scored and can be used to identify suppressors (soc, suppressor of Clr) that reduce EGL-15 signaling efficiency. These suppressors define an EGL-15 signaling pathway necessary for fluid homeostasis, which involves the activation of the Ras/MAPK cascade via the SEM-5/Grb2 adaptor protein, the let-341/sos-1 Sos-like guanine nucleotide exchange factor, and a PTP-2-SOC-1/Shp2-Gab1 cassette (Borland et al. 2001).Mutations in egl-15 have been identified on the basis of their effects on either fluid homeostasis or the guidance of the migrating SMs (DeVore et al. 1995; Goodman et al. 2003). Most of these alleles fall into an allelic series and affect the general aspects of EGL-15 signaling (Goodman et al. 2003). The strongest of these alleles confers an early larval arrest phenotype, whereas weaker alleles confer either a scrawny body morphology (Scr) or just the ability to suppress the Clr phenotype (Soc). While the weakest of these alleles does not affect egg laying, more highly compromised mutants show an egg-laying defect due to the mispositioning of the SMs. Four egl-15 alleles specifically affect SM migration; when homozygous, these mutations cause dramatic mispositioning of the SMs, but do not cause a Soc phenotype (Goodman et al. 2003). Three of these are nonsense mutations in exon 5A and eliminate the 5A EGL-15 isoform. The phenotype of these mutants highlights the specific requirement of the 5A isoform for SM migration guidance. The fourth mutation in this class, egl-15(n1457), is a nonsense mutation that truncates the carboxy-terminal domain, specifically implicating the CTD in SM migration guidance.Immediately following their birth at the end of the first larval stage (L1), the two bilaterally symmetric SMs undergo anteriorly directed migrations to final positions that flank the precise center of the gonad (Sulston and Horvitz 1977). In the middle of the third larval stage, the SMs divide to generate 16 cells that differentiate into the egg-laying muscles. Multiple mechanisms help guide the migrations of the SMs (Chen and Stern 1998; Branda and Stern 2000), including a chemoattraction mediated by EGL-15 that guides the SMs to their precise final positions (Burdine et al. 1998). The EGL-17 FGF serves as the chemoattractive cue, emanating from central gonadal cells (Branda and Stern 2000). In the absence of this chemoattraction, SMs are posteriorly displaced (Stern and Horvitz 1991). While mispositioned SMs still generate sex muscles, these muscles end up too far posterior to attach properly, causing the animal to be defective in egg laying (Egl).The signal transduction pathway downstream of EGL-15 that mediates SM chemoattraction is not well established. Several lines of evidence implicate SEM-5/Grb2, LET-341/Sos, and LET-60/Ras in SM chemoattraction. The roles of components in the Ras-MAPK cascade in this event are less clear (Sundaram et al. 1996; Chen et al. 1997; Chen and Stern 1998). A crucial gap in our understanding lies in the link between activated EGL-15 and the downstream signaling components. Here we show that SEM-5, the C. elegans GRB2 ortholog, appears to bind directly to SH2 binding sites within the carboxy terminal tail of EGL-15. These interactions are required for SM chemoattraction, but not for the essential function of EGL-15.     

成纤维细胞生长因子与其受体相互作用及其抑制剂的研究进展

成纤维细胞生长因子（FGF）有许多重要的生理功能，并与肿瘤的形成有关.为了弄清FGF与成纤维细胞生长因子受体(FGFR)相互作用的机制，人们对FGF和FGFR的各个结合结构域进行了深入、细致的研究，定位了aFGF、bFGF的肝素结合区、bFGF的受体结合区、FGF受体的肝素结合区、配体结合区和FGF受体相互结合区，提出了两个FGF与FGFR相互作用的模型，在此基础上设计了FGF的核酸类、糖类和多肽类抑制剂，为寻找新一代抗癌药物打下了理论基础.     

Activated EGL-15 FGF receptor promotes protein degradation in muscles of Caenorhabditis elegans

Signaling by fibroblast growth factors (FGFs) and their receptors has been previously implicated in control of cell proliferation, differentiation and migration. Here we report a novel role for signaling by the EGL-15 FGFR of Caenorhabditis elegans in controlling protein degradation in differentiated muscle. Activation of EGL-15, by means of a reduction of function mutation (clr-1) affecting an inhibitory phosphatase, triggers protein degradation in adult muscle cells using a pre-existing proteolytic system. This activation is not suppressed by mutation in either of the known genes encoding FGF ligands (egl-17 or let-756) but is well suppressed when both are mutated, indicating that either ligand is sufficient and at least one is necessary for FGFR activation. Activity of the Ras pathway through mitogen-activated protein kinase (MAPK) is required to trigger protein degradation. This is the first report that degradation of intracellular protein can be triggered by a growth factor receptor using an identified signal transduction pathway. The data raise the possibility that FGF-triggered proteolysis may be relevant to muscle remodeling or dedifferentiation.     

Mannose Phosphate Isomerase Regulates Fibroblast Growth Factor Receptor Family Signaling and Glioma Radiosensitivity

Asparagine-linked glycosylation is an endoplasmic reticulum co- and post- translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.     

Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation,Not Catalytic Activity

We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca²⁺ dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.     

Fibroblast growth factor signaling in Caenorhabditis elegans.

