Ras oncogene at 85D


EVOLUTIONARY HOMOLOGS

Table of contents

Ras and Grb-2, a vertebrate SH3: SH2: SH3 adaptor protein homologous to Drosophila Drk

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. In rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signaling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and also stimulates in turn mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells (Rozakis-Adcock, 1993).

Endothelin-1 (ET-1) induces cell proliferation and differentiation through multiple G-protein-linked signaling systems, including p21(ras) activation. Whereas p21(ras) activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21(ras) activation induced by engagement of G-protein-coupled receptors remains to be fully elucidated. ET-1 induces a biphasic activation of p21(ras) in rat glomerular mesangial cells. The first peak of activation of p21(ras), at 2-5 min, is mediated by immediate association of phosphorylated Shc with the guanosine exchange factor Sos1 via the adaptor protein Grb2. This initial activation of p21(ras) results in activation of the extracellular signal-regulated kinase (ERK) cascade. ET-1 signaling elicits a negative feedback mechanism, modulating p21(ras) activity through ERK-dependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21(ras) and ERK deactivation, Sos1 reverts to the non-phosphorylated condition, enabling it to bind again to the Grb2/Shc complex, which is stabilized by persistent Shc phosphorylation. However, the resulting secondary activation of p21(ras) at 30 min does not lead to ERK activation, correlating with intensive, ET-1-induced expression of MAP kinase phosphatase-1, but does result in increased p21(ras)-associated phosphatidylinositol 3-kinase activity. These data provide evidence that ET-1-induced biphasic p21(ras) activation causes sequential stimulation of divergent downstream signaling pathways (Foschi, 1997).

GRB2/Sem-5 is a 25-kDa adaptor protein which contains a central Src homology type 2 (SH2) domain flanked by two Src homology type 3 (SH3) domains. GRB2/Sem-5 was first identified due to the essential role of the sem-5 gene product in the vulval induction pathway in Caenorhabditis elegans. The SH2 domain of GRB2/Sem-5 binds to a number of tyrosine phosphorylated proteins, most notably the epidermal growth factor receptor, the insulin receptor substrate IRS-1 and another putative adaptor protein, Shc. The SH3 domains bind to Sos, a guanine nucleotide exchange factor for Ras proteins. GRB2/Sem-5 brings together Sos and tyrosine phosphoproteins into a complex and thereby may regulate the nucleotide exchange rate of Ras and hence its activation state (Downward, 1994).

The recently cloned Ash/Grb-2 protein, a 25-28 kDa molecule composed entirely of SH2 and SH3 domains, is a mammalian homolog of the C. elegans Sem-5 protein, which communicates between a receptor protein tyrosine kinase and a Ras protein. In the present study the function of Ash/Grb-2 was investigated by microinjecting cells with an anti-Ash antibody. The antibody abolishes both S phase entry and the reorganization of actin assembly to ruffle formation upon stimulation with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). In contrast, anti-Ash antibody had no effect on S phase entry or actin stress fiber formation induced by either serum or lysophosphatidic acid. Since the induction of DNA synthesis, ruffle induction and stress fiber formation involve the respective function of Ras, Rac activation and Rho activation, the findings strongly suggest that Ash plays a critical role in the signaling of both pathways downstream from growth factor receptors to Ras and Rac. Consistent with this, Ash co-precipitated with EGF receptor from EGF-stimulated cells. Other proteins of approximately 21, 29, 135 and 160 kDa were also detected in the anti-Ash antibody immunoprecipitates, suggesting a role of Ash as a linker molecule in signal transduction downstream of growth factor receptors (Matuoka, 1993).

A lipid-anchored Grb2-binding protein links FGF-receptor activation to the Ras/MAPK signaling pathway. Activation of the Ras/MAPK signaling cascade is essential for growth factor-induced cell proliferation and differentiation. A novel protein, designated FRS2 is tyrosine phosphorylated and binds to Grb2/Sos in response to FGF or NGF stimulation. FRS2 is myristylated and this modification is essential for membrane localization, tyrosine phosphorylation, Grb2/Sos recruitment, and MAPK activation. FRS2 functions as a lipid-anchored docking protein that targets signaling molecules to the plasma membrane in response to FGF stimulation to link receptor activation with the MAPK and other signaling pathways essential for cell growth and differentiation. FRS2 is closely related and probably indentical to SNT (suc1-associated neurotrophic factor target), the long-sought target of FGF and NGF receptors (Kouhara, 1997).

In the case of growth factors such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), the steps leading to activation of MAPK require the function of the adaptor protein Grb2 (growth factor receptor binding protein 2), which can bind either directly or indirectly via its Src homology 2 domain to activated receptor tyrosine kinases. A cell-permeable mimetic of the EGF receptor Grb2 binding site has been investigated for its ability to inhibit biological responses stimulated by a variety of growth factors. Pretreatment of cells with this peptide results in the accumulation of the peptide in cells and its association with Grb2. This is associated with a complete inhibition of the mitogenic response stimulated by EGF and PDGF. In contrast, the peptide has no effect on the mitogenic response stimulated by fibroblast growth factor. The peptide could also inhibit the phosphorylation of MAPK stimulated with EGF and PDGF in the absence of an effect on the fibroblast growth factor response. These data demonstrate that cell-permeable mimetics of Src homology 2 binding sites can selectively inhibit growth factor-stimulated mitogenesis, and also directly demonstrate specificity in the coupling of activated receptor tyrosine kinases to the MAPK cascade (Williams, 1997).

Antibody- or laminin-induced ligation of a laminin receptor involved in morphogenesis and tumor progression, the hemidesmosomal integrin alpha 6 beta 4, causes tyrosine phosphorylation of the beta 4 subunit in intact cells. This event is mediated by a protein kinase(s) physically associated with the integrin. Co-immunoprecipitation and GST fusion protein binding experiments show that the adaptor protein Shc forms a complex with the tyrosine-phosphorylated beta 4 subunit. Shc is then phosphorylated on tyrosine residues and recruits the adaptor Grb2, thereby potentially linking alpha 6 beta 4 to the ras pathway. The beta 4 subunit is phosphorylated at multiple tyrosine residues in vivo, including a tyrosine-based activation motif (TAM) resembling those found in T and B cell receptors. Phenylalanine substitutions at the beta 4 TAM disrupt association of alpha 6 beta 4 with hemidesmosomes, but do not interfere with tyrosine phosphorylation of Shc and recruitment of Grb2. Signal transduction by the alpha 6 beta 4 integrin is mediated by an associated tyrosine kinase and phosphorylation of distinct sites in the beta 4 tail mediate assembly of the hemidesmosomal cytoskeleton and recruitment of Shc/Grb2 (Mainiero, 1995).

The signaling pathways linking integrins to nuclear events are incompletely understood. Intracellular signaling by the alpha6beta4 integrin has been studied. alpha6beta4 is a laminin receptor expressed in basal keratinocytes and other cells. Ligation of alpha6beta4 in primary human keratinocytes causes tyrosine phosphorylation of Shc, recruitment of Grb2, activation of Ras and stimulation of the MAP kinases Erk and Jnk. beta4 is known to be phosphorylated by integrin associated kinase. In contrast, ligation of the laminin- and collagen-binding integrins alpha3beta1 and alpha2beta1 does not cause these events. While the stimulation of Erk by alpha6beta4 is suppressed by dominant-negative Shc, Ras and RhoA, the activation of Jnk is inhibited by dominant-negative Ras and Rac1 and by the phosphoinositide 3-kinase inhibitor Wortmannin. Adhesion mediated by alpha6beta4 induces transcription from the Fos serum response element and promoted cell cycle progression in response to mitogens. In contrast, alpha3beta1- and alpha2beta1-dependent adhesion did not induce these events. These findings suggest that the coupling of alpha6beta4 integrin to the control of cell cycle progression mediated by Shc regulates the proliferation of basal keratinocytes and possibly other cells that are in contact with the basement membrane in vivo. The results of this study support the notion that the recruitment of Shc to the alpha6beta4 is mediated by the cytoplasmic domain of beta4 (Mainiero, 1997).

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. The nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK: Drosophila homolog Focal adhesion kinase-like) can be independently activated after fibronectin (FN) stimulation; their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction. The time course of FN-stimulated ERK2 activation parallels that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibits FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibits c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 result in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity is not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promotes Shc binding to FAK. In vitro, FAK directly phosphorylates Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc is observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promotes Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc is tyrosine phosphorylated after FN stimulation of human 293T cells. Its expression does not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also does not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 is inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. These studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation (Schlaepfer, 1998).

Focal adhesion kinase (FAK) overexpression enhances ras-dependent integrin signaling to ERK2/mitogen-activated protein kinase through interactions with and activation of c-Src. Focal adhesion kinase associates with integrin receptors, and FN-stimulated phosphorylation of FAK at Tyr-397 and Tyr-925 promotes the binding of Src family protein tyrosine kinases (PTKs) and Grb2, respectively. To investigate the mechanisms by which FAK, c-Src, and Grb2 function in Fibronectin-stimulated signaling events to ERK2, wild type and mutant forms of FAK were expressed in human 293 epithelial cells by transient transfection. FAK overexpression enhances FN-stimulated activation of ERK2 approximately 4-fold. This is blocked by co-expression of the dominant negative Asn-17 mutant Ras, indicating that FN stimulation of ERK2 is Ras-dependent. FN-stimulated c-Src PTK activity is enhanced by wild type FAK expression, whereas FN-stimulated activation of ERK2 is blocked by expression of the c-Src binding site Phe-397 mutant of FAK. Expression of the Grb2 binding site Phe-925 mutant of FAK enhances activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK does not. Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enhances p130(Cas) association with FAK, Shc tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation. FN-induced Grb2-Shc association is another pathway leading to activation of ERK2 via Ras. The inhibitory effects of Tyr-397 FAK expression show that FAK-mediated association and activation of c-Src is essential for maximal signaling to ERK2. Moreover, multiple signaling pathways are activated upon the formation of a FAK.c-Src complex, and several of these can lead to Ras-dependent ERK2 mitogen-activated protein kinase activation (Schlaepfer, 1997).

B-cell antigen receptor (BCR) stimulation induces tyrosine phosphorylation of the Shc adaptor protein and BCF's association with Grb2. The Shc/Grb2 complex may be involved in Ras activation, since Grb2 interacts with the guanine nucleotide exchange factor Sos. There is an additional complexity of the BCR-induced Shc/Grb2 complex: it contains tyrosine phosphorylated proteins of 130, 110 and 75 kDa. The 130 kDa molecule inducibly associates with Shc, while the 75 kDa protein interacts with the carboxy-terminal SH3 domain of Grb2. The 110 kDa molecule is defined as Cbl (See Drosophila Cbl), the product of the c-cbl oncogene, which is strongly phosphorylated on tyrosine upon BCR stimulation. Cbl constitutively interacts with the SH3 domains of Grb2, with a preference for the amino-terminal domain, and is in this way recruited to Shc upon BCR stimulation. Grb2-associated Cbl can be phosphorylated by BCR-induced tyrosine kinases independent of a Shc/Grb2 interaction. This indicates that the BCR can also couple to a Grb2 complex without the involvement of Shc. Cbl not only interacts with Grb2, but also with the adaptor protein Crk. In contrast to its constitutive interaction with Grb2, tyrosine-phosphorylated Cbl only associates with Crk after BCR stimulation. In summary, the BCR activates Shc/Grb2-, Grb2- and Crk adaptor complexes of distinct composition, which may allow selective coupling to different signal transduction cascades. Cbl participates in all three adaptor complexes and is tyrosine phosphorylated upon BCR stimulation, pointing to a central role for this molecule in the regulation of antigen receptor-induced B cell responses (Smit, 1996).

Ras is a necessary downstream element of Abelson murine leukemia virus (Ab-MLV)-mediated transformation. Ras can be activated by the phosphotyrosine-dependent association of Shc with the Grb2-mSos complex. Shc is tyrosine-phosphorylated and associates with Grb2 in v-Abl-transformed cells (See Drosophila Abl oncogene, whereas Shc in NIH3T3 cells is phosphorylated solely on serine and is not Grb2-associated. In addition, Shc coprecipitates with P120 v-Abl and P70 v-Abl, which lacks the carboxyl terminus. Surprisingly, a kinase-defective mutant of P120 also binds Shc, demonstrating that Shc/v-Abl association is a phosphotyrosine-independent interaction. Shc from both NIH3T3 and v-Abl-transformed cells binds to the Abl SH2 domain and P120 v-Abl binds to a region in the amino terminus of Shc. Consistent with these data, a v-Abl mutant encoding only the Gag and SH2 regions is able to bind Shc in vivo. The unique non-phosphotyrosine-mediated binding of Shc may allow direct tyrosine phosphorylation of Shc by v-Abl and subsequent activation of the Ras pathway through assembly of a signaling complex with Grb2-mSos (Raffel, 1996).

