Signaling adapter that couples activated growth factor receptors to signaling pathways. Participates in a signaling cascade initiated by activated KIT and KITLG/SCF.
Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1.
Isoform
p66Shc
Acts as a downstream target of the tumor suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. The expression of isoform p66Shc has been correlated with life span (By similarity). Participates in signaling downstream of the angiopoietin receptor TEK/TIE2, and plays a role in the regulation of endothelial cell migration and sprouting angiogenesis.
Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1.
Isoforms
p52Shc
and
p46Shc
Once phosphorylated, couple activated receptor tyrosine kinases to Ras via the recruitment of the GRB2/SOS complex and are implicated in the cytoplasmic propagation of mitogenic signals.
Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1.
Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1.
Isoform
p66Shc
Does not mediate Ras activation, but is involved in signal transduction pathways that regulate the cellular response to oxidative stress and life span.
Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1.
Eph receptors and their ligands (ephrins) play an important role in axonal guidance, topographic mapping, and angiogenesis. The signaling pathways mediating these activities are starting to emerge and are highly cell- and receptor-type specific. Here we demonstrate that activated EphB1 recruits the adaptor proteins Grb2 and p52Shc and promotes p52Shc and c-Src tyrosine phosphorylation as well as MAPK/extracellular signal-regulated kinase (ERK) activation. EphB1-mediated increase of cell migration was abrogated by the MEK inhibitor PD98059 and Src inhibitor PP2. In contrast, cell adhesion, which we previously showed to be c-jun NH2-terminal kinase (JNK) dependent, was unaffected by ERK1/2 and Src inhibition. Expression of dominant-negative c-Src significantly reduced EphB1-dependent ERK1/2 activation and chemotaxis. Site-directed mutagenesis experiments demonstrate that tyrosines 600 and 778 of EphB1 are required for its interaction with c-Src and p52Shc. Furthermore, phosphorylation of p52Shc by c-Src is essential for its recruitment to EphB1 signaling complexes through its phosphotyrosine binding domain. Together these findings highlight a new aspect of EphB1 signaling, whereby the concerted action of c-Src and p52Shc activates MAPK/ERK and regulates events involved in cell motility.
The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.
Evidence
2:
Inferred from Physical InteractionUniProtKB
J. Biol. Chem. 270, 23258-23262 (1995)[PubMed:7559478]
Insulin receptor substrate 1 (IRS-1) and src homology and collagen protein (SHC) are signaling proteins which are rapidly phosphorylated on tyrosines after insulin receptor (IR) activation. We have recently shown that both SHC and IRS-1 interact with the tyrosine-phosphorylated NPEY motif of the IR and insulin-like growth factor I receptor via non-SH2 domains (Gustafson, T. A., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J. (1995) Mol. Cell. Biol. 15, 2500-2508; O'Neill, T. J., Craparo, A., and Gustafson, T. A. (1994) Mol. Cell. Biol. 14, 6433-6442; Craparo, A., O'Neill, T. J., and Gustafson, T. A. (1995) J. Biol. Chem. 270, 15639-15643). In this study we characterize these interactions by examining the effects of 18 amino acid substitutions within and around the IR NPEY motif upon interaction with SHC and IRS-1. We confirm that Tyr-960 within the NPEY motif of the IR is essential for both IRS-1 and SHC interaction and that Asn-957 and Pro-958 are essential for IRS-1 interaction and important but not critical for SHC interaction. Additional mutations surrounding the NPEY motif revealed completely distinct patterns of interaction for SHC and IRS-1. Specifically, mutation of Leu-952 or Tyr-953 (at positions -7 and -8 from Tyr-960) markedly reduced IRS-1 interaction but had no effect upon SHC interaction. Likewise, mutation of Ala-963 (+3) reduced IRS-1 but not SHC interaction. Conversely, substitution of Leu-961 (+1) with either Ala or Arg reduced SHC interaction by 70 and 90%, respectively, yet had no effect upon interaction with IRS-1. Our data show that the sequences within and surrounding the NPEY contribute differentially to either SHC or IRS-1 recognition. Our findings suggest mechanisms by which the differential interaction of known receptors with IRS-1 and SHC may be mediated.
