Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.     

Differential regulation of cell migration and cell cycle progression by FAK complexes with Src, PI3K, Grb7 and Grb2 in focal contacts.

Focal adhesion kinase (FAK) is a key mediator of integrin signaling, which has been implicated in the regulation of cell migration and cell cycle progression. Using chimeric molecules that fuse the focal adhesion targeting (FAT) sequence directly to several signaling molecules, we investigated the potential role of FAK recruitments of signaling molecules to focal contacts in the regulation of cell migration and cell cycle progression. We found that fusion of FAT to Src, the p85 subunit of phosphatidylinositol 3-kinase, Grb7 and Grb2 resulted in the efficient focal adhesion targeting of these signaling molecules. We showed that expression of Src-FAT, p85-FAT, or Grb7-FAT, but not Grb2-FAT, each stimulated cell migration. Interestingly, tyrosine phosphorylation of paxillin, but not p130cas, was induced by expression of Src-FAT, suggesting a potential role of paxillin in mediating stimulation of cell migration by the chimeric molecule. In contrast, targeting of Grb2, but not Src, p85, or Grb7, to focal contacts increased cell cycle progression. Biochemical analyses correlated Erk activation by Grb2-FAT with its stimulation of cell cycle progression. Together, these results suggest that at least part of the role of FAK interaction with these signaling molecules is to recruit them to focal contacts and that distinct FAK signaling complexes are involved in the regulation of cell migration vs. cell cycle progression.     

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.     

Association of Grb7 with phosphoinositides and its role 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.     

Role of Grb7 targeting to focal contacts and its phosphorylation by focal adhesion kinase in regulation of 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.     

Analysis of FAK-associated signaling pathways in the regulation of cell cycle progression

Focal adhesion kinase (FAK) is an important mediator of signal transduction pathways initiated by integrins in cell migration, survival and cell cycle regulation. The ability of FAK to mediate integrin signaling in the regulation of cell cycle progression depends on the phosphorylation of Tyr397, which implies a functional significance for the formation of FAK signaling complexes with Src, phosphatidylinositol-3-kinase (PI3K) and Grb7. We have previously described a FAK mutant, D395A, that selectively disrupts FAK binding to PI3K, but allows FAK association with Src. Using this mutation in a mislocalized FAK mutant background, we show here that formation of a FAK/PI3K complex is not sufficient for cell cycle progression but the formation of a FAK/Src complex plays an essential role. We also show that mutation of D395 to A disrupted FAK association with Grb7. This suggests that a FAK/Grb7 complex is not involved in the cell cycle regulation either, which is supported by direct analysis of cells expressing a dominant negative Grb7 construct. Finally, we provide evidence that the Src-dependent association of FAK with Grb2 and p130(Cas) are both required for the regulation of cell cycle progression by FAK. Together, these studies identify important FAK downstream signaling pathways in cell cycle regulation.     

The v-Src SH3 domain facilitates a cell adhesion-independent association with focal adhesion kinase

Integrins facilitate cell attachment to the extracellular matrix, and these interactions generate cell survival, proliferation, and motility signals. Integrin signals are relayed in part by focal adhesion kinase (FAK) activation and the formation of a transient signaling complex initiated by Src homology 2 (SH2)-dependent binding of Src family protein-tyrosine kinases to the FAK Tyr-397 autophosphorylation site. Here we show that in viral Src (v-Src)-transformed NIH3T3 fibroblasts, an adhesion-independent FAK-Src signaling complex occurs. Co-expression studies in human 293T cells showed that v-Src could associate with and phosphorylate a Phe-397 FAK mutant at Tyr-925 promoting Grb2 binding to FAK in suspended cells. In vitro, glutathione S-transferase fusion proteins of the v-Src SH3 but not c-Src SH3 domain bound to FAK in lysates of NIH3T3 fibroblasts. The v-Src SH3-binding sites were mapped to known proline-X-X-proline (PXXP) SH3-binding motifs in the FAK N- (residues 371-377) and C-terminal domains (residues 712-718 and 871-882) by in vitro pull-down assays, and these sites are composed of a PXXPXXPhi (where Phi is a hydrophobic residue) v-Src SH3 binding consensus. Sequence comparisons show that residues in the RT loop region of the c-Src and v-Src SH3 domains differ. Substitution of c-Src RT loop residues (Arg-97 and Thr-98) for those found in the v-Src SH3 domain (Trp-97 and Ile-98) enhanced the binding of distinct NIH3T3 cellular proteins to a glutathione S-transferase fusion protein of the c-Src (Trp-97 + Ile-98) SH3 domain. FAK was identified as a c-Src (Trp-97 + Ile-98) SH3 domain target in fibroblasts, and co-expression studies in 293T cells showed that full-length c-Src (Trp-97 + Ile-98) could associate in vivo with Phe-397 FAK in an SH2-independent manner. These studies establish a functional role for the v-Src SH3 domain in stabilizing an adhesion-independent signaling complex with FAK.     

Identification of p130Cas as a Mediator of Focal Adhesion Kinase–promoted Cell Migration

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130^Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130^Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130^Cas and its subsequent binding to several SH2 domains, which depended on both the p130^Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130^Cas further increased cell migration on FN and coexpression of the p130^Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130^Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130^Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.     

SRC catalytic but not scaffolding function is needed for integrin-regulated tyrosine phosphorylation, cell migration, and cell spreading

Src family kinases (SFKs) are crucial for signaling through a variety of cell surface receptors, including integrins. There is evidence that integrin activation induces focal adhesion kinase (FAK) autophosphorylation at Y397 and that Src binds to and is activated by FAK to carry out subsequent phosphorylation events. However, it has also been suggested that Src functions as a scaffolding molecule through its SH2 and SH3 domains and that its kinase activity is not necessary. To examine the role of SFKs in integrin signaling, we have expressed various Src molecules in fibroblasts lacking other SFKs. In cells plated on fibronectin, FAK could indeed autophosphorylate at Y397 independently of Src but with lower efficiency than when Src was present. This step was promoted by kinase-inactive Src, but Src kinase activity was required for full rescue. Src kinase activity was also required for phosphorylation of additional sites on FAK and for other integrin-directed functions, including cell migration and spreading on fibronectin. In contrast, Src mutations in the SH2 or SH3 domain greatly reduced binding to FAK, Cas, and paxillin but had little effect on tyrosine phosphorylation or biological assays. Furthermore, our indirect evidence indicates that Src kinase activity does not need to be regulated to promote cell migration and FAK phosphorylation. Although Src clearly plays important roles in integrin signaling, it was not concentrated in focal adhesions. These results indicate that the primary role of Src in integrin signaling is as a kinase. Indirect models for Src function are proposed.     

Grb2 Promotes Integrin-Induced Focal Adhesion Kinase (FAK) Autophosphorylation and Directs the Phosphorylation of Protein Tyrosine Phosphatase α by the Src-FAK Kinase Complex

The integrin-activated Src-focal adhesion kinase (FAK) kinase complex phosphorylates PTPα at Tyr789, initiating PTPα-mediated signaling that promotes cell migration. Recruitment of the BCAR3-Cas complex by PTPα-phospho-Tyr789 at focal adhesions is one mechanism of PTPα signaling. The adaptor protein Grb2 is also recruited by PTPα-phospho-Tyr789, although the role of the PTPα-Grb2 complex in integrin signaling is unknown. We show that silencing Grb2 expression in fibroblasts abolishes PTPα-Tyr789 phosphorylation and that this is due to two unexpected actions of Grb2. First, Grb2 promotes integrin-induced autophosphorylation of FAK-Tyr397. This is impaired in Grb2-depleted cells and prohibits FAK activation and formation of the Src-FAK complex. Grb2-depleted cells contain less paxillin, and paxillin overexpression rescues FAK-Tyr397 phosphorylation, suggesting that the FAK-activating action of Grb2 involves paxillin. A second distinct role for Grb2 in PTPα-Tyr789 phosphorylation involves Grb2-mediated coupling of Src-FAK and PTPα. This requires two phosphosites, FAK-Tyr925 and PTPα-Tyr789, for Grb2-Src homology 2 (SH2) binding. We propose that a Grb2 dimer links FAK and PTPα, and this positions active Src-FAK in proximity with other, perhaps integrin-clustered, molecules of PTPα to enable maximal PTPα-Tyr789 phosphorylation. These findings identify Grb2 as a new FAK activator and reveal its essential role in coordinating PTPα tyrosine phosphorylation to enable downstream integrin signaling and migration.     

Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells.

Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated to play a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. In this study, we show that Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. We find that WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds significantly stronger than the C-terminal SH3 domain to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein Nck, remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton.     

Grb7 binds to Hax‐1 and undergoes an intramolecular domain association that offers a model for Grb7 regulation

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. Copyright © 2010 John Wiley & Sons, Ltd.     

Grb7 signal transduction protein mediates metastatic progression of esophageal carcinoma

We have previously reported the association of tumor cell invasion with expression of growth factor receptor-bound protein 7 (Grb7). This molecule contains a Src homology 2 (SH2) domain and shares structural homology with a cell migration molecule designated Mig-10 found in Caenorhabditis elegans. In the present study, Grb7 expression was analyzed in human esophageal carcinomas with or without metastatic spread. The Grb7 protein was overexpressed in 14 of 31 esophageal carcinomas as compared to the adjacent normal mucosa (45%) and this finding was significantly correlated with the presence of lymph node metastases. We also identified that Grb7 protein in esophageal carcinoma cells was phosphorylated on tyrosine by epidermal growth factor as well as attachment to extracellular matrix proteins including fibronectin. Such fibronectin-dependent phosphorylation of Grb7 was regulated by integrin signaling that leads to the interaction with focal adhesion kinase protein. Furthermore, ectopic expression of a Grb7-SH2 dominant-negative fragment inhibited the fibronectin-dependent phosphorylation of endogenous Grb7, and reduced migration of esophageal carcinoma cells into fibronectin. Our results suggest a role of Grb7 mediated signal transduction in generation of an invasive cell phenotype against extracellular matrix, and thus contributes to metastatic progression of human esophageal carcinoma.     

Grb7 protein RA domain oligomerization

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl‐1, Mec‐3 (LIM) domains isoform 2 and filamin α. These interactions are believed to play a role in regulating Grb7‐mediated cell migration function. The full‐length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the ras associating and pleckstrin homology domains, and the importance of this oligomerization in Grb7 function, is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, nuclear magnetic resonance, nuclear relaxation studies, glutaraldehyde cross linking, and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.     

The BPS domain of Grb10 inhibits the catalytic activity of the insulin and IGF1 receptors

Grb7, Grb10 and Grb14 comprise a family of adaptor proteins that interact with numerous receptor tyrosine kinases upon receptor activation. Between the pleckstrin homology (PH) domain and the Src homology 2 (SH2) domain of these proteins is a region of approximately 50 residues known as the BPS (between PH and SH2) domain. Here we show, using purified recombinant proteins, that the BPS domain of Grb10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and the insulin-like growth factor 1 (IGF1) receptor. Although inhibition by the BPS domain is dependent on tyrosine phosphorylation of the kinase activation loop, peptide competition experiments indicate that the BPS domain does not bind directly to phosphotyrosine. These studies provide a molecular mechanism by which Grb10 functions as a negative regulator of insulin- and/or IGF1-mediated signaling.     

The adapter protein Crkl links Cbl to C3G after integrin ligation and enhances cell migration

Crkl, an SH2-SH3-SH3 adapter protein, is one of the major tyrosine phosphoproteins detected in cells from patients with chronic myelogenous leukemia. Crkl binds to BCR/ABL through its N-terminal SH3 domain and is known to interact with several signaling proteins that have been implicated in integrin signaling, including Cbl, Cas, Hef-1, and paxillin. We have previously shown that overexpression of Crkl enhances adhesion to extracellular matrix proteins through beta(1) integrins. In this study, the effects of Crkl on spontaneous and chemokine-directed migration of the hematopoietic cell line Ba/F3 were examined. Full-length, SH2-, and SH3(N)-domain deletion mutants of Crkl were expressed transiently as fusion proteins with green fluorescent protein. Successfully transfected cells were isolated by fluorescence-activated cell sorting. The ability of these cells to migrate across a fibronectin-coated membrane, either spontaneously or in response to the chemokine stromal-derived factor-1alpha, was determined. Cells expressing green fluorescent protein alone were not distinguishable from untransfected or mock transfected Ba/F3 cells. However, Ba/F3 cells overexpressing full-length Crkl were found to have an increase in spontaneous migration of 2.8 +/- 0.6-fold in seven independent assays. The enhancement of migration required both the SH2 domain and the N-terminal SH3 domain. Migration in response to stromal-derived factor-1alpha was not significantly enhanced by overexpression of Crkl. Overexpression of Crkii also augmented spontaneous migration but to a lesser degree than did Crkl. Because the SH2 domain was required for enhanced migration, we looked for changes in phosphotyrosine containing proteins coprecipitating with Crkl, but not Crkl DeltaSH2, after integrin cross-linking. Full-length Crkl, but not CrklDeltaSH2, coprecipitated with a single major tyrosine phosphoprotein with an M(r) of approximately 120 kDa, identified as Cbl. The major Crkl SH3-binding protein in these cells was found to be the guanine nucleotide exchange factor, C3G. Interestingly, overexpression of C3G also enhanced migration, suggesting that a Cbl-Crkl-C3G complex may be involved in migration signaling in Ba/F3 cells. These data suggest that Crkl is involved in signaling pathways that regulate migration, possibly through a complex with Cbl and C3G.     