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi subunit of hetertrimeric G protein, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. Agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, a 100 kDa tyrosine-phosphorylated protein (p100) is detected that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. These results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway. The identity of p100 is not known, but p100 is not Cbl and neither is it dynamin, a Grb2 binding GTPase mediating endocytosis (Kranenburg, 1997)

The mammalian Grb2 adaptor protein binds pTyr-X-Asn motifs through its SH2 domain, and engages downstream targets such as Sos1 and Gab1 through its SH3 domains. Grb2 thereby couples receptor tyrosine kinases to the Ras-MAP kinase pathway, and potentially to phosphatidylinositol (PI) 3'-kinase. By creating a null (Delta) allele of mouse Grb2, it has been shown that Grb2 is required for endoderm differentiation at embryonic day 4.0. Grb2 likely has multiple embryonic and postnatal functions. To address this issue, a hypomorphic mutation, first characterized in the Caenorhabditis elegans Grb2 ortholog Sem-5, was engineered into the mouse Grb2 gene. This mutation (E89K) reduces phosphotyrosine binding by the SH2 domain. Embryos that are compound heterozygous for the null and hypomorphic alleles exhibit defects in placental morphogenesis and in the survival of a subset of migrating neural crest cells required for branchial arch formation. Furthermore, animals homozygous for the hypomorphic mutation die perinatally because of clefting of the palate, a branchial arch-derived structure. Analysis of E89K/Delta Grb2 mutant fibroblasts reveal a marked defect in ERK/MAP kinase activation and Gab1 tyrosine phosphorylation following growth factor stimulation. An allelic series has been created within mouse Grb2, which has revealed distinct functions for phosphotyrosine-Grb2 signaling in tissue morphogenesis and cell viability necessary for mammalian development. The placental defects in E89K/Delta mutant embryos are reminiscent of those seen in receptor tyrosine kinase-, Sos1-, and Gab1-deficient embryos, consistent with the finding that endogenous Grb2 is required for efficient RTK signaling to the Ras-MAP kinase and Gab1 pathways (Saxton, 2001).

Cell migration and outgrowth are thought to be based on analogous mechanisms that require repeated cycles of process extension, reading and integration of multiple directional signals, followed by stabilization in a preferred direction, and renewed extension. A C. elegans gene, unc-53, appears to act cell autonomously in the migration and outgrowth of muscles, axons and excretory canals. Abrogation of unc-53 function disrupts anteroposterior outgrowth in those cells that normally express the gene. Conversely, overexpression of unc-53 in bodywall muscles leads to exaggerated outgrowth. UNC-53 is a novel protein conserved in vertebrates that contains putative SH3- and actin-binding sites. unc-53 interacts genetically with sem-5 and there is a direct interaction in vitro between UNC-53 and the SH2-SH3 adaptor protein SEM-5/GRB2. Thus, unc-53 is involved in longitudinal navigation and might act by linking extracellular guidance cues to the intracellular cytoskeleton (Stringham, 2002).

Molecular analyses revealed multiple unc-53 transcripts. The longest encodes a predicted protein of 1583 amino acids containing a CH domain, two LKK motifs, two SH3-binding domains, two coiled-coil regions and a nucleotide-binding domain. Sequence analysis of two unc-53 alleles, n152 and e2432 shows that they probably correspond to severely truncated proteins, lacking the conserved nucleotide binding domain, the C-terminal LKK motif and the two coiled-coil regions. These modules are therefore likely to be functionally important. Since the regions associated with a putative function make up only about 25% of the protein, molecular analysis of additional mutations may pinpoint further domains involved in specific molecular interactions (Stringham, 2002).