J. Biol. Chem. 270, 15639-15643 (1995)[PubMed:7541045]
Insulin receptor substrate-1 (IRS-1) and SHC become rapidly phosphorylated upon tyrosines after insulin-like growth factor I receptor (IGFIR) activation. In this study we demonstrate that IRS-1, SHC, and the p85 subunit of phosphatidylinositol 3-kinase interact directly and specifically with the IGFIR. The interaction of all three proteins is dependent upon IGFIR kinase activity and, furthermore, substitution of Tyr-950 with Phe within the NPEY motif of the IGFIR eliminated interaction with both SHC and IRS-1 but had no effect upon p85 interaction. We show that residues 160-516 of IRS-1 and 1-238 of SHC are sufficient and necessary for receptor interaction in the yeast two-hybrid assay. We also demonstrate a direct in vitro interaction between the IGFIR and a fusion protein containing SHC amino acids 1-238. No interaction was observed with a SHC protein containing only the SH2 domain. We conclude that SHC and IRS-1 interact with the tyrosine-phosphorylated NPEY motif of the IGFIR, and that both proteins interact via related motifs located in their amino termini. We conclude that the interactions of SHC and IRS-1 with the IGFIR are similar to those which we have previously defined with the insulin receptor.
We identify a novel alternative TrkA splice variant, TrkAIII, with deletion of exons 6, 7, and 9 and functional extracellular IG-C1 and N-glycosylation domains, that exhibits expression restricted to undifferentiated early neural progenitors, human neuroblastomas (NBs), and a subset of other neural crest-derived tumors. This NGF-unresponsive isoform is oncogenic in NIH3T3 cells and promotes tumorigenic NB cell behavior in vitro and in vivo (cell survival, xenograft growth, angiogenesis) resulting from spontaneous tyrosine kinase activity and IP3K/Akt/NF-kappaB but not Ras/MAPK signaling. TrkAIII antagonizes NGF/TrkAI signaling, which is responsible for NB growth arrest and differentiation through Ras/MAPK, and its expression is promoted by hypoxia at the expense of NGF-responsive receptors, providing a mechanism for converting NGF/TrkA/Ras/MAPK antioncogenic signals to TrkAIII/IP3K/Akt/NF-kappaB tumor-promoting signals during tumor progression.
Tetraspanins is a large family of membrane proteins that are implicated in cell proliferation, differentiation and tumor invasion. Specifically, the tetraspanin CD81 has been involved in cell proliferation but the mechanism is unknown. Here, we show that CD81 clustering stimulates ERK/MAPKinase activity and tyrosine phosphorylation of the adapter protein Shc in Huh7 cancer cells. In addition, overexpression of CD81 in HepG2 cells, NIH3T3 cells, and murine fibroblasts GD25 lacking the beta1 family of integrins induces cell proliferation and ERK/MAPKinase activation. Linked with this event, we observed an increase in CD81-associated type II phosphatidylinositol 4-kinase activity. A mutant in the PTB domain of Shc failed to interact with phosphoinositides and localize to the plasma membrane thus blocking CD81-induced ERK/MAPKinase activation. Therefore, we conclude that CD81 stimulates synthesis of phosphoinositides with the recruitment of Shc to the plasma membrane via PTB domain, and this sequence of events induces activation of ERK/MAPKinase. These findings define a novel mechanism of ERK/MAPKinase activation and tumor cell proliferation.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
Evidence for p52Shc
J. Biol. Chem. 270, 15639-15643 (1995)[PubMed:7541045]
Insulin receptor substrate-1 (IRS-1) and SHC become rapidly phosphorylated upon tyrosines after insulin-like growth factor I receptor (IGFIR) activation. In this study we demonstrate that IRS-1, SHC, and the p85 subunit of phosphatidylinositol 3-kinase interact directly and specifically with the IGFIR. The interaction of all three proteins is dependent upon IGFIR kinase activity and, furthermore, substitution of Tyr-950 with Phe within the NPEY motif of the IGFIR eliminated interaction with both SHC and IRS-1 but had no effect upon p85 interaction. We show that residues 160-516 of IRS-1 and 1-238 of SHC are sufficient and necessary for receptor interaction in the yeast two-hybrid assay. We also demonstrate a direct in vitro interaction between the IGFIR and a fusion protein containing SHC amino acids 1-238. No interaction was observed with a SHC protein containing only the SH2 domain. We conclude that SHC and IRS-1 interact with the tyrosine-phosphorylated NPEY motif of the IGFIR, and that both proteins interact via related motifs located in their amino termini. We conclude that the interactions of SHC and IRS-1 with the IGFIR are similar to those which we have previously defined with the insulin receptor.
Evidence
2:
Inferred from Physical InteractionIntAct
Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded muLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cgamma (PLCgamma), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6.
Evidence
3:
Inferred from Physical InteractionUniProtKB
J. Biol. Chem. 270, 23258-23262 (1995)[PubMed:7559478]
Insulin receptor substrate 1 (IRS-1) and src homology and collagen protein (SHC) are signaling proteins which are rapidly phosphorylated on tyrosines after insulin receptor (IR) activation. We have recently shown that both SHC and IRS-1 interact with the tyrosine-phosphorylated NPEY motif of the IR and insulin-like growth factor I receptor via non-SH2 domains (Gustafson, T. A., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J. (1995) Mol. Cell. Biol. 15, 2500-2508; O'Neill, T. J., Craparo, A., and Gustafson, T. A. (1994) Mol. Cell. Biol. 14, 6433-6442; Craparo, A., O'Neill, T. J., and Gustafson, T. A. (1995) J. Biol. Chem. 270, 15639-15643). In this study we characterize these interactions by examining the effects of 18 amino acid substitutions within and around the IR NPEY motif upon interaction with SHC and IRS-1. We confirm that Tyr-960 within the NPEY motif of the IR is essential for both IRS-1 and SHC interaction and that Asn-957 and Pro-958 are essential for IRS-1 interaction and important but not critical for SHC interaction. Additional mutations surrounding the NPEY motif revealed completely distinct patterns of interaction for SHC and IRS-1. Specifically, mutation of Leu-952 or Tyr-953 (at positions -7 and -8 from Tyr-960) markedly reduced IRS-1 interaction but had no effect upon SHC interaction. Likewise, mutation of Ala-963 (+3) reduced IRS-1 but not SHC interaction. Conversely, substitution of Leu-961 (+1) with either Ala or Arg reduced SHC interaction by 70 and 90%, respectively, yet had no effect upon interaction with IRS-1. Our data show that the sequences within and surrounding the NPEY contribute differentially to either SHC or IRS-1 recognition. Our findings suggest mechanisms by which the differential interaction of known receptors with IRS-1 and SHC may be mediated.
Evidence
4:
Inferred from Physical InteractionIntAct
beta-Amyloid precursor protein (APP) is a widely expressed transmembrane protein of unknown function that is involved in the pathogenesis of Alzheimer's disease. The cytoplasmic tail of APP interacts with phosphotyrosine binding (PTB) domain containing proteins (Fe65, X11, mDab-1, and JIP-1) and may modulate gene expression and apoptosis. We now identify Shc A and Shc C, PTB-containing adapter proteins that signal to cellular differentiation and survival pathways, as novel APP-interacting proteins. The APP cytoplasmic tail contains a PTB-binding motif (Y(682)ENPTY(687)) that, when phosphorylated on Tyr(682), precipitated the PTB domain of Shc A and Shc C, as well as endogenous full-length Shc A. APP and Shc C were physically associated in adult mouse brain homogenates. Increase in phosphorylation of APP by overexpression of the nerve growth factor receptor Trk A in 293T cells promoted the interaction of transfected APP and endogenous Shc A. Pervanadate treatment of N2a neuroblastoma cells resulted in tyrosine phosphorylation and association of endogenous APP and Shc A. Thus, APP and Shc proteins interact in vitro, in cells, and in the mouse brain. Tyrosine phosphorylation of APP may promote the interaction with Shc proteins.
Evidence
5:
Inferred from Physical InteractionUniProtKB
Grb2-associated binder (Gab)2 functions downstream of a variety of receptor and cytoplasmic tyrosine kinases as a docking platform for specific signal transducers and performs important functions in both normal physiology and oncogenesis. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear. We now demonstrate that growth factor-induced phosphorylation of Gab2 on two residues, S210 and T391, leads to recruitment of 14-3-3 proteins. Together, these events mediate negative-feedback regulation, as Gab2(S210A/T391A) exhibits sustained receptor association and signalling and promotes cell proliferation and transformation. Importantly, introduction of constitutive 14-3-3-binding sites into Gab2 renders it refractory to receptor activation, demonstrating that site-selective binding of 14-3-3 proteins is sufficient to terminate Gab2 signalling. Furthermore, this is associated with reduced binding of Grb2. This leads to a model where signal attenuation occurs because 14-3-3 promotes dissociation of Gab2 from Grb2, and thereby uncouples Gab2 from the receptor complex. This represents a novel regulatory mechanism with implications for diverse tyrosine kinase signalling systems.
Evidence
6:
Inferred from Physical InteractionUniProtKB
GRB2, a small protein comprising one SH2 domain and two SH3 domains, represents the human homologue of the Caenorhabditis elegans protein, sem-5. Both GRB2 and sem-5 have been implicated in a highly conserved mechanism that regulates p21ras signalling by receptor tyrosine kinases. In this report we show that in response to insulin, GRB2 forms a stable complex with two tyrosine-phosphorylated proteins. One protein is the major insulin receptor substrate IRS-1 and the second is the SH2 domain-containing oncogenic protein, Shc. The interactions between GRB2 and these two proteins require ligand activation of the insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS-1 and Shc. Although GRB2 associates with IRS-1 and Shc, it is not tyrosine-phosphorylated after insulin stimulation, implying that GRB2 is not a substrate for the insulin receptor. Furthermore, we have identified a short sequence motif (YV/IN) present in IRS-1, EGFR and Shc, which specifically binds the SH2 domain of GRB2 with high affinity. Interestingly, both GRB2 and phosphatidylinositol-3 (PI-3) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS-1 molecule, suggesting a mechanism whereby IRS-1 could provide the core for a large signalling complex. We propose a model whereby insulin stimulation leads to formation of multiple protein--protein interactions between GRB2 and the two targets IRS-1 and Shc. These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules.
Evidence
7:
Inferred from Physical InteractionIntAct
Signaling pathways are commonly organized through inducible protein-protein interactions, mediated by adaptor proteins that link activated receptors to cytoplasmic effectors. However, we have little quantitative data regarding the kinetics with which such networks assemble and dissolve to generate specific cellular responses. To address this deficiency, we designed a mass spectrometry method, affinity purification-selected reaction monitoring (AP-SRM), which we used to comprehensively and quantitatively investigate changes in protein interactions with GRB2, an adaptor protein that participates in a remarkably diverse set of protein complexes involved in multiple aspects of cellular function. Our data reliably define context-specific and time-dependent networks that form around GRB2 after stimulation, and reveal core and growth factor-selective complexes comprising 90 proteins identified as interacting with GRB2 in HEK293T cells. Capturing a key hub protein and dissecting its interactions by SRM should be equally applicable to quantifying signaling dynamics for a range of hubs in protein interaction networks.
Evidence
8:
Inferred from Physical InteractionIntAct
It is prevailingly thought that estrogen signaling is not involved in development of estrogen receptor (ER)-negative breast cancer. However, there is evidence indicating that ovariectomy prevents the development of both ER-positive and -negative breast cancer, suggesting that estrogen signaling is involved in the development of ER-negative breast cancer. Previously, our laboratory cloned a variant of ER-α, ER-α36, and found that ER-α36 mediated nongenomic estrogen signaling and is highly expressed in ER-negative breast cancer cells. In this study, we found that ER-α36 was highly expressed in 10/12 cases of triple-negative breast cancer. We investigated the role of mitogenic estrogen signaling mediated by ER-α36 in malignant growth of triple-negative breast cancer MDA-MB-231 and MDA-MB-436 cells that express high levels of ER-α36 and found that these cells strongly responded to mitogenic estrogen signaling both in vitro and in vivo. Knockdown of ER-α36 expression in these cells using the small hairpin RNA method diminished their responsiveness to estrogen. ER-α36 physically interacted with the EGFR/Src/Shc complex and mediated estrogen-induced phosphorylation of epidermal growth factor receptor (EGFR) and Src. EGFR signaling activated ER-α36 transcription through an AP1 site in the ER-α36 promoter, and ER-α36 expression was able to stabilize EGFR protein. Our results, thus demonstrated that ER-α36 mediates nongenomic estrogen signaling through the EGFR/Src/ERK signaling pathway in ER-negative breast cancer cells and suggested that a subset of ER-negative breast tumors that expresses ER-α36, retains responsiveness to mitogenic estrogen signaling.
Evidence
9:
Inferred from Physical InteractionIntAct
The tyrosine kinase Bcr-Abl causes chronic myeloid leukemia and is the cognate target of tyrosine kinase inhibitors like imatinib. We have charted the protein-protein interaction network of Bcr-Abl by a 2-pronged approach. Using a monoclonal antibody we have first purified endogenous Bcr-Abl protein complexes from the CML K562 cell line and characterized the set of most tightly-associated interactors by MS. Nine interactors were subsequently subjected to tandem affinity purifications/MS analysis to obtain a molecular interaction network of some hundred cellular proteins. The resulting network revealed a high degree of interconnection of 7 "core" components around Bcr-Abl (Grb2, Shc1, Crk-I, c-Cbl, p85, Sts-1, and SHIP-2), and their links to different signaling pathways. Quantitative proteomics analysis showed that tyrosine kinase inhibitors lead to a disruption of this network. Certain components still appear to interact with Bcr-Abl in a phosphotyrosine-independent manner. We propose that Bcr-Abl and other drug targets, rather than being considered as single polypeptides, can be considered as complex protein assemblies that remodel upon drug action.
Evidence
10:
Inferred from Physical InteractionIntAct
Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
Evidence
11:
Inferred from Physical InteractionIntAct
Multiple SRC-family kinases (SFKs) are commonly activated in carcinoma and appear to have a role in metastasis through incompletely understood mechanisms. Recent studies have shown that CDCP1 (CUB (complement C1r/C1s, Uegf, Bmp1) Domain-Containing Protein-1) is a transmembrane protein and an SRC substrate potentially involved in metastasis. Here we show that increased SFK and CDCP1 tyrosine phosphorylation is, surprisingly, associated with a decrease in FAK phosphorylation. This appears to be true in human tumors as shown by our correlation analysis of a mass spectrometric data set of affinity-purified phosphotyrosine peptides obtained from normal and cancer lung tissue samples. Induction of tyrosine phosphorylation of CDCP1 in cell culture, including by a mAb that binds to its extracellular domain, promoted changes in SFK and FAK tyrosine phosphorylation, as well as in PKC(TM), a protein known to associate with CDCP1, and these changes are accompanied by increases in adhesion and motility. Thus, signaling events that accompany the CDCP1 tyrosine phosphorylation observed in cell lines and human lung tumors may explain how the CDCP1/SFK complex regulates motility and adhesion.
Evidence
12:
Inferred from Physical InteractionIntAct
Translationally controlled tumor protein (TCTP) is implicated in cell growth and malignant transformation. TCTP has been found to interact directly with the third cytoplasmic domain of the α subunit of Na,K-ATPase, but whether this interaction has a role in tumorigenesis is unclear. In this study, we examined TCTP-induced tumor progression signaling networks in human breast epithelial cells, using adenoviral infection. We found that TCTP (a) induces Src release from Na,K-ATPase α subunit and Src activation; (b) phosphorylates tyrosine residues 845, 992, 1086, 1148 and 1173 on anti-epidermal growth factor receptor (EGFR); (c) activates PI3K (phosphatidylinositol 3-kinase )-AKT, Ras-Raf-MEK-ERK1/2, Rac-PAK1/2, MKK3/6-p38 and phospholipase C (PLC)-γ pathways; (d) enhances NADPH oxidase-dependent reactive oxygen species (ROS) generation; (e) stimulates cytoskeletal remodeling and cell motility and (f) upregulates matrix metalloproteinase (MMP) 3 and 13. These findings suggest that TCTP induces tumorigenesis through distinct multicellular signaling pathways involving Src-dependent EGFR transactivation, ROS generation and MMP expression.
The binding activity of a molecule that brings together a transmembrane receptor protein tyrosine kinase and one or more other molecules, permitting them to function in a coordinated way.
Tetraspanins is a large family of membrane proteins that are implicated in cell proliferation, differentiation and tumor invasion. Specifically, the tetraspanin CD81 has been involved in cell proliferation but the mechanism is unknown. Here, we show that CD81 clustering stimulates ERK/MAPKinase activity and tyrosine phosphorylation of the adapter protein Shc in Huh7 cancer cells. In addition, overexpression of CD81 in HepG2 cells, NIH3T3 cells, and murine fibroblasts GD25 lacking the beta1 family of integrins induces cell proliferation and ERK/MAPKinase activation. Linked with this event, we observed an increase in CD81-associated type II phosphatidylinositol 4-kinase activity. A mutant in the PTB domain of Shc failed to interact with phosphoinositides and localize to the plasma membrane thus blocking CD81-induced ERK/MAPKinase activation. Therefore, we conclude that CD81 stimulates synthesis of phosphoinositides with the recruitment of Shc to the plasma membrane via PTB domain, and this sequence of events induces activation of ERK/MAPKinase. These findings define a novel mechanism of ERK/MAPKinase activation and tumor cell proliferation.
A process that is carried out at the cellular level which results in dynamic structural changes to the arrangement of constituent parts of cytoskeletal structures comprising actin filaments and their associated proteins.
Tetraspanins is a large family of membrane proteins that are implicated in cell proliferation, differentiation and tumor invasion. Specifically, the tetraspanin CD81 has been involved in cell proliferation but the mechanism is unknown. Here, we show that CD81 clustering stimulates ERK/MAPKinase activity and tyrosine phosphorylation of the adapter protein Shc in Huh7 cancer cells. In addition, overexpression of CD81 in HepG2 cells, NIH3T3 cells, and murine fibroblasts GD25 lacking the beta1 family of integrins induces cell proliferation and ERK/MAPKinase activation. Linked with this event, we observed an increase in CD81-associated type II phosphatidylinositol 4-kinase activity. A mutant in the PTB domain of Shc failed to interact with phosphoinositides and localize to the plasma membrane thus blocking CD81-induced ERK/MAPKinase activation. Therefore, we conclude that CD81 stimulates synthesis of phosphoinositides with the recruitment of Shc to the plasma membrane via PTB domain, and this sequence of events induces activation of ERK/MAPKinase. These findings define a novel mechanism of ERK/MAPKinase activation and tumor cell proliferation.
The process whose specific outcome is the progression of the heart over time, from its formation to the mature structure. The heart is a hollow, muscular organ, which, by contracting rhythmically, keeps up the circulation of the blood.
An intracellular protein kinase cascade containing at least a MAPK, a MAPKK and a MAP3K. The cascade can also contain two additional tiers: the upstream MAP4K and the downstream MAP Kinase-activated kinase (MAPKAPK). The kinases in each tier phosphorylate and activate the kinases in the downstream tier to transmit a signal within a cell.
Tetraspanins is a large family of membrane proteins that are implicated in cell proliferation, differentiation and tumor invasion. Specifically, the tetraspanin CD81 has been involved in cell proliferation but the mechanism is unknown. Here, we show that CD81 clustering stimulates ERK/MAPKinase activity and tyrosine phosphorylation of the adapter protein Shc in Huh7 cancer cells. In addition, overexpression of CD81 in HepG2 cells, NIH3T3 cells, and murine fibroblasts GD25 lacking the beta1 family of integrins induces cell proliferation and ERK/MAPKinase activation. Linked with this event, we observed an increase in CD81-associated type II phosphatidylinositol 4-kinase activity. A mutant in the PTB domain of Shc failed to interact with phosphoinositides and localize to the plasma membrane thus blocking CD81-induced ERK/MAPKinase activation. Therefore, we conclude that CD81 stimulates synthesis of phosphoinositides with the recruitment of Shc to the plasma membrane via PTB domain, and this sequence of events induces activation of ERK/MAPKinase. These findings define a novel mechanism of ERK/MAPKinase activation and tumor cell proliferation.
Tetraspanins is a large family of membrane proteins that are implicated in cell proliferation, differentiation and tumor invasion. Specifically, the tetraspanin CD81 has been involved in cell proliferation but the mechanism is unknown. Here, we show that CD81 clustering stimulates ERK/MAPKinase activity and tyrosine phosphorylation of the adapter protein Shc in Huh7 cancer cells. In addition, overexpression of CD81 in HepG2 cells, NIH3T3 cells, and murine fibroblasts GD25 lacking the beta1 family of integrins induces cell proliferation and ERK/MAPKinase activation. Linked with this event, we observed an increase in CD81-associated type II phosphatidylinositol 4-kinase activity. A mutant in the PTB domain of Shc failed to interact with phosphoinositides and localize to the plasma membrane thus blocking CD81-induced ERK/MAPKinase activation. Therefore, we conclude that CD81 stimulates synthesis of phosphoinositides with the recruitment of Shc to the plasma membrane via PTB domain, and this sequence of events induces activation of ERK/MAPKinase. These findings define a novel mechanism of ERK/MAPKinase activation and tumor cell proliferation.
A new SH2-containing sequence, SHC, was isolated by screening cDNA libraries with SH2 representative DNA probes. The SHC cDNA is predicted to encode overlapping proteins of 46.8 and 51.7 kd that contain a single C-terminal SH2 domain, and an adjacent glycine/proline-rich motif with regions of homology with the alpha 1 chain of collagen, but no identifiable catalytic domain. Anti-SHC antibodies recognized three proteins of 46, 52, and 66 kd in a wide range of mammalian cell lines. These SHC proteins complexed with and were phosphorylated by activated epidermal growth factor receptor. The physical association of SHC proteins with activated receptors was recreated in vitro by using a bacterially expressed SHC SH2 domain. NIH 3T3 mouse fibroblasts that constitutively overexpressed SHC acquired a transformed phenotype in culture and formed tumors in nude mice. These results suggest that the SHC gene products couple activated growth factor receptors to a signaling pathway that regulates the proliferation of mammalian cells.
Any process that modulates the frequency, rate or extent of the growth of all or part of an organism so that it occurs at its proper speed, either globally or in a specific part of the organism's development.
IEAUniProtKB KW
Pathways
According to KEGG, this protein belongs to the following pathways:
Bacterial invasion of epithelial cells hsa05100+6464
Protein involved in angiogenesis, the sprouting or splitting of capillaries from pre-existing vasculature. Angiogenesis plays an important role for example during embryonic development, normal growth of tissues and maintenance of the normal vasculature, wound healing, tumor growth and metastasis.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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