Structural basis for dimerization of the Grb10 Src homology 2 domain. Implications for ligand specificity

Grb7, Grb10, and Grb14 are members of a distinct family of adapter proteins that interact with various receptor tyrosine kinases upon receptor activation. Proteins in this family contain several modular signaling domains including a pleckstrin homology (PH) domain, a BPS (between PH and SH2) domain, and a C-terminal Src homology 2 (SH2) domain. Although SH2 domains are typically monomeric, we show that the Grb10 SH2 domain and also full-length Grb10 gamma are dimeric in solution under physiologic conditions. The crystal structure of the Grb10 SH2 domain at 1.65-A resolution reveals a non-covalent dimer whose interface comprises residues within and flanking the C-terminal alpha helix, which are conserved in the Grb7/Grb10/Grb14 family but not in other SH2 domains. Val-522 in the BG loop (BG3) and Asp-500 in the EF loop (EF1) are positioned to interfere with the binding of the P+3 residue of a phosphopeptide ligand. These structural features of the Grb10 SH2 domain will favor binding of dimeric, turn-containing phosphotyrosine sequences, such as the phosphorylated activation loops in the two beta subunits of the insulin and insulin-like growth factor-1 receptors. Moreover, the structure suggests the mechanism by which the Grb7 SH2 domain binds selectively to pTyr-1139 (pYVNQ) in Her2, which along with Grb7 is co-amplified in human breast cancers.     

Association of fibroblast growth factor receptor 1 with the adaptor protein Grb14. Characterization of a new receptor binding partner

Using the cytoplasmic domain of fibroblast growth factor receptor 1 (FGFR1) as bait in a yeast two-hybrid screen, Grb14 was identified as a FGFR1 binding partner. A kinase-inactive mutant of FGFR1 failed to interact with Grb14, indicating that activation of FGFR1 is necessary for binding. Deletion of the C-tail or mutation of both C-tail tyrosine residues of FGFR1 to phenylalanine abolished binding, and deletion of the juxtamembrane domain of the receptor reduced binding, suggesting that Grb14 binds to FGFR1 at multiple sites. Co-immunoprecipitation and in vitro binding assays demonstrated that binding of Grb14 to FGFR1 in mammalian cells was dependent on receptor activation by fibroblast growth factor-2 (FGF-2). Deletion of the Src homology 2 (SH2) domain of Grb14 reduced but did not block binding to FGFR1 and eliminated dependence on receptor activation. The SH2 domain alone bound both FGFR1 and platelet-derived growth factor receptor, whereas full-length Grb14 bound only FGFR1, suggesting that regions upstream of the SH2 domain confer specificity for FGFR1. Grb14 was phosphorylated on serine and threonine residues in unstimulated cells, and treatment with FGF-2 enhanced this phosphorylation. Expression of exogenous Grb14 inhibited FGF-2-induced cell proliferation, whereas a point-mutated form of Grb14 incapable of binding to FGFR1 enhanced FGF-2-induced mitogenesis. These data demonstrate an interaction between activated FGFR1 and Grb14 and suggest a role for Grb14 in FGF signaling.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

p21-activated kinase 1 (PAK1) interacts with the Grb2 adapter protein to couple to growth factor signaling

A variety of intracellular signaling pathways are linked to cell surface receptor signaling through their recruitment by Src homology 2 (SH2)/SH3-containing adapter molecules. p21-activated kinase 1 (PAK1) is an effector of Rac/Cdc42 GTPases that has been implicated in the regulation of cytoskeletal dynamics, proliferation, and cell survival signaling. In this study, we describe the specific interaction of PAK1 with the Grb2 adapter protein both in vitro and in vivo. We identify the site of this interaction as the second proline-rich SH3 binding domain of PAK1. Stimulation of the epidermal growth factor receptor (EGFR) in HaCaT cells enhances the level of EGFR-associated PAK1 and Grb2, although the PAK1-Grb2 association is itself independent of this stimulation. A cell-permeant TAT-tagged peptide encompassing the second proline-rich SH3 binding domain of PAK1 simultaneously blocked Grb2 and activated EGFR association with PAK1, in vitro and in vivo, indicating that Grb2 mediates the interaction of PAK1 with the activated EGFR. Blockade of this interaction decreased the epidermal growth factor-induced extension of membrane lamellae. Thus Grb2 may serve as an important mechanism for linking downstream PAK signaling to various upstream pathways.