Growth factor receptor tyrosine kinases (RTKs), such as the fibroblast growth factor receptor (FGFR), play a major role in how cells communicate with their environment. FGFR signaling is crucial for normal development, and its misregulation in humans has been linked to developmental abnormalities and cancer. The precise molecular mechanisms by which FGFRs transduce extracellular signals to effect specific biologic responses is an area of intense research. Genetic analyses in model organisms have played a central role in our evolving understanding of these signal transduction cascades. Genetic studies in the nematode C. elegans have contributed to our knowledge of FGFR signaling by identifying genes involved in FGFR signal transduction and linking their gene products together into signaling modules. This review will describe FGFR-mediated signal transduction in C. elegans and focus on how these studies have contributed to our understanding of how FGFRs orchestrate the assembly of intracellular signaling pathways.     

A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma

Herein, we have identified cross-talk between the Hippo and fibroblast growth factor receptor (FGFR) oncogenic signaling pathways in cholangiocarcinoma (CCA). Yes-associated protein (YAP) nuclear localization and up-regulation of canonical target genes was observed in CCA cell lines and a patient-derived xenograft (PDX). Expression of FGFR1, -2, and -4 was identified in human CCA cell lines, driven, in part, by YAP coactivation of TBX5. In turn, FGFR signaling in a cell line with minimal basal YAP expression induced its cellular protein expression and nuclear localization. Treatment of YAP-positive CCA cell lines with BGJ398, a pan-FGFR inhibitor, resulted in a decrease in YAP activation. FGFR activation of YAP appears to be driven largely by FGF5 activation of FGFR2, as siRNA silencing of this ligand or receptor, respectively, inhibited YAP nuclear localization. BGJ398 treatment of YAP-expressing cells induced cell death due to Mcl-1 depletion. In a YAP-associated mouse model of CCA, expression of FGFR 1, 2, and 4 was also significantly increased. Accordingly, BGJ398 treatment was tumor-suppressive in this model and in a YAP-positive PDX model. These preclinical data suggest not only that the YAP and Hippo signaling pathways culminate in an Mcl-1-regulated tumor survival pathway but also that nuclear YAP expression may be a biomarker to employ in FGFR-directed therapy.     

Basolateral Localization of the Caenorhabditis elegans Epidermal Growth Factor Receptor in Epithelial Cells by the PDZ Protein LIN-10

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.     

Regulation of Kinase Activity in the Caenorhabditis elegans EGF Receptor,LET-23

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Structural Analysis of the Human Fibroblast Growth Factor Receptor 4 Kinase

The family of fibroblast growth factor receptors (FGFRs) plays an important and well-characterized role in a variety of pathological disorders. FGFR4 is involved in myogenesis and muscle regeneration. Mutations affecting the kinase domain of FGFR4 may cause cancer, for example, breast cancer or rhabdomyosarcoma. Whereas FGFR1–FGFR3 have been structurally characterized, the structure of the FGFR4 kinase domain has not yet been reported. In this study, we present four structures of the kinase domain of FGFR4, in its apo-form and in complex with different types of small-molecule inhibitors. The two apo-FGFR4 kinase domain structures show an activation segment similar in conformation to an autoinhibitory segment observed in the hepatocyte growth factor receptor kinase but different from the known structures of other FGFR kinases. The structures of FGFR4 in complex with the type I inhibitor Dovitinib and the type II inhibitor Ponatinib reveal the molecular interactions with different types of kinase inhibitors and may assist in the design and development of FGFR4 inhibitors.     

Nectin-1 Binds and Signals through the Fibroblast Growth Factor Receptor

Nectins belong to a family of immunoglobulin (Ig)-like cell-adhesion molecules comprising four members, nectin-1 through nectin-4. Nectins are involved in formation of the mechanical adhesive puncta adherentia junctions of synapses. Nectins share the same overall structural topology with an extracellular region containing three Ig modules, a transmembrane region, and a cytoplasmic region. In nectin-1, the first and second Ig module in the extracellular region are necessary for the trans-interaction with nectin-3 and formation of cis-dimers, respectively. The function of the third Ig module of nectin-1 remains unknown. We here report the structure in solution of the third, membrane-proximal Ig module of mouse nectin-1 (nectin-1 Ig3) solved by means of nuclear magnetic resonance (NMR) spectroscopy. It belongs to the C1 set of the Ig superfamily. Nectin-1 Ig3 was produced as a recombinant protein and induced neurite outgrowth in primary cultures of hippocampal and cerebellar granule neurons, an effect abolished by treatment with the fibroblast growth factor receptor (FGFR) inhibitor SU5402, or by transfection with a dominant-negative FGFR1 construct. We showed by surface plasmon resonance (SPR) analysis that nectin-1 Ig3 directly interacted with various isoforms of FGFR. Nectin-1 Ig3 induced phosphorylation of FGFR1c in the same manner as the whole nectin-1 ectodomain, and promoted survival of cerebellar granule neurons induced to undergo apoptosis. Finally, we constructed a peptide, nectide, by employing in silico modeling of various FGFR ligand-binding sites. Nectide mimicked all the effects of nectin-1 Ig3. We suggest that FGFR is a downstream signaling partner of nectin-1.     

The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling.