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.     

The adaptor protein Gab1 couples the stimulation of vascular endothelial growth factor receptor-2 to the activation of phosphoinositide 3-kinase

Phosphoinositide 3-kinase (PI3K) mediates essential functions of vascular endothelial growth factor (VEGF), including the stimulation of endothelial cell proliferation and migration. Nevertheless, the mechanisms coupling the receptor VEGFR-2 to PI3K remain obscure. We observed that the Grb2-bound adapter Gab1 is tyrosine-phosphorylated and relocated to membrane fractions upon VEGF stimulation of endothelial cells. We could detect the PI3K regulatory subunit p85 in immunoprecipitates of endogenous Gab1, and vice versa, and measure a Gab1-associated lipid kinase activity upon VEGF stimulation. Furthermore, transfection of the Gab1-YF3 mutant lacking all p85-binding sites strongly repressed PI3K activation measured in vitro. Moreover, Gab1-YF3 severely decreased the cellular amount of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generated in response to VEGF. Furthermore, adenoviral expression of Gab1-YF3 suppressed both Akt phosphorylation and recovery of wounded human umbilical vein endothelial cell monolayers, a VEGF-dependent process involving cell migration and proliferation under PI3K control. Transfection of other Gab1 mutants, lacking Grb2-binding sites or the pleckstrin homology (PH) domain, also prevented Akt activation, further demonstrating Gab1 involvement in PI3K activation. These mutants were also used to show that interactions with both Grb2 and PtdIns(3,4,5)P3 mediate Gab1 recruitment by VEGFR-2. Importantly, Gab1 mobilization was impaired by (i) PI3K inhibitors, (ii) deletion of Gab1 PH domain, (iii) PTEN (phosphatase and tensin homolog deleted on chromosome 10) overexpression to repress PtdIns(3,4,5)P3 production, and (iv) overexpression of a competitor PH domain for PtdIns(3,4,5)P3 binding, which altogether demonstrated that PI3K is also an upstream regulator of Gab1. Gab1 thus appears as a primary actor in coupling VEGFR-2 to PI3K/Akt, recruited through an amplification loop involving PtdIns(3,4,5)P3 and its PH domain.     

Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action

The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism.     

Differential roles of PI3-kinase and Kit tyrosine 821 in Kit receptor-mediated proliferation, survival and cell adhesion in mast cells.

The pleiotropic effects of the Kit receptor system are mediated by Kit-Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3'-kinase (PI3-kinase), p21ras and mitogen-activated protein kinase (MAPK). To define the role of PI3-kinase, p21ras and MAPK in Kit-mediated cell proliferation, survival and adhesion in bone marrow-derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c-kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL-induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3-kinase, p21ras and MAPK, and induced expression of c-fos, junB, c-myc and c-myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3-kinase activation, diminished c-fos and junB induction, and impaired KL-induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3-kinase, p21ras and MAPK activation, and induction of c-myc, c-myb, c-fos and c-jun mRNA, while KL-induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3-kinase in Kit-mediated cell adhesion, wortmannin blocked Kit-mediated cell adhesion at concentrations known to specifically inhibit PI3-kinase. We conclude, that association of Kit with p85PI3-K, and thus with PI3-kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit-mediated mitogenesis and survival, but not cell adhesion.     

New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway

Most, if not all, cytokines activate phosphatidylinositol 3-kinase (PI-3K). Although many cytokine receptors have direct binding sites for the p85 subunit of PI-3K, others, such as the interleukin-3 (IL-3) receptor beta common chain (betac) and the IL-2 receptor beta chain (IL-2Rbeta), lack such sites, leaving the mechanism by which they activate PI-3K unclear. Here, we show that the protooncoprotein Shc, which promotes Ras activation by recruiting the Grb2-Sos complex in response to stimulation of cytokine stimulation, also signals to the PI-3K/Akt pathway. Analysis of Y-->F and "add-back" mutants of betac shows that Y577, the Shc binding site, is the major site required for Gab2 phosphorylation in response to cytokine stimulation. When fused directly to a mutant form of IL-2Rbeta that lacks other cytoplasmic tyrosines, Shc can promote Gab2 tyrosyl phosphorylation. Mutation of the three tyrosyl phosphorylation sites of Shc, which bind Grb2, blocks the ability of the Shc chimera to evoke Gab2 tyrosyl phosphorylation. Overexpression of mutants of Grb2 with inactive SH2 or SH3 domains also blocks cytokine-stimulated Gab2 phosphorylation. The majority of cytokine-stimulated PI-3K activity associates with Gab2, and inducible expression of a Gab2 mutant unable to bind PI-3K markedly impairs IL-3-induced Akt activation and cell growth. Experiments with the chimeric receptors indicate that Shc also signals to the PI-3K/Akt pathway in response to IL-2. Our results suggest that cytokine receptors lacking direct PI-3K binding sites activate Akt via a Shc/Grb2/Gab2/PI-3K pathway, thereby regulating cell survival and/or proliferation.     

Akt activation through the phosphorylation of erythropoietin receptor at tyrosine 479 is required for myeloproliferative disorder-associated JAK2 V617F mutant-induced cellular transformation

The disruption of Janus kinase 2 (JAK2) signaling regulation by its point mutation, V617F, is involved in various myeloproliferative disorders (MPDs). JAK2 V617F mutant induced constitutive activation of Akt when erythropoietin receptor (EpoR) was coexpressed; however, the physiological role of Akt activation in MPDs has not been elucidated. LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, inhibited Akt activation and induced apoptotic cell death in cells expressing JAK2 V617F mutant and EpoR. Previously, it has been shown that the phosphorylation at Y479 in EpoR is critical for the interaction with PI3K, an upstream molecule of Akt. Hence, EpoR mutant with a point mutation of Y479F, which fails to activate Akt, is useful for addressing the role of Akt activation in JAK2 V617F mutant-induced tumorigenesis. Interestingly, under the expression of EpoR Y479F mutant, JAK2 V617F mutant failed to exhibit potent anti-apoptotic activity. In addition, JAK2 V617F mutant-induced phosphorylation of CREB and GSK-3β was significantly decreased in cells expressing EpoR Y479F mutant, resulting in the downregulation of Bcl-XL and Mcl-1 expression. Furthermore, compared with when nude mice were inoculated with cells expressing JAK2 V617F mutant and EpoR, the lifespan of nude mice inoculated with cells expressing JAK2 V617F mutant and EpoR Y479F mutant was effectively prolonged. Taken together, it was clarified that PI3K-Akt activation through the phosphorylation of EpoR at Y479 is required for oncogenic signaling of JAK2 V617F mutant and that targeted disruption of this pathway has therapeutic utility.     

The adaptor protein Grb14 regulates the localization of 3-phosphoinositide-dependent kinase-1

The metabolic actions of insulin are transduced through the phosphatidylinositol 3-kinase pathway. A critical component of this pathway is 3-phosphoinositide-dependent kinase-1 (PDK-1), a PH domain-containing enzyme that catalyzes the activating phosphorylation for many AGC kinases, including Akt and protein kinase C isozymes. We used a directed proteomics-based approach to identify the adaptor protein Grb14, which binds the insulin receptor through an SH2 domain, as a novel PDK-1 binding partner. Interaction of these two proteins is constitutive and mediated by a PDK-1 binding motif on Grb14. Disruption of this motif by point mutation or deletion of the Grb14 SH2 domain prevents the insulin-triggered membrane translocation of PDK-1. The interaction of PDK-1 with Grb14 facilitates Akt function: disruption of the interaction by overexpression of a construct of Grb14 mutated in the PDK-1 binding motif significantly decreases insulin-dependent activation of Akt. Thus, Grb14 serves as an adaptor protein to recruit PDK-1 to activated insulin receptor, thus promoting Akt phosphorylation and transduction of the insulin signal.     

Scaffolding adapter Grb2-associated binder 2 requires Syk to transmit signals from FcepsilonRI

Scaffolding adapter Grb2-associated binder 2 (Gab2) is a key component of FcepsilonRI signaling in mast cells, required for the activation of PI3K. To understand how Gab2 is activated in FcepsilonRI signaling, we asked which protein tyrosine kinase is required for Gab2 phosphorylation. We found that Gab2 tyrosyl phosphorylation requires Lyn and Syk. In agreement with published results, we found that Fyn also contributes to Gab2 tyrosyl phosphorylation. However, Syk activation is defective in Fyn(-/-) mast cells, suggesting that Syk is the proximal kinase responsible for Gab2 tyrosyl phosphorylation. Then, we asked which domains in Gab2 are required for Gab2 tyrosyl phosphorylation. We found that the Grb2-Src homology 3 (SH3) binding sites are required for, whereas the pleckstrin homology (PH) domain contributes to, Gab2 tyrosyl phosphorylation. Using a protein/lipid overlay assay, we determined that the Gab2 PH domain preferentially binds the PI3K lipid products, PI3, 4,5P3 and PI3, 4P2. Furthermore, the Grb2-SH3 binding sites and PH domain binding to PI3K lipid products are required for Gab2 function in FcepsilonRI-evoked degranulation and Akt activation. Our data strongly suggest a model for Gab2 action in FcepsilonRI signaling. The Grb2 SH3 binding sites play a critical role in bringing Gab2 to FcepsilonRI, whereupon Gab2 becomes tyrosyl-phosphorylated in a Syk-dependent fashion. Phosphorylated Gab2 results in recruitment and activation of PI3K, whose lipid products bind the PH domain of Gab2 and acts in positive feedback loop for sustained PI3K recruitment and phosphatidylinositol-3,4,5-trisphosphate production, required for FcepsilonRI-evoked degranulation of mast cells.     

Molecular and cellular analysis of Grb2 SH3 domain mutants: interaction with Sos and dynamin.

Quantitative analysis of Grb2/dynamin interaction through plasmon resonance analysis (BIAcore) using Grb2 mutants showed that the high affinity measured between Grb2 and dynamin is essentially mediated by the N-SH3 domain of Grb2. In order to study the interactions between Grb2 and either dynamin or Sos in more detail, Grb2 N-SH3 domains containing different mutations have been analysed. Two mutations were located on the hydrophobic platform binding proline-rich peptides (Y7V and P49L) and one (E40T) located in a region that we had previously shown to be essential for Grb2/dynamin interactions. Through NMR analysis, we have clearly demonstrated that the structure of the P49L mutant is not folded, while the other E40T and Y7V mutants adopt folded structures that are quite similar to that described for the reference domain. Nevertheless, these point mutations were shown to alter the overall stability of these domains by inducing an equilibrium between a folded and an unfolded form. The complex formed between the peptide VPPPVPPRRR, derived from Sos, and the E40T mutant was shown to have the same 3D structure as that described for the wild-type SH3 domain. However, the VPPPVPPRRR peptide adopts a slightly different orientation when it is complexed with the Y7V mutant. Finally, the affinity of the proline-rich peptide GPPPQVPSRPNR, derived from dynamin, for the Grb2 N-SH3 domain was too low to be analyzed by NMR. Thus, the interaction between either Sos or dynamin and the SH3 mutants were tested on a cellular homogenate by means of a far-Western blot analysis. In these conditions, the P49L mutant was shown to be devoid of affinity for Sos as well as for dynamin. The Y7V SH3 mutant displayed a decrease of affinity for both Sos and dynamin, while the E40T mutant exhibited a decrease of affinity only for dynamin. These results support the existence of two binding sites between dynamin and the Grb2 N-SH3 domain.     

Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor

Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain. We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.     