Genetic evidence has suggested that sem-5 and unc-53 co-operate to control sex myoblast migration. SEM-5 is the C. elegans GRB2 homolog and consists exclusively of SH2 and SH3 domains. The biochemical experiments of the present study suggest that the interaction between unc-53 and sem-5 is direct. UNC-53 protein physically associates with SEM-5 and its mammalian homolog GRB2 in vitro, presumably via its SH3-binding domain (Stringham, 2002).

Although functional actin-binding modules cannot always be accurately predicted, UNC-53 does contain a conserved CH domain and LKK motifs. The CH domain is found in a variety of cytoskeletal and signal transduction molecules implicated in the regulation of cell shape dynamics. The actin cross-linking proteins alpha-actinin, ß-spectrin and dystrophin each contain two relatively dissimilar CH domains, while UNC-53, in common with Vav and calponin, contains only one. Preliminary experiments suggest that UNC-53 is able to co-sediment actin in vitro. It is therefore possible that UNC-53 could link SEM-5 to the actin cytoskeleton (Stringham, 2002).

Two models for the action of UNC-53 can be proposed. Within the cell, activation of UNC-53 by SEM-5 could lead to the recruitment of UNC-53 to the actin cytoskeleton and then regulate, perhaps via nucleotide binding, the crosslinking of actin molecules to stabilize a growth cone spike and promote extension in a specific direction. Alternatively, UNC-53 may act as a signal relay, associated with the cytoskeleton, but not directly responsible for the modifications of the actin cytoskeleton associated with growth cone extension. In both models, the unc-53 pathway integrates signals received at the cell surface and determines the direction and rate of growth cone extension. Preliminary experiments have suggested that UNC-53 does indeed localize to the cytoskeleton. Further characterization of the interactions between UNC-53 and its molecular partners may shed more light on the mechanisms of cell migration and growth cone extension (Stringham, 2002).

Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. Addition of NGF to PC-12 cells results in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induces the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity is associated with Gab1. PC-12 cells that overexpress Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacks the binding sites for PI 3-kinase enhances apoptosis and diminishes the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF (Holgado-Madruga, 1997).

Two of the positive regulators of the Notch pathway of Drosophila are encoded by the Suppressor of hairless ([Su(H)]) and deltex (dx) genes. Drosophila dx encodes a ubiquitous, novel cytoplasmic protein of unknown biochemical function. A human deltex homolog has been cloned and characterized in parallel with its Drosophila counterpart, in biochemical assays to assess Deltex function. Both human and Drosophila Deltex bind to Notch across species and carry putative SH3-binding domains. Using the yeast interaction trap system, it has been found that Drosophila and human Deltex bind to the human SH3-domain containing protein Grb2. Results from two different reporter assays demonstrate the association of Deltex with Notch-dependent transcriptional events. Evidence is presented linking Deltex to the modulation of basic helix-loop-helix (bHLH) transcription factor activity (Matsuno, 1998).

Disabled-2 (Dab2), a mammalian structural homolog of Drosophila Disabled (Dab), is a mitogen-responsive phosphoprotein. It has been speculated to be a negative regulator of growth since its expression is lost in ovarian carcinomas. Dab2 contains a C-terminal proline-rich domain with sequences similar to those found in Sos, a guanine nucleotide exchange factor for Ras. The proline-rich sequences of Sos mediate the interaction of Sos with Grb2, an adaptor protein which coupled tyrosine kinase receptors to Sos. The possibility that Dab2 interacts with Grb2 has been investigated. In experiments of co-immunoprecipitation from BAC1.2F5 macrophage cell lysates, significant quantities of Grb2 are associated with both Sos and Dab2, although Dab2 and Sos were not present in the same complex. Transfection of Dab2 into a Dab2-negative cell line (293 cells) decreases the amount of Grb2 associated with Sos, suggesting that Dab2 competes with Sos for binding to Grb2. Proline-rich peptides corresponding to Dab2 (#661-669) and to Sos (#1146-1161) inhibit the binding of Dab2 to Grb2, but are less effective in disrupting the Grb2-Sos complex. The expressed proline-rich domain of Dab2 (#600-730) binds Grb2, but other regions of Dab2 fail to bind Grb2. Both of the individual SH3 domains of Grb2 bind to Sos (the N-terminal SH3 domain of Grb2 has a greated affinity than C-terminal SH3 domain), but binding to Dab2 requires the intact Grb2, suggesting cooperative binding using both SH3 domains of Grb2. These data indicate that Dab2 binds to the SH3 domains of Grb2 via its C-terminal proline-rich sequences. Dab2 may modulate growth factor/Ras pathways by competing with Sos for binding to Grb2. While the N-terminal region of Dab2 and Drosophila Dab show significant homology to one another (both contain the PID domain), the C-terminal proline-rich domain of Dab2 differes significantly from that of Drosophila Dab. However, the C-terminal half of Drosophila Dab also contains several stretches of proline-rich sequences, which might function analgously to those in Dab2. Thus, it is possible that Drosophila Dab binds to a homologous SH2-containing Drosophila proteins, such as the Grb2 homolog Drk2 (Xu, 1998).

