Adapter protein that interacts with the cytoplasmic domain of numerous receptor kinases and modulates down-stream signaling. Promotes activation of down-stream protein kinases, including STAT3, AKT1, MAPK1 and/or MAPK3. Promotes activation of HRAS. Plays a role in signal transduction in response to EGF. Plays a role in the regulation of cell proliferation and cell migration. Plays a role in the assembly and stability of RNA stress granules. Binds to the 5'UTR of target mRNA molecules and represses translation of target mRNA species, when not phosphorylated. Phosphorylation impairs RNA binding and promotes stress granule disassembly during recovery after cellular stress (By similarity).
EphB1 is a member of the Eph family of receptor tyrosine kinases that play important roles in diverse biological processes including nervous system development, angiogenesis, and neural synapsis formation and maturation. Grb7 is an adaptor molecule implicated in the regulation of cell migration. Here we report identification of an interaction between Grb7 and the cytoplasmic domain of EphB1 by using Grb7 as a "bait" in a yeast two-hybrid screening. Co-immunoprecipitation was used to confirm the interaction of Grb7 with the cytoplasmic domain of EphB1 as well as the full-length receptor in intact cells. This interaction is mediated by the SH2 domain of Grb7 and requires tyrosine autophosphorylation of EphB1. Furthermore, Tyr-928 of EphB1 was identified as the primary binding site for Grb7. Stimulation of endogenous EphB1 in embryonal carcinoma P19 cells with its ligand ephrinB1 increased its association with Grb7, which is consistent with a role for the autophosphorylation of EphB1. We also found that EphB1 could phosphorylate Grb7 and mutation of either Tyr-928 or Tyr-594 to Phe decreased this activity. Finally, we show that EphB1 could stimulate fibroblast motility on extracellular matrix in a kinase-dependent manner, which also correlated with its association with Grb7. Consistent with this, co-expression of Grb7 with EphB1 further enhanced cell motility, whereas co-expression of the Grb7 SH2 domain abolished EphB1-stimulated cell migration. Together, our results identified a novel interaction between EphB1 with the adaptor molecule Grb7 and suggested that this interaction may play a role in the regulation of cell migration by EphB1.
We have previously described Grb7 association with focal adhesion kinase (FAK) and its possible roles in cell migration. In this paper, we investigated the mechanisms by which Grb7 and its association with FAK regulate cell migration. We found that deletion of the Grb7 SH2 domain eliminated partial Grb7 localization to focal contacts and its ability to stimulate cell migration. Replacement of the SH2 domain with the focal adhesion targeting sequence from FAK resulted in the focal contacts localization of the chimeric molecule and restored its activity to stimulate cell migration. We also found that Grb7 could be phosphorylated by FAK, which was dependent on the FAK kinase activity but not the presence of the Src family kinases. Cell adhesion also enhanced Grb7 phosphorylation in FAK+/+ cells but not FAK-/- cells, suggesting that Grb7 is a physiological substrate of FAK. Furthermore, both Grb7 and the chimeric molecule did not increase migration of FAK-/- cells, although the chimeric molecule was targeted to the focal contacts. Last, we showed that other Grb7 family members could not stimulate cell migration under similar experimental conditions. Together, these results demonstrate a role for Grb7 targeting to focal contacts and its phosphorylation by FAK in the regulation of cell migration.
Grb7 is the prototype of a family of adaptor molecules that also include Grb10 and Grb14 that share a conserved molecular architecture including Src homology 2 (SH2) and pleckstrin homology (PH) domains. Grb7 has been implicated as a downstream mediator of integrin-FAK signal pathways in the regulation of cell migration, although the molecular mechanisms are still not well understood. In this paper, we investigated the potential role and mechanisms of PH domain in Grb7 in the regulation of cell migration. We found that the PH domain mediated Grb7 binding to phospholipids both in vitro and in intact cells. Furthermore, both Grb7 and its PH domain preferentially interacted with phosphatidylinositol phosphates showing strongest affinity to the D3- and D5-phosphoinositides. The PH domain interaction with phosphoinositides was shown to play a role in the stimulation of cell migration by Grb7. It was also shown to be necessary for Grb7 phosphorylation by FAK, although it was not required for Grb7 interaction with FAK or recruitment to the focal contacts. Last, we found that PI 3-kinase activity played a role in both Grb7 association with phosphoinositides and its stimulation of cell migration. In addition, both FAK binding to PI 3-kinase via its autophosphorylated Tyr(397) and integrin-mediated cell adhesion increased Grb7 association with phosphoinositides. Together, these results identified the Grb7 PH domain interaction with phosphoinositides and suggested a potential mechanism by which several signaling molecules including Grb7, FAK, and PI 3-kinase and their interactions cooperate to mediate signal transduction pathways in integrin-mediated cell migration.