In brain, Axl recruits Grb2 and the p85 regulatory subunit of PI3 kinase; in vitro mutagenesis defines the requisite binding sites for downstream Akt activation

Axl is a receptor tyrosine kinase implicated in cell survival following growth factor withdrawal and other stressors. The binding of Axl's ligand, growth arrest-specific protein 6 (Gas6), results in Axl autophosphorylation, recruitment of signaling molecules, and activation of downstream survival pathways. Pull-down assays and immunoprecipitations using wildtype and mutant Axl transfected cells determined that Axl directly binds growth factor receptor-bound protein 2 (Grb2) at pYVN and the p85 subunit of phosphatidylinositol-3 kinase (PI3 kinase) at two pYXXM sites (pY779 and pY821). Also, p85 can indirectly bind to Axl via an interaction between p85's second proline-rich region and the N-terminal SH3 domain of Grb2. Further, Grb2 and p85 can compete for binding at the pY821VNM site. Gas6-stimulation of Axl-transfected COS7 cells recruited activated PI3 kinase and phosphorylated Akt. An interaction between Axl, p85 and Grb2 was confirmed in brain homogenates, enriched populations of O4+ oligodendrocytes, and O4− flow-through prepared from day 10 mouse brain, indicating that cells with active Gas6/Axl signal through Grb2 and the PI3 kinase/Akt pathways.     

PIKE GTPase-mediated nuclear signalings promote cell survival

The nuclear GTPase PIKE (PI 3-kinase Enhancer) binds PI 3-kinase and enhances it lipid kinase activity. PIKE predominantly distributes in the brain, and nerve growth factor stimulation triggers PIKE activation by provoking nuclear translocation of PLC-gamma1, which acts as a physiologic guanine nucleotide exchange factor (GEF) for PIKE through its SH3 domain. PIKE contains GTPase and ArfGAP domains, which are separated by a PH domain. C-terminal ArfGAP domain activates its internal GTPase activity, and this process is regulated by the interaction between phosphatidylinositols and PH domain. PI 3-kinase occurs in the nuclei of a broad range of cell types, and various stimuli elicit its nuclear translocation. The nuclei from NGF-treated PC12 cells are resistant to DNA fragmentation initiated by activated cell-free apoptosome, for which PIKE/nuclear PI 3-kinase signaling through nuclear PI(3,4,5)P(3) and Akt plays an essential role. As a nuclear receptor for PI(3,4,5)P(3,) B23 binds to PI(3,4,5)P(3) in an NGF-dependent way. The PI(3,4,5)P(3)/B23 complex inhibits DNA fragmentation activity of CAD. Nuclear Akt regulation of apoptosis is dependent on its phosphorylation of key substrates in the nucleus, but the identities of these substrates are unknown. Identification of its nuclear substrates will further our understanding of the physiological roles of nuclear PI 3-kinase/Akt signaling.     

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.     

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.     

Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling.

SH2-containing inositol 5'-phosphatase (SHIP) plays a negative regulatory role in hematopoietic cells. We have now cloned the rat SHIP isozyme (SHIP2) cDNA from skeletal muscle, which is one of the most important target tissue of insulin action. Rat SHIP2 cDNA encodes a 1183-amino-acid protein that is 45% identical with rat SHIP. Rat SHIP2 contains an amino-terminal SH2 domain, a central 5'-phosphoinositol phosphatase activity domain, and a phosphotyrosine binding (PTB) consensus sequence and a proline-rich region at the carboxyl tail. Specific antibodies to SHIP2 were raised and the function of SHIP2 was studied by stably overexpressing rat SHIP2 in Rat1 fibroblasts expressing human insulin receptors (HIRc). Endogenous SHIP2 underwent insulin-mediated tyrosine phosphorylation and phosphorylation was markedly increased when SHIP2 was overexpressed. Although overexpression of SHIP2 did not affect insulin-induced tyrosine phosphorylation of the insulin receptor beta-subunit and Shc, subsequent association of Shc with Grb2 was inhibited, possibly by competition between the SH2 domains of SHIP2 and Grb2 for the Shc phosphotyrosine. As a result, insulin-stimulated MAP kinase activation was reduced in SHIP2-overexpressing cells. Insulin-induced tyrosine phosphorylation of IRS-1, IRS-1 association with the p85 subunit of PI3-kinase, and PI3-kinase activation were not affected by overexpression of SHIP2. Interestingly, although both PtdIns-(3,4,5)P3 and PtdIns(3,4)P2 have been implicated in the regulation of Akt activity in vitro, overexpression of SHIP2 inhibited insulin-induced Akt activation, presumably by its 5'-inositol phosphatase activity. Furthermore, insulin-induced thymidine incorporation was decreased by overexpression of SHIP2. These results indicate that SHIP2 plays a negative regulatory role in insulin-induced mitogenesis, and regulation of the Shc. Grb2 complex and of the downstream products of PI3-kinase provides possible mechanisms of SHIP2 action in insulin signaling.     

