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1.
Dmitry S Kanibolotsky Oleksandra V Novosyl'na Catherine M Abbott Boris S Negrutskii Anna V El'skaya 《BMC structural biology》2008,8(1):1-16
Background
Eukaryotic translation elongation factor eEF1A directs the correct aminoacyl-tRNA to ribosomal A-site. In addition, eEF1A is involved in carcinogenesis and apoptosis and can interact with large number of non-translational ligands. There are two isoforms of eEF1A, which are 98% similar. Despite the strong similarity, the isoforms differ in some properties. Importantly, the appearance of eEF1A2 in tissues in which the variant is not normally expressed can be coupled to cancer development. We reasoned that the background for the functional difference of eEF1A1 and eEF1A2 might lie in changes of dynamics of the isoforms.Results
It has been determined by multiple MD simulation that eEF1A1 shows increased reciprocal flexibility of structural domains I and II and less average distance between the domains, while increased non-correlated diffusive atom motions within protein domains characterize eEF1A2. The divergence in the dynamic properties of eEF1A1 and eEF1A2 is caused by interactions of amino acid residues that differ between the two variants with neighboring residues and water environment. The main correlated motion of both protein isoforms is the change in proximity of domains I and II which can lead to disappearance of the gap between the domains and transition of the protein into a "closed" conformation. Such a transition is reversible and the protein can adopt an "open" conformation again. This finding is in line with our earlier experimental observation that the transition between "open" and "closed" conformations of eEF1A could be essential for binding of tRNA and/or other biological ligands. The putative calmodulin-binding region Asn311-Gly327 is less flexible in eEF1A1 implying its increased affinity for calmodulin. The ability of eEF1A1 rather than eEF1A2 to interact with Ca2+/calmodulin is shown experimentally in an ELISA-based test.Conclusion
We have found that reversible transitions between "open" and "close" conformations of eEF1A provide a molecular background for the earlier observation that the eEF1A molecule is able to change the shape upon interaction with tRNA. The ability of eEF1A1 rather than eEF1A2 to interact with calmodulin is predicted by MD analysis and showed experimentally. The differential ability of the eEF1A isoforms to interact with signaling molecules discovered in this study could be associated with cancer-related properties of eEF1A2. 相似文献2.
Lamberti A Sanges C Chambery A Migliaccio N Rosso F Di Maro A Papale F Marra M Parente A Caraglia M Abbruzzese A Arcari P 《Biochimie》2011,93(10):1738-1746
The eukaryotic translation elongation factor 1A (eEF1A), besides to its canonical role in protein synthesis, is also involved in several other cellular processes, depending on changes in cellular location, cell type, concentration of ligands, substrates or cofactors. Therefore eEF1A is a moonlighting protein that participates to a network of molecular interactions involving its structural domains. Since the identification of novel protein–protein interactions represents important tasks in post-genomic era, the interactome of eEF1A1 M-domain was investigated by using a proteomic approach. To this purpose, the eEF1A1 M-domain was fused with glutathione-S-transferase (GST) and Strep-tag (ST) at it’s N- and C-terminal, respectively. The recombinant protein (GST-M-ST) was purified and incubated with a mouse embryo lysate by applying an affinity chromatography strategy. The interacting proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Besides the known partners, the pool of interacting proteins contained sorbin, a polypeptide of 153 amino acids present in SH3 domain-containing adaptor proteins, such as SORBS2. This interaction was also assessed by Western blot on immunoprecipitate from mouse embryo or H1355 cell lysates with anti-eEF1A or anti-SORBS2 antibodies and on eEF1A1-His pull-down from H1355 cell lysate with antibody anti-SORBS2. Furthermore, the interaction between eEF1A and SORBS2 was also confirmed by confocal microscopy and FRET analysis. Interestingly, a co-localization of SORBS2 and eEF1A was evidenced at level of plasma membrane, thus suggesting the involvement of eEF1A1 in novel key signal transduction complexes. 相似文献
3.
Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1. 总被引:6,自引:8,他引:6
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S Richard D Yu K J Blumer D Hausladen M W Olszowy P A Connelly A S Shaw 《Molecular and cellular biology》1995,15(1):186-197
src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules. 相似文献
4.
p46(Shc) and p52(Shc) become heavily tyrosine phosphorylated in response to interleukin 3 (IL-3) treatment. We have investigated the potential of Shc to integrate IL-3 signalling pathways and demonstrate that Shc associates with the beta subunits of the human (betac) and murine (Aic2A) IL-3 receptors, SHIP and Gab2 following IL-3 stimulation. The interaction between Shc and the IL-3 receptor beta chains was direct, mediated by both the SH2 and PTB domains. Interaction with SHIP was via the Shc PTB domain and the Shc SH2 domain mediated the interaction with Gab2. Phosphopeptide competition studies suggest that the SH2 domain interacts primarily with tyrosine 612 of betac (610 of Aic2A), and the PTB domain with tyrosine 577 of betac (575 of Aic2A). PTB binding to IL-3R beta chains was of highest affinity, and appeared to play the primary role in binding. These findings suggest that Shc may play an important role in coordinately integrating IL-3 signalling pathways. 相似文献
5.
Mansilla F Dominguez CA Yeadon JE Corydon TJ Burden SJ Knudsen CR 《Journal of cellular biochemistry》2008,105(3):847-858
Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation. 相似文献
6.
7.
Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3''-kinase SH2 domains: a model for SH2-mediated receptor-target interactions. 总被引:30,自引:8,他引:22
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M Reedijk X Liu P van der Geer K Letwin M D Waterfield T Hunter T Pawson 《The EMBO journal》1992,11(4):1365-1372
Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. 相似文献
8.
Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality 总被引:1,自引:0,他引:1
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Ke Y Zhang EE Hagihara K Wu D Pang Y Klein R Curran T Ranscht B Feng GS 《Molecular and cellular biology》2007,27(19):6706-6717
The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation. 相似文献
9.
Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells. 总被引:20,自引:1,他引:19
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S J Holland N W Gale G D Gish R A Roth Z Songyang L C Cantley M Henkemeyer G D Yancopoulos T Pawson 《The EMBO journal》1997,16(13):3877-3888
Eph-related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, we have expressed the EphB2 receptor (formerly Nuk/Cek5/Sek3) in neuronal NG108-15 cells, and have observed the tyrosine phosphorylation of multiple cellular proteins upon activation of EphB2 by its ligand, ephrin-B1 (formerly Elk-L/Lerk2). The activated EphB2 receptor induced the tyrosine phosphorylation of a 62-64 kDa protein (p62[dok]), which in turn formed a complex with the Ras GTPase-activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also bound through its SH2 domains to tyrosine-phosphorylated EphB2 in vitro, and complexed with activated EphB2 in vivo. We have localized an in vitro RasGAP-binding site to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2, and demonstrated that substitution of these amino acids abolishes ephrin-B1-induced signalling events in EphB2-expressing NG108-15 cells. These tyrosine residues are followed by proline at the + 3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2-activated signalling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture. 相似文献
10.
Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses 总被引:22,自引:0,他引:22
Schaeper U Gehring NH Fuchs KP Sachs M Kempkes B Birchmeier W 《The Journal of cell biology》2000,149(7):1419-1432
Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met-specific branching morphogenesis. It associates directly with c-Met via the c-Met-binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met-binding site is localized to a 13-amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met-binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling. 相似文献
11.
It has recently been observed that G protein-coupled receptors (GPCRs) can interact with SH3 domains through polyproline motifs. These interactions appear to be involved in receptor internalization and MAPK signalling. Here we report that the third cytoplasmic loop of the dopamine D3 receptor can interact in vitro with the adaptor protein Grb2. While the amino- and carboxy-terminal SH3 domains of Grb2 separately did not interact with the D3 receptor loop, the interaction is at least partially maintained with a Grb2 mutant for the amino-terminal SH3 domain, but disrupted for a Grb2 mutant with a nonfunctional carboxy-terminal SH3 domain. The data indicate the need of structural integrity of the entire Grb2 protein for the interaction and dominant role of the carboxy-terminal SH3 domain in the interaction. Disruption of the PXXP motifs in the D3 receptor did not affect the interaction with Grb2. These results indicate that GPCRs may contain SH3 ligands that do not contain the postulated minimal consensus sequence PXXP. 相似文献
12.
We present here a computational, rule-based model to study the function of the SH2 domain-containing protein tyrosine phosphatase, Shp2, in intracellular signal transduction. The two SH2 domains of Shp2 differentially regulate the enzymatic activity by a well-characterized mechanism, but they also affect the targeting of Shp2 to signaling receptors in cells. Our kinetic model integrates these potentially competing effects by considering the intra- and intermolecular interactions of the Shp2 SH2 domains and catalytic site as well as the effect of Shp2 phosphorylation. Even for the isolated Shp2/receptor system, which may seem simple by certain standards, we find that the network of possible binding and phosphorylation states is composed of over 1000 members. To our knowledge, this is the first kinetic model to fully consider the modular, multifunctional structure of a signaling protein, and the computational approach should be generally applicable to other complex intermolecular interactions. 相似文献
13.
The cytoplasmic domain of the Fc gamma receptor IIB (FcgammaRIIB) can be successfully displayed on the surface of filamentous phage, and after phosphorylation in vitro, can interact specifically with the SH2 domains of SHP-2, a cytoplasmic tyrosine phosphatase. When full-length FcgammaRIIB is expressed on phage, however, this interaction is greatly compromised, illustrating that characteristics of the full-length sequence are not well tolerated by the phage display system. Many associations in cell physiology are driven by similar interactions involving small modular binding domains or ligands, and so a fragmented cDNA library will facilitate display of such domains free of sequences which compromise their expression. A fragmented leukocyte cDNA display library of 10(8) clones was constructed. This library was phosphorylated in vitro with fyn kinase and was selected against the tandem SH2 domains of SHP-2 in the search for additional ligands. A depletion strategy to remove non-specific clones was employed, using SHP-2 Sepharose, prior to in vitro phosphorylation and selection. This permitted the emergence of clones encoding the cytoplasmic domain of PECAM-1, another natural ligand for SHP-2. The importance of dual phosphorylation of tyrosine residues at positions 663 and 686 was confirmed in competition ELISA experiments using phosphorylated phage and synthetic peptides. Thus, phage display of fragmented cDNA libraries permits the identification and characterisation of phosphorylated ligands of modular binding domains based on their functional interaction. 相似文献
14.
F Kohlhuber N C Rogers D Watling J Feng D Guschin J Briscoe B A Witthuhn S V Kotenko S Pestka G R Stark J N Ihle I M Kerr 《Molecular and cellular biology》1997,17(2):695-706
Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains. 相似文献
15.
The cellular Bcr protein consists of an N-terminal serine/threonine kinase domain, a central guanine nucleotide exchange factor homology region and a C-terminal GTPase-activating protein domain. Previous work in our laboratory established that Bcr is a major transformation-related substrate for the v-Fps tyrosine kinase, and tyrosine phosphorylation of Bcr induces Bcr-Grb-2/SOS association in vivo through the Src homology 2 (SH2) domain of Grb-2. In the present study, we mapped the region of Bcr tyrosine phosphorylation by c-Fes, the human homologue of v-Fps, to Bcr N-terminal amino acids 162-413 by using a baculovirus/Sf-9 cell co-expression system. Tyrosine phosphorylation of Bcr by Fes greatly enhanced the binding of Bcr to the SH2 domains of multiple signalling molecules in vitro, including Grb-2, Ras GTPase activating protein, phospholipase C-gamma, the 85,000 M(r) subunit of phosphatidylinositol 3'-kinase, and the Abl tyrosine kinase. In contrast with SH2 binding, tyrosine phosphorylation of Bcr reduced its ability to associate with the 14-3-3 protein Bap-1 (Bcr-associated protein-1), a Bcr substrate and member of a family of phosphoserine-binding adaptor proteins. These experiments provide in vitro evidence that tyrosine phosphorylation may modulate the interaction of Bcr with multiple growth-regulatory signalling pathways. 相似文献
16.
Zamal Ahmed Roger George Chi-Chuan Lin Kin Man Suen James A. Levitt Klaus Suhling John E. Ladbury 《Cellular signalling》2010,22(1):23-33
The adaptor protein Grb2 is recruited to intracellular early signalling complexes of many receptor tyrosine kinases and plays an important role transducing signals leading to MAP kinase activation. To date the SH2 domain of Grb2 has been shown to mediate receptor interactions with phosphorylated tyrosine residues sited directly on the receptor or on auxiliary docking proteins. Here we report that FGFR2 recruits Grb2 through its C-terminal SH3 domain. The binding site of this domain was mapped to the proline-rich C-terminus of the receptor. Deletion of the last 10 amino acids of FGFR2 abrogates interaction with Grb2. Synthetic peptides based on the C-terminus of FGFR2 bind to full length Grb2 with low micromolar affinity. The function of this novel mode of Grb2 binding provides resistance to site-specific Shp2-mediated receptor dephosphorylation. 相似文献
17.
At the vertebrate neuromuscular junction (NMJ), postsynaptic aggregation of muscle acetylcholine receptors (AChRs) depends on the activation of MuSK, a muscle-specific tyrosine kinase that is stimulated by neural agrin and regulated by muscle-intrinsic tyrosine kinases and phosphatases. We recently reported that Shp2, a tyrosine phosphatase containing src homology two domains, suppressed MuSK-dependent AChR clustering in cultured myotubes, but how this effect of Shp2 is controlled has remained unclear. In this study, biochemical assays showed that agrin-treatment of C2 mouse myotubes enhanced the tyrosine phosphorylation of signal regulatory protein alpha1 (SIRPalpha1), a known activator of Shp2, and promoted SIRPalpha1's interaction with Shp2. Moreover, in situ experiments revealed that treatment of myotubes with the Shp2-selective inhibitor NSC-87877 increased spontaneous and agrin-induced AChR clustering, and that AChR clustering was also enhanced in myotubes ectopically expressing inactive (dominant-negative) Shp2; in contrast, AChR clustering was reduced in myotubes expressing constitutively active Shp2. Significantly, expression of truncated (nonShp2-binding) and full-length (Shp2-binding) forms of SIRPalpha1 in myotubes also increased and decreased AChR clustering, respectively, and coexpression of truncated SIRPalpha1 with active Shp2 and full-length SIRPalpha1 with inactive Shp2 reversed the actions of the exogenous Shp2 proteins on AChR clustering. These results suggest that SIRPalpha1 is a novel downstream target of MuSK that activates Shp2, which, in turn, suppresses AChR clustering. We propose that an inhibitory loop involving both tyrosine kinases and phosphatases sets the level of agrin/MuSK signaling and constrains it spatially to help generate high-density AChR clusters selectively at NMJs. 相似文献
18.
Borthwick EB Korobko IV Luke C Drel VR Fedyshyn YY Ninkina N Drobot LB Buchman VL 《Journal of molecular biology》2004,343(4):1135-1146
Ruk/CIN85/SETA/CD2BP3 and CD2AP/CMS/METS-1 comprise a new family of proteins involved in such fundamental processes as clustering of receptors and rearrangement of the cytoskeleton in regions of specialised cell-cell contacts, ligand-activated internalisation and targeting to lysosome degradation pathway of receptor tyrosine kinases, and apoptotic cell death. As typical adapter proteins they execute these functions by interacting with other signalling molecules via multiple protein-protein interaction interfaces: SH3 domains, Pro-rich region and coiled-coil domain. It has been previously demonstrated that Ruk is able to interact with the p85alpha regulatory subunit of PI 3-kinase and that the SH3 domain of p85alpha is required for this interaction. However, later observations hinted at a more complex mechanism than simple one-way SH3-Pro-rich interaction. Because interaction with p85alpha was suggested to be important for pro-apoptotic activity of the long isoform of Ruk, Ruk(l)/CIN85, we carried out detailed studies of the mechanism of this interaction and demonstrated that multiple domains are involved; SH3 domains of Ruk are required and sufficient for efficient interaction with full-length p85alpha but the SH3 domain of p85alpha is vital for their "activation" by ousting them from intramolecular interaction with the Pro-rich region of Ruk. Our data also suggest that homodimerisation via C-terminal coiled-coil domain affects both intra- and intermolecular interactions of Ruk proteins. 相似文献
19.
FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors
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Ong SH Guy GR Hadari YR Laks S Gotoh N Schlessinger J Lax I 《Molecular and cellular biology》2000,20(3):979-989
The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases. 相似文献
20.
During T cell signaling, Itk selectively phosphorylates a tyrosine within its own SH3 domain and a tyrosine within PLCgamma1. We find that the remote SH2 domain in each of these substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk and Tec. Additionally, we detect a stable interaction between the substrate SH2 domains and the kinase domain of Itk and find that addition of specific, exogenous SH2 domains to the in vitro kinase assay competes directly with substrate phosphorylation. On the basis of these results, we show that the kinetic parameters of a generic peptide substrate of Itk are significantly improved via fusion of the peptide substrate to the SH2 domain of PLCgamma1. This work is the first characterization of a substrate docking mechanism for the Tec kinases and provides evidence of a novel, phosphotyrosine-independent regulatory role for the ubiquitous SH2 domain. 相似文献