Co-amplification and co-overexpression of ErbB2 and Grb7 are frequently found in various cancers, including breast cancer. Biochemical and functional correlations of the two molecules have identified Grb7 to be a pivotal mediator downstream of ErbB2-mediated oncogenesis. However, it remains largely unknown how Grb7 is involve in the ErbB2-mediated tumorigenesis. In this study, we show that Grb7-mediated cell proliferation and growth are essential for the tumorigenesis that occurs in ErbB2-Grb7-overexpressing breast cancer cells. Intrinsically, EGF-induced de novo Grb7 tyrosine phosphorylation/activation recruits and activates Ras-GTPases and subsequently promotes the phosphorylation of ERK1/2, thereby stimulating tumor growth. Furthermore, we also found the anti-tumor effect could be synergized by co-treatment with Herceptin plus Grb7 knockdown in Sk-Br3 breast cancer cells. Our findings illustrate an underlying mechanism by which Grb7 promotes tumorigenesis through the formation of a novel EGFR-Grb7-Ras signaling complex, thereby highlighting the potential strategy of targeting Grb7 as an anti-breast cancer therapy.
Growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is co-overexpressed and forms a tight complex with the ErbB2 receptor in a number of breast tumours and breast cancer cell lines. The interaction of Grb7 with the ErbB2 receptor is mediated via its Src homology 2 (SH2) domain. Whilst most SH2 domains exist as monomers, recently reported studies have suggested that the Grb7-SH2 domain exists as a homodimer. The self-association properties of the Grb7-SH2 domain were therefore studied using sedimentation equilibrium ultracentrifugation. Analysis of the data demonstrated that the Grb7-SH2 domain is dimeric with a dissociation constant of approximately 11 muM. We also demonstrate, using size-exclusion chromatography, that mutation of phenylalanine 511 to an arginine produces a monomeric form of the Grb7-SH2 domain. This mutation represents the first step in the engineering of a Grb7-SH2 domain with good solution properties for further biophysical and structural investigation.
Interacting selectively and non-covalently with any inositol-containing glycerophospholipid, i.e. phosphatidylinositol (PtdIns) and its phosphorylated derivatives.
Grb7 is the prototype of a family of adaptor molecules that also include Grb10 and Grb14 that share a conserved molecular architecture including Src homology 2 (SH2) and pleckstrin homology (PH) domains. Grb7 has been implicated as a downstream mediator of integrin-FAK signal pathways in the regulation of cell migration, although the molecular mechanisms are still not well understood. In this paper, we investigated the potential role and mechanisms of PH domain in Grb7 in the regulation of cell migration. We found that the PH domain mediated Grb7 binding to phospholipids both in vitro and in intact cells. Furthermore, both Grb7 and its PH domain preferentially interacted with phosphatidylinositol phosphates showing strongest affinity to the D3- and D5-phosphoinositides. The PH domain interaction with phosphoinositides was shown to play a role in the stimulation of cell migration by Grb7. It was also shown to be necessary for Grb7 phosphorylation by FAK, although it was not required for Grb7 interaction with FAK or recruitment to the focal contacts. Last, we found that PI 3-kinase activity played a role in both Grb7 association with phosphoinositides and its stimulation of cell migration. In addition, both FAK binding to PI 3-kinase via its autophosphorylated Tyr(397) and integrin-mediated cell adhesion increased Grb7 association with phosphoinositides. Together, these results identified the Grb7 PH domain interaction with phosphoinositides and suggested a potential mechanism by which several signaling molecules including Grb7, FAK, and PI 3-kinase and their interactions cooperate to mediate signal transduction pathways in integrin-mediated cell migration.
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
Grb7 is a member of a family of molecular adapters which are able to contribute positively but also negatively to signal transduction and whose precise roles remain obscure. Rnd1 is a member of the Rho family, but, as opposed to usual GTPases, it is constitutively bound to GTP. We show here that Rnd1 and Grb7 interact, in two-hybrid assays, in vitro, and in pull-down experiments performed with SK-BR3, a breast cancer cell line that overexpresses Grb7. This interaction involves switch II loop of Rnd1, a region crucial for guanine nucleotide exchange in all GTPases, and a Grb7 SH2 domain, a region crucial for Grb7 interaction with several activated receptors. The contribution of the interaction between Rnd1 and Grb7 to their respective functions and properties is discussed.