Structural Basis for the Interaction of the Adaptor Protein Grb14 with Activated Ras

Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region, and a C-terminal Src-homology-2 (SH2) domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V). The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM) and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.     

Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells.

T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One of the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal protein containing multiple proline-rich motifs that are potential binding sites for proteins containing Src homology 3 (SH3) domains. We report here that in cultured Jurkat T cells, Cbl is coprecipitated with antibody against the adapter protein Grb2. Upon activation of Jurkat T cells via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but after cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally to its SH2 domain. Grb2 antisera also precipitated p85 from serum-starved cells, while TCR activation increased p85 and tyrosine-phosphorylated Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinositol (PI) 3-kinase activity was immunoprecipitated from serum-starved cells with Cbl and to a lesser extent with Grb2 antisera, and TCR cross-linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activity that could be precipitated by p85 antisera. The Ras exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitates from activated cells, and Cbl was not detectable in anti-Sos-1 precipitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are present as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation.     

Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase

Proximal signaling events and protein-protein interactions initiated after activation of the c-Ret receptor tyrosine kinase by its ligand, glial cell line-derived neurotrophic factor (GDNF), were investigated in cells carrying native and mutated forms of this receptor. Mutation of Tyr-1062 (Y1062F) in the cytoplasmic tail of c-Ret abolished receptor binding and phosphorylation of the adaptor Shc and eliminated activation of Ras by GDNF. Phosphorylation of Erk kinases was also greatly attenuated but not eliminated by this mutation. This residual wave of Erk phosphorylation was independent of the kinase activity of c-Ret. Mutation of Tyr-1096 (Y1096F), a binding site for the adaptor Grb2, had no effect on Erk activation by GDNF. Activation of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt was also reduced in the Y1062F mutant but not completely abolished unless Tyr-1096 was also mutated. Ligand stimulation of neuronal cells induced the assembly of a large protein complex containing c-Ret, Grb2, and tyrosine-phosphorylated forms of Shc, p85(PI3K), the adaptor Gab2, and the protein-tyrosine phosphatase SHP-2. In agreement with Ras-independent activation of PI3K by GDNF in neuronal cells, survival of sympathetic neurons induced by GDNF was dependent on PI3K but was not affected by microinjection of blocking anti-Ras antibodies, which did compromise neuronal survival by nerve growth factor, suggesting that Ras is not required for GDNF-induced survival of sympathetic neurons. These results indicate that upon ligand stimulation, at least two distinct protein complexes assemble on phosphorylated Tyr-1062 of c-Ret via Shc, one leading to activation of the Ras/Erk pathway through recruitment of Grb2/Sos and another to the PI3K/Akt pathway through recruitment of Grb2/Gab2 followed by p85(PI3K) and SHP-2. This latter complex can also assemble directly onto phosphorylated Tyr-1096, offering an alternative route to PI3K activation by GDNF.     

The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor.