Sprouty is a general inhibitor of receptor tyrosine kinase signaling.

Sprouty was originally identified as an inhibitor of Drosophila FGF receptor signaling during tracheal development. By following the capacity of ectopic Sprouty to abolish the pattern of activated MAP kinase in embryos, we show that Sprouty can inhibit other receptor tyrosine kinase (RTK) signaling pathways, namely the Heartless FGF receptor and the EGF receptor. Similarly, in wing imaginal discs, ectopic Sprouty abolishes activated MAP kinase induced by the EGF receptor pathway. Sprouty expression is induced by the EGFR pathway in some, but not all, tissues in which EGFR is activated, most notably in follicle cells of the ovary, the wing imaginal disc and the eye disc. In the ovary, induction of sprouty expression follows the pattern of EGFR activation in the follicle cells. Generation of homozygous sprouty mutant follicle-cell clones demonstrates an essential role for Sprouty in restricting EGFR activation throughout oogenesis. At the stage when dorso-ventral polarity of the follicle cells is established, Sprouty limits the ventral expansion of the activating Gurken signal. Later, when dorsal appendage fates are determined, reduction of signaling by Sprouty facilitates the induction of inter-appendage cell fates. The capacity of Sprouty to reduce or eliminate accumulation of activated MAP kinase indicates that in vivo it intersects with the pathway upstream to MAP kinase. The ability of ectopic Sprouty to rescue lethality caused by activated Raf suggests that it may impinge upon the pathway by interacting with Raf or downstream to it.     

Chromosomal localization of an SH2-containing tyrosine phosphatase (PTPN6).

We have used panels of somatic cell hybrids and fluorescent in situ hybridization to determine the chromosomal localization of the novel nontransmembrane tyrosine phosphatase PTPN6 (protein tyrosine phosphatase, nonreceptor type 6), which contains two SH2 domains. PTPN6 maps to 12p13, a region commonly involved in leukemia-associated chromosomal abnormalities. Since PTPN6 is expressed at high levels in hematopoietic cells of all lineages and its expression is induced early in hematopoietic differentiation, altered expression and/or structure of PTPN6 may play a role in leukemogenesis.     

CD150 association with either the SH2-containing inositol phosphatase or the SH2-containing protein tyrosine phosphatase is regulated by the adaptor protein SH2D1A

CD150 (SLAM/IPO-3) is a cell surface receptor that, like the B cell receptor, CD40, and CD95, can transmit positive or negative signals. CD150 can associate with the SH2-containing inositol phosphatase (SHIP), the SH2-containing protein tyrosine phosphatase (SHP-2), and the adaptor protein SH2 domain protein 1A (SH2D1A/DSHP/SAP, also called Duncan's disease SH2-protein (DSHP) or SLAM-associated protein (SAP)). Mutations in SH2D1A are found in X-linked lymphoproliferative syndrome and non-Hodgkin's lymphomas. Here we report that SH2D1A is expressed in tonsillar B cells and in some B lymphoblastoid cell lines, where CD150 coprecipitates with SH2D1A and SHIP. However, in SH2D1A-negative B cell lines, including B cell lines from X-linked lymphoproliferative syndrome patients, CD150 associates only with SHP-2. SH2D1A protein levels are up-regulated by CD40 cross-linking and down-regulated by B cell receptor ligation. Using GST-fusion proteins with single replacements of tyrosine at Y269F, Y281F, Y307F, or Y327F in the CD150 cytoplasmic tail, we found that the same phosphorylated Y281 and Y327 are essential for both SHP-2 and SHIP binding. The presence of SH2D1A facilitates binding of SHIP to CD150. Apparently, SH2D1A may function as a regulator of alternative interactions of CD150 with SHP-2 or SHIP via a novel TxYxxV/I motif (immunoreceptor tyrosine-based switch motif (ITSM)). Multiple sequence alignments revealed the presence of this TxYxxV/I motif not only in CD2 subfamily members but also in the cytoplasmic domains of the members of the SHP-2 substrate 1, sialic acid-binding Ig-like lectin, carcinoembryonic Ag, and leukocyte-inhibitory receptor families.     