Evidence
2:
Inferred from Physical InteractionIntAct
EphB1 is a member of the Eph family of receptor tyrosine kinases that play important roles in diverse biological processes including nervous system development, angiogenesis, and neural synapsis formation and maturation. Grb7 is an adaptor molecule implicated in the regulation of cell migration. Here we report identification of an interaction between Grb7 and the cytoplasmic domain of EphB1 by using Grb7 as a "bait" in a yeast two-hybrid screening. Co-immunoprecipitation was used to confirm the interaction of Grb7 with the cytoplasmic domain of EphB1 as well as the full-length receptor in intact cells. This interaction is mediated by the SH2 domain of Grb7 and requires tyrosine autophosphorylation of EphB1. Furthermore, Tyr-928 of EphB1 was identified as the primary binding site for Grb7. Stimulation of endogenous EphB1 in embryonal carcinoma P19 cells with its ligand ephrinB1 increased its association with Grb7, which is consistent with a role for the autophosphorylation of EphB1. We also found that EphB1 could phosphorylate Grb7 and mutation of either Tyr-928 or Tyr-594 to Phe decreased this activity. Finally, we show that EphB1 could stimulate fibroblast motility on extracellular matrix in a kinase-dependent manner, which also correlated with its association with Grb7. Consistent with this, co-expression of Grb7 with EphB1 further enhanced cell motility, whereas co-expression of the Grb7 SH2 domain abolished EphB1-stimulated cell migration. Together, our results identified a novel interaction between EphB1 with the adaptor molecule Grb7 and suggested that this interaction may play a role in the regulation of cell migration by EphB1.
Evidence
3:
Inferred from Physical InteractionIntAct
We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243-256) predicted to form an alpha-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Delta and Grb7VDelta) have less capacity to be associated to membranes. Grb7Delta maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin beta1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Delta instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Delta and Grb7VDelta lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.
Evidence
4:
Inferred from Physical InteractionIntAct
Adaptor proteins mediate signal transduction from cell surface receptors to downstream signaling pathways. The Grb7 protein family of adaptor proteins is constituted by Grb7, Grb10, and Grb14. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7-mediated cell migration has been shown to proceed through a focal adhesion kinase (FAK)/Grb7 pathway, although the specific participants downstream of Grb7 in cell migration signaling have not been fully determined. In this study, we report that Grb7 interacts with Hax-1, a cytoskeletal-associated protein found overexpressed in metastatic tumors and cancer cell lines. Additionally, in yeast 2-hybrid assays, we show that the interaction is specific to the Grb7-RA and -PH domains. We have also demonstrated that full-length Grb7 and Hax-1 interact in mammalian cells and that Grb7 is tyrosine phosphorylated. Isothermal titration calorimetry measurements demonstrate the Grb7-RA-PH domains bind to the Grb7-SH2 domain with micromolar affinity, suggesting full-length Grb7 can exist in a head-to-tail conformational state that could serve a self-regulatory function.
Interacting selectively and non-covalently with a protein kinase, any enzyme that catalyzes the transfer of a phosphate group, usually from ATP, to a protein substrate.
Grb7 is the prototype of a family of adaptor molecules that also include Grb10 and Grb14 that share a conserved molecular architecture including Src homology 2 (SH2) and pleckstrin homology (PH) domains. Grb7 has been implicated as a downstream mediator of integrin-FAK signal pathways in the regulation of cell migration, although the molecular mechanisms are still not well understood. In this paper, we investigated the potential role and mechanisms of PH domain in Grb7 in the regulation of cell migration. We found that the PH domain mediated Grb7 binding to phospholipids both in vitro and in intact cells. Furthermore, both Grb7 and its PH domain preferentially interacted with phosphatidylinositol phosphates showing strongest affinity to the D3- and D5-phosphoinositides. The PH domain interaction with phosphoinositides was shown to play a role in the stimulation of cell migration by Grb7. It was also shown to be necessary for Grb7 phosphorylation by FAK, although it was not required for Grb7 interaction with FAK or recruitment to the focal contacts. Last, we found that PI 3-kinase activity played a role in both Grb7 association with phosphoinositides and its stimulation of cell migration. In addition, both FAK binding to PI 3-kinase via its autophosphorylated Tyr(397) and integrin-mediated cell adhesion increased Grb7 association with phosphoinositides. Together, these results identified the Grb7 PH domain interaction with phosphoinositides and suggested a potential mechanism by which several signaling molecules including Grb7, FAK, and PI 3-kinase and their interactions cooperate to mediate signal transduction pathways in integrin-mediated cell migration.
Interacting selectively and non-covalently and simultaneously with one or more signal transduction molecules, usually acting as a scaffold to bring these molecules into close proximity either using their own SH2/SH3 domains (e.g. Grb2) or those of their target molecules (e.g. SAM68).
The cDNAs of a putative growth factor-bound (Grb) 7 signal transduction molecule and Grb7V novel splice variant were isolated from an invasive human esophageal carcinoma. Although both Grb7 isoforms share homology with the Mig-10 cell migration gene, the Grb7V isoform lacks 88 base pairs in the C terminus; the resultant frame shift leads to substitution of an SH2 domain with a short hydrophobic sequence. The wild-type Grb7 protein, but not the Grb7V isoform, is rapidly tyrosyl phosphorylated in response to EGF stimulation in esophageal carcinoma cells. Analysis of human esophageal tumor tissues and regional lymph nodes with metastases revealed that Grb7V was expressed in 40% of Grb7-positive esophageal carcinomas. More importantly, Grb7V expression was enhanced after metastatic spread to lymph nodes as compared to the original tumor tissues. Finally, transfection of an antisense Grb7 RNA expression construct lowered endogenous Grb7 protein levels and suppressed the invasive phenotype exhibited by esophageal carcinoma cells. These findings suggest that Grb7 isoforms are involved in cell invasion and metastatic progression of human esophageal carcinomas.
A series of molecular signals initiated by binding of a ligand to the tyrosine kinase receptor EGFR (ERBB1) on the surface of a cell. The pathway ends with regulation of a downstream cellular process, e.g. transcription.
The cDNAs of a putative growth factor-bound (Grb) 7 signal transduction molecule and Grb7V novel splice variant were isolated from an invasive human esophageal carcinoma. Although both Grb7 isoforms share homology with the Mig-10 cell migration gene, the Grb7V isoform lacks 88 base pairs in the C terminus; the resultant frame shift leads to substitution of an SH2 domain with a short hydrophobic sequence. The wild-type Grb7 protein, but not the Grb7V isoform, is rapidly tyrosyl phosphorylated in response to EGF stimulation in esophageal carcinoma cells. Analysis of human esophageal tumor tissues and regional lymph nodes with metastases revealed that Grb7V was expressed in 40% of Grb7-positive esophageal carcinomas. More importantly, Grb7V expression was enhanced after metastatic spread to lymph nodes as compared to the original tumor tissues. Finally, transfection of an antisense Grb7 RNA expression construct lowered endogenous Grb7 protein levels and suppressed the invasive phenotype exhibited by esophageal carcinoma cells. These findings suggest that Grb7 isoforms are involved in cell invasion and metastatic progression of human esophageal carcinomas.
Any process that stops, prevents, or reduces the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of proteins by the translation of mRNA.
Grb7 is the prototype of a family of adaptor molecules that also include Grb10 and Grb14 that share a conserved molecular architecture including Src homology 2 (SH2) and pleckstrin homology (PH) domains. Grb7 has been implicated as a downstream mediator of integrin-FAK signal pathways in the regulation of cell migration, although the molecular mechanisms are still not well understood. In this paper, we investigated the potential role and mechanisms of PH domain in Grb7 in the regulation of cell migration. We found that the PH domain mediated Grb7 binding to phospholipids both in vitro and in intact cells. Furthermore, both Grb7 and its PH domain preferentially interacted with phosphatidylinositol phosphates showing strongest affinity to the D3- and D5-phosphoinositides. The PH domain interaction with phosphoinositides was shown to play a role in the stimulation of cell migration by Grb7. It was also shown to be necessary for Grb7 phosphorylation by FAK, although it was not required for Grb7 interaction with FAK or recruitment to the focal contacts. Last, we found that PI 3-kinase activity played a role in both Grb7 association with phosphoinositides and its stimulation of cell migration. In addition, both FAK binding to PI 3-kinase via its autophosphorylated Tyr(397) and integrin-mediated cell adhesion increased Grb7 association with phosphoinositides. Together, these results identified the Grb7 PH domain interaction with phosphoinositides and suggested a potential mechanism by which several signaling molecules including Grb7, FAK, and PI 3-kinase and their interactions cooperate to mediate signal transduction pathways in integrin-mediated cell migration.
We have previously described Grb7 association with focal adhesion kinase (FAK) and its possible roles in cell migration. In this paper, we investigated the mechanisms by which Grb7 and its association with FAK regulate cell migration. We found that deletion of the Grb7 SH2 domain eliminated partial Grb7 localization to focal contacts and its ability to stimulate cell migration. Replacement of the SH2 domain with the focal adhesion targeting sequence from FAK resulted in the focal contacts localization of the chimeric molecule and restored its activity to stimulate cell migration. We also found that Grb7 could be phosphorylated by FAK, which was dependent on the FAK kinase activity but not the presence of the Src family kinases. Cell adhesion also enhanced Grb7 phosphorylation in FAK+/+ cells but not FAK-/- cells, suggesting that Grb7 is a physiological substrate of FAK. Furthermore, both Grb7 and the chimeric molecule did not increase migration of FAK-/- cells, although the chimeric molecule was targeted to the focal contacts. Last, we showed that other Grb7 family members could not stimulate cell migration under similar experimental conditions. Together, these results demonstrate a role for Grb7 targeting to focal contacts and its phosphorylation by FAK in the regulation of cell migration.
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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