Proteins with SH2 and SH3 domains link tyrosine kinases to intracellular pathways. To investigate the biological functions of a mammalian SH2/SH3 adaptor, a null mutation has been introduced into the mouse gene for Grb2. Analysis of mutant embryonic stem cells, embryos, and chimeras reveals that Grb2 is required during embyrogenesis for the differentiation of endodermal cells and formation of the epiblast. Grb2 acts physiologically as an adaptor, since replacing the C terminus of the Ras activator Sos1 with the Grb2 SH2 domain yields a fusion protein that largely rescues the defects caused by the Grb2 mutation. Furthermore, Grb2 is rate limiting for mammary carcinomas induced by polyomavirus middle T antigen. These data provide genetic evidence for a mammalian Grb2-Ras signaling pathway, mediated by SH2/SH3 domain interactions, that has multiple functions in embryogenesis and cancer (Cheng, 1998).

Proteins of the Wiskott-Aldrich Syndrome protein (WASp) family connect signaling pathways to the actin polymerization-driven cell motility. The ubiquitous homolog of WASp, N-WASp, is a multidomain protein that interacts with the Arp2/3 complex and G-actin via its C-terminal WA domain to stimulate actin polymerization. The activity of N-WASp is enhanced by the binding of effectors like Cdc42-guanosine 5'-3-O-(thio)triphosphate, phosphatidylinositol bisphosphate, or the Shigella IcsA protein. The SH3-SH2-SH3 adaptor Grb2 is another activator of N-WASp; Grb2 stimulates actin polymerization by increasing the amount of N-WASp. Arp2/3 complex. The concentration dependence of N-WASp activity, sedimentation velocity and cross-linking experiments together suggest that N-WASp is subject to self-association, and Grb2 enhances N-WASp activity by binding preferentially to the active N-WASp monomeric form. Use of peptide inhibitors, mutated Grb2, and isolated SH3 domains demonstrate that the effect of Grb2 is mediated by the interaction of its C-terminal SH3 domain with the proline-rich region of N-WASp. Cdc42 and Grb2 bind simultaneously to N-WASp and enhance actin polymerization synergistically. Grb2 shortens the delay preceding the onset of Escherichia coli (IcsA) actin-based reconstituted movement. These results suggest that Grb2 may activate Arp2/3 complex-mediated actin polymerization downstream from the receptor tyrosine kinase signaling pathway (Carlier, 2000).

Sprouty as a suppressor of Ras signaling

Shp2/Ptpn11 tyrosine phosphatase is a general regulator of the RTK pathways. By genetic ablation, it was demonstrated that Shp2 is required for lacrimal gland budding, lens cell proliferation, survival and differentiation. Shp2 deletion disrupts ERK signaling and cell cycle regulation, which could be partially compensated by activated Kras signaling, confirming that Ras signaling is the main downstream target of Shp2 in lens and lacrimal gland development. It was also shown that Sprouty2, a general suppressor of Ras signaling, is regulated by Shp2 positively at the transcriptional level and negatively at the post-translational level. Only in the absence of Sprouty2 can activated Kras signaling robustly rescue the lens proliferation and lacrimal-gland-budding defects in the Shp2 mutants. It is proposed that the dynamic regulation of Sprouty by Shp2 might be important not only for modulating Ras signaling in lens and lacrimal gland development, but also for RTK signaling in general (Pan, 2010).

Evolutionary homologs: Table of contents

Ras85D: Biological Overview | Regulation | Protein Interactions | Effects of Mutation | References

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