Insulin-like growth factor-I inhibits the stress-activated protein kinase/c-Jun N-terminal kinase.

The pathways involved in the cellular responses to the insulin-like growth factors (IGFs) are numerous and vary according to cell type. Following activation of the IGF-I receptor, the mitogen-activated protein kinase and phosphatidylinositide 3'-kinase (PI3'K) pathways are activated and result in cellular proliferation and inhibition of apoptosis. In this study, we analyzed the IGF-I effect on the stress-activated protein kinase/c-Jun N-terminal kinase (JNK) activity using human embryonic kidney 293 cells, 293 cells transiently expressing hemagglutinin-JNK, and 293 cells stably expressing a hemagglutinin-JNK transgene. In all cell types, endogenous or transfected JNK activity was strongly stimulated by anisomycin or tumor necrosis factor-alpha, and 10 nM IGF-I pretreatment suppressed the induced JNK activity. To determine whether the effect of IGF-I on JNK activity involves the mitogen-activated protein kinase or PI3'K pathway, we used the specific MEK1 inhibitor PD098059 and the PI3'K inhibitor LY 294002. PD098059 did not alter the IGF-I suppressive effect on stressor-induced JNK activity, but LY 294002 suppressed the IGF-I effect. Moreover, in transiently transfected parental 293 cells expressing dominant-negative Akt, anisomycin-increased JNK activity was not suppressed by pretreatment with IGF-I. Our results demonstrate that the action of IGF-I on JNK in these cells is via PI3'K and Akt.

Replacement of tyrosine 1251 in the carboxyl terminus of the insulin-like growth factor-I receptor disrupts the actin cytoskeleton and inhibits proliferation and anchorage-independent growth.

Insulin-like growth factor (IGF)-I signaling through the IGF-I receptor modulates cellular adhesion and proliferation and the transforming ability of cells overexpressing the IGF-I receptor. Tyrosine phosphorylation of intracellular proteins is essential for this transduction of the IGF-I-induced mitogenic and tumorigenic signals. IGF-I induces specific cytoskeletal structure and the phosphorylation of proteins in the associated focal adhesion complexes. The determination of the exact pathways emanating from the IGF-I receptor that are involved in mediating these signals will contribute greatly to the understanding of IGF-I action. We have previously shown that replacement of tyrosine residues 1250 and 1251 in the carboxyl terminus of the IGF-I receptor abrogates IGF-I-induced cellular proliferation and tumor formation in nude mice. In this study, replacement of either tyrosine 1250 or 1251 similarly reduces the cells ability to grow in an anchorage-independent manner. The actin cytoskeleton and cellular localization of vinculin are disrupted by replacement of tyrosine 1251. Tyrosine residues 1250 and 1251 are not essential for tyrosine phosphorylation of two known substrates; insulin receptor substrate-1 and SHC, nor association of known downstream adaptor proteins to these substrates. In addition, these mutant IGF-I receptors do not affect IGF-I-stimulated p42/p44 mitogen-activated protein kinase activation or phosphatidylinositol (PI) 3'-kinase activity. Thus, it appears that in fibroblasts expressing tyrosine 1250 and 1251 mutant IGF-I receptors, the signal transduction pathways impacting on mitogenesis and tumorigenesis do not occur exclusively through the PI 3'-kinase or mitogen-activated protein kinase pathways.

Stimulation of tumor growth by recombinant human insulin-like growth factor-I (IGF-I) is dependent on the dose and the level of IGF-I receptor expression.

The insulin-like growth factors (IGF) I and II regulate metabolism, mitogenesis, differentiation, and apoptosis. The therapeutic uses of IGF-I have been discussed extensively; however, excessive activity of the IGF ligands and IGF-I receptor has been suggested as a factor in tumorigenesis. The inhibition of apoptosis by IGF-I is believed to be particularly important for the stimulation of tumor growth. This study examined whether systemic recombinant human IGF-I (rhIGF-I) therapy affects the growth of fibrosarcomas derived from fibroblasts expressing the IGF-I receptor at high or naturally occurring densities (1.9 x 10(5) compared with 1.6 x 10(4) IGF-I receptors/cell) in athymic nude mice. Treatment with 4 or 10 mg/kg rhIGF-I resulted in a marked reduction in the tumor latency and stimulated the growth of fibrosarcomas that overexpressed the IGF-I receptor. The latency and growth of fibrosarcomas expressing parental levels of the IGF-I receptor were not affected by rhIGF-I therapy. Analysis of mitosis by histone H3 mRNA in situ hybridization and of apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling assay indicated that rhIGF-I-stimulated tumor growth was associated with a marked increase in mitogenesis; however, there was no evidence for any significant effect on apoptosis. These data imply that: (a) systemic rhIGF-I can stimulate the growth of tumors directly by stimulating mitosis; and (b) a reasonable level of IGF-I receptor expression is required for stimulation of tumor growth by systemic rhIGF-I.

Expression of a kinase-deficient IGF-I-R suppresses tumorigenicity of rhabdomyosarcoma cells constitutively expressing a wild type IGF-I-R.

Previous results have shown that the insulin-like growth factor type I receptor (IGF-I-R) plays a critical role in the control of rhabdomyosarcoma (RMS) growth. The purpose of this study was to investigate whether a mutated IGF-I-R, when expressed in RMS cells, may interfere with the function of the endogenous wild-type IGF-I-R. We also examined whether the expression of a mutated IGF-I-R may induce phenotypic changes in RMS cells. We used here the mutated IGF-I-R with a lysine to arginine residue 1003 substitution, called IGF-I-KR, which carries a mutation in the ATP-binding domain of the intracellular beta subunit, while the extracellular, ligand binding alpha subunit remains unchanged. We observed that the expression of this mutated IGF-I-KR markedly decreased the response of RMS cells to stimulation with IGF-I. While stimulation with IGF-I increases the autophosphorylation of IGF-I-R in the parent cells, stimulation with IGF-I failed to produce a comparable increase in autophosphorylation in the cells expressing the mutated IGF-I-KR. We also observed a decreased plating efficiency of cells expressing the mutated IGF-I-KR. Consistently, a decrease of RMS growth in vivo was observed in an animal model. Our data suggest that the IGF/IGF-I-R signaling pathway may be inhibited by expressing a mutated IGF-I-KR and that such a mutant gene could be utilized in developing novel therapeutic strategies to suppress RMS growth. 1998.

Interaction in vitro of the product of the c-Crk-II proto-oncogene with the insulin-like growth factor I receptor.

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in vitro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in vitro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. Mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in vitro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in vitro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which region of the IGF-I receptor interacts with Crk-II, a peptide association assay was used in vitro. Different domains of the IGF-I receptor were expressed as (His)6-tagged fusion peptides, phosphorylated with activated wheat germ agglutinin-purified IGF-I receptors and tested for association with GST-Crk-II fusion proteins. Using wild-type as well as mutated peptides, we showed that the SH2 domain of Crk-II preferentially binds the peptide encoding the juxtamembrane region of the IGF-I receptor. Phosphorylation of Tyr-950 and Tyr-943 of the receptor is important for this interaction. These findings allow us to propose a model of direct interaction of Crk-II and the IGF-I receptor in vivo. On activation of the IGF-I receptor, Crk-II binds to phosphorylated tyrosine residues, especially in the juxtamembrane region. As a result of this binding, the IGF-I receptor kinase phosphorylates Tyr-221 of Crk-II, resulting in a change in intramolecular folding and binding of the SH2 domain to the phosphorylated Tyr-221, which causes rapid disassociation of the Crk-II-IGF-I receptor complex.

The constitutively active mutant Galpha13 transforms mouse fibroblast cells deficient in insulin-like growth factor-I receptor.

Insulin-like growth factor-I (IGF-I) receptor plays an important role in normal cell cycle progression and tumor growth, and it is thought to be essential for cellular transformation. To test this hypothesis, we stably transfected a GTPase-deficient mutant human Galpha13, which is highly oncogenic when overexpressed in vitro, into R- fibroblasts derived from IGF-I receptor-deficient mice. Northern blots of multiple clones revealed the expression of a 1.8-kilobase pair mutant Galpha13 transcript in transfected cells, in addition to the 6-kilobase pair endogenous mRNA. The transfection resulted in a doubling of the expression of Galpha13 protein in these cells as assessed by Western blot analysis. The transforming ability of the mutant Galpha13 was tested using the soft agar assay. Nontransfected R- cells cultured with 10% fetal bovine serum failed to form colonies after 3 weeks. Most of the mutant Galpha13-expressing clones formed significant numbers of colonies (11-50 colonies/1000 cells plated). Overexpression of the IGF-I receptor enabled R- cells to form colonies (27 colonies), and co-transfection of the mutant Galpha13 caused a further increase in colony formation (117-153 colonies) in three of five clones analyzed. Apparently Galpha13 works through pathways other than mitogen-activated protein kinase and c-Jun N-terminal kinase in transforming R- cells, because their activities were not significantly altered by the mutant Galpha13 expression. These results demonstrate that Galpha13 can induce cellular transformation through pathways apparently independent of the IGF-I receptor and that activation of the IGF-I receptor signaling pathways, although not essential for the transforming phenotype, enhances the effect of other pathways.

In vivo regulation of CrkII and CrkL proto-oncogenes in the uterus by insulin-like growth factor-I. Differential effects on tyrosine phosphorylation and association with paxillin.

Changes in CrkII and CrkL phosphorylation are associated with insulin-like growth factor receptor activation in cultured cells. We examined whether similar changes also occur following administration of recombinant human insulin-like growth factor-I to the intact animal. In female rats starved overnight, CrkL phosphorylation was significantly increased 12 min after insulin-like growth factor-I administration. Tyrosine phosphorylation of CrkII was not detectable in either control or treated animals. Paxillin, a 65-70-kDa phosphoprotein containing high affinity binding sites common for the Src homology 2 (SH2) domains of CrkII and CrkL, was observed in both CrkII and CrkL immunoprecipitates. Insulin-like growth factor-I treatment stimulated the association of CrkII with paxillin. In contrast, the same treatment resulted in the dissociation of the CrkL-paxillin complex. Similar effects of insulin-like growth factor-I treatment on the association of CrkL