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.     

SH2-containing 5'-inositol phosphatase, SHIP2, regulates cytoskeleton organization and ligand-dependent down-regulation of the epidermal growth factor receptor

Phosphoinositide lipid second messengers are integral components of signaling pathways mediated by insulin, growth factors, and integrins. SHIP2 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate generated by the activated phosphatidylinositol 3'-kinase. SHIP2 down-regulates insulin signaling and is present at higher levels in diabetes and obesity. SHIP2 associates with p130Cas and filamin, regulators of cell adhesion/migration and cytoskeleton, influencing cell adhesion/spreading. Type I collagen specifically induces Src-mediated tyrosine phosphorylation of SHIP2. To better understand SHIP2 function, we employed RNA interference (RNAi) approach to silence the expression of the endogenous SHIP2 in HeLa cells. Suppression of SHIP2 levels caused severe F-actin deformities characterized by weak cortical actin and peripheral actin spikes. SHIP2 RNAi cells displayed cell-spreading defects involving a notable absence of focal contact structures and the formation of multiple slender membrane protrusions capped by actin spikes. Furthermore, decreased SHIP2 levels altered distribution of early endocytic antigen 1 (EEA1)-positive endocytic vesicles and of vesicles containing internalized epidermal growth factor (EGF) and transferrin. EGF treatment of SHIP2 RNAi cells led to the following: enhanced EGF receptor (EGFR) degradation; increased EGFR ubiquitination; and increased association of EGFR with c-Cbl ubiquitin ligase. Taken together, these experiments demonstrate that SHIP2 functions in the maintenance and dynamic remodeling of actin structures as well as in endocytosis, having a major impact on ligand-induced EGFR internalization and degradation. Accordingly, we suggest that, in HeLa cells, SHIP2 plays a distinct role in signaling pathways mediated by integrins and growth factor receptors.     

Regulation of colony-stimulating factor 1 receptor signaling by the SH2 domain-containing tyrosine phosphatase SHPTP1.

SHPTP1 (PTP1C, HCP, SHP) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hematopoietic cells. A frameshift mutation in the SHPTP1 gene causes the motheaten (me/me) mouse. These mice are essentially SHPTP1 null and display multiple hematopoietic abnormalities, most prominently hyperproliferation and inappropriate activation of granulocytes and macrophages. The me/me phenotype suggests that SHPTP1 negatively regulates macrophage proliferative pathways. Using primary bone marrow-derived macrophages from me/me mice and normal littermates, we examined the role of SHPTP1 in regulating signaling by the major macrophage mitogen colony-stimulating factor 1 (CSF-1) (also known as macrophage colony-stimulating factor). Macrophages from me/me mice hyperproliferate in response to CSF-1. In the absence of SHPTP1, the CSF-1 receptor (CSF-1R) is hyperphosphorylated upon CSF-1 stimulation, suggesting that SHPTP1 dephosphorylates the CSF-1R. At least some CSF-1R-associated proteins also are hyperactivated. SHPTP1 is associated constitutively, via its SH2 domains, with an unidentified 130-kDa phosphotyrosyl protein (P130). P130 and SHPTP1 are further tyrosyl phosphorylated upon CSF-1 stimulation. Tyrosyl-phosphorylated SHPTP1 binds to Grb2 via the Grb2 SH2 domain. Moreover, in me/me macrophages, Grb2 is associated, via its SH3 domains, with several tyrosyl phosphoproteins. These proteins are hyperphosphorylated on tyrosyl residues in me/me macrophages, suggesting that Grb2 may recruit substrates for SHPTP1. Our results indicate that SHPTP1 is a critical negative regulator of CSF-1 signaling in vivo and suggest a potential new function for Grb2.     

Anaplastic lymphoma kinase is activated through the pleiotrophin/receptor protein-tyrosine phosphatase beta/zeta signaling pathway: an alternative mechanism of receptor tyrosine kinase activation

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) first discovered as the constitutively active nucleophosmin-ALK oncoprotein in anaplastic large cell lymphomas (ALCL). Full-length ALK has a critical role in normal development and differentiation. Activated full-length ALK also is found in different malignant cancers. Nevertheless, the ligand to activate ALK remained unknown until recently, when ALK was proposed to be the physiological receptor of the cytokine pleiotrophin (PTN, Ptn). However, earlier studies had demonstrated that receptor protein tyrosine phosphatase (RPTP) beta/zeta is a physiological PTN receptor. We now demonstrate that phosphorylation of ALK in PTN-stimulated cells is mediated through the PTN/RPTPbeta/zeta signaling pathway. ALK is phosphorylated independently of a direct interaction of PTN with ALK. The data thus support a unique model of ALK activation. In cells not stimulated by PTN, RPTPbeta/zeta dephosphorylates ALK at the site(s) in ALK that is undergoing autophosphorylation through autoactivation. In contrast, when RPTPbeta/zeta is inactivated in PTN-stimulated cells, the sites that are autophosphorylated in ALK no longer can be dephosphorylated by RPTPbeta/zeta; thus, autoactivation and tyrosine phosphorylation of ALK rapidly increase. The data indicate that the PTN/RPTPbeta/zeta signaling pathway is a critical regulator of the steady state levels of tyrosine phosphorylation and activation of ALK; the data support the conclusion that ALK phosphorylation and activation in PTN-stimulated cells are increased through a unique "alternative mechanism of RTK activation."     

Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites

BACKGROUND: Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated. RESULTS: Using mass spectrometry to analyze the KSR1-scaffolding complex, we identify the serine/threonine protein phosphatase PP2A as a KSR1-associated protein and show that PP2A is a critical regulator of KSR1 activity. We find that the enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, we find that PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation. CONCLUSIONS: These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites.     

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.     

Shp2 protein tyrosine phosphatase inhibitor activity of estramustine phosphate and its triterpenoid analogs

Shp2 protein tyrosine phosphate (PTP) is a novel target for anticancer drug discovery. We identified estramustine phosphate as a Shp2 PTP inhibitor from the National Cancer Institute Approved Oncology Drug set. A focused structure-activity relationship study indicated that the 17-phosphate group is required for the Shp2 PTP inhibitor activity of estramustine phosphate. A search for estramustine phosphate analogs led to identification of two triterpenoids, enoxolone, and celastrol, having Shp2 PTP inhibitor activity. With the previously reported PTP1B inhibitor trodusquemine, our study reveals steroids and triterpenoids with negatively charged phosphate, carboxylate, or sulfonate groups as novel pharmacophores of selective PTP inhibitors.     

Hepatocyte growth factor receptor tyrosine kinase met is a substrate of the receptor protein-tyrosine phosphatase DEP-1

The receptor protein-tyrosine phosphatase (PTP) DEP-1 (CD148/PTP-eta) has been implicated in the regulation of cell growth, differentiation, and transformation, and most recently has been identified as a potential tumor suppressor gene mutated in colon, lung, and breast cancers. We have generated constructs comprising the cytoplasmic segment of DEP-1 fused to the maltose-binding protein to identify potential substrates and thereby suggest a physiological function for DEP-1. We have shown that the substrate-trapping mutant form of DEP-1 interacted with a small subset of tyrosine-phosphorylated proteins from lysates of the human breast tumor cell lines MDA-MB-231, T-47D, and T-47D/Met and have identified the hepatocyte growth factor/scatter factor receptor Met, the adapter protein Gab1, and the junctional component p120 catenin as potential substrates. Following ligand stimulation, phosphorylation of specific tyrosyl residues in Met induces mitogenic, motogenic, and morphogenic responses. When co-expressed in 293 cells, the full-length substrate-trapping mutant form of DEP-1 formed a stable complex with the chimeric receptor colony stimulating factor 1 (CSF)-Met and wild type DEP-1 dephosphorylated CSF-Met. Furthermore, we observed that DEP-1 preferentially dephosphorylated a Gab1 binding site (Tyr(1349)) and a COOH-terminal tyrosine implicated in morphogenesis (Tyr(1365)), whereas tyrosine residues in the activation loop of Met (Tyr(1230), Tyr(1234), and Tyr(1235)) were not preferred targets of the PTP. The ability of DEP-1 preferentially to dephosphorylate particular tyrosine residues that are required for Met-induced signaling suggests that DEP-1 may function in controlling the specificity of signals induced by this PTK, rather than as a simple "off-switch" to counteract PTK activity.     

Tyrosine phosphorylation of an SH2-containing protein tyrosine phosphatase is coupled to platelet thrombin receptor via a pertussis toxin-sensitive heterotrimeric G-protein.

SH-PTP1 is a protein tyrosine phosphatase (PTP) predominantly expressed in haematopoietic cells and containing two src homology-2 (SH2) domains. Here we report that SH-PTP1 is phosphorylated on both serine and tyrosine residues in response to thrombin or phorbol myristate acetate (PMA), which increased by 60 and 40%, respectively, SH-PTP1 activity. Thrombin-induced phosphorylation of SH-PTP1 is an early signalling event (maximal within 10 s) involving neither integrin signalling, nor calcium, nor release of ADP or thromboxane A2. Moreover, in contrast with PMA, the effect of thrombin on the tyrosine phosphorylation of SH-PTP1 was hardly affected by GF109203X, a specific protein kinase C (PKC) inhibitor. Finally, phosphorylation of SH-PTP1 could be provoked in permeabilized platelets by thrombin or GTP gamma S. This was abolished by pertussis toxin, the specificity of this effect being verified with the megakaryocytic cell line Dami cell. Our data thus identify SH-PTP1 as an in vivo substrate of a putative protein tyrosine kinase linked to the thrombin receptor by a Gi protein. This might offer some clue to unravel the mechanism of thrombin not only in platelets but also in nucleated cells, where its mitogenic effect is known to involve pertussis toxin-sensitive G-proteins, tyrosine phosphorylation and the ras pathway.     

CD5 negatively regulates the T-cell antigen receptor signal transduction pathway: involvement of SH2-containing phosphotyrosine phosphatase SHP-1

The negative regulation of T- or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca2+ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3zeta, ZAP-70, Syk, and phospholipase Cgammal but not the Src family tyrosine kinase p56(lck). By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.     

Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila

Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. However, factors that regulate endosomal sorting of TKRs are largely unknown. Here, we demonstrate that Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) is one such factor. Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active epidermal growth factor (EGF) and Torso TKRs, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling.     

The linker between SH2 and kinase domains positively regulates catalysis of the Tec family kinases

Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead.     

The SH2-containing 5'-inositol phosphatase (SHIP) is tyrosine phosphorylated after Fc gamma receptor clustering in monocytes.

Current models of Fc gamma R signal transduction in monocytes describe a molecular cascade that begins upon clustering of Fc gamma R with the phosphorylation of critical tyrosine residues in the cytoplasmic domains of Fc gamma RIIa or the gamma-chain subunit of Fc gamma RI and Fc gamma RIIIa. The cascade engages several other tyrosine-phosphorylated molecules, either enzymes or adapters, to manifest ultimately an array of biological responses, including phagocytosis, cell killing, secretion of a variety of inflammatory mediators, and activation. Continuing to assess systematically the molecules participating in the cascade, we have found that the SH2-containing 5'-inositol phosphatase (SHIP) is phosphorylated on tyrosine early and transiently after Fc gamma R clustering. This molecule in other systems, such as B cells and mast cells, mediates an inhibitory signal. We find that clustering of either Fc gamma RIIa or Fc gamma RI is effective in inducing SHIP phosphorylation, that SHIP binds in vitro to a phosphorylated immunoreceptor tyrosine-based activation motif, peptide from the cytoplasmic domain of Fc gamma RIIa in activation-independent fashion, although SHIP binding increases upon cell activation, and that Fc gamma RIIb and Fc gamma RIIc are not responsible for the observed SHIP phosphorylation. These findings prompt us to propose that SHIP inhibits Fc gamma R-mediated signal transduction by engaging immunoreceptor tyrosine-based activation motif-containing cytoplasmic domains of Fc gamma RIIa and Fc gamma RI-associated gamma-chain.     

Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2

Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.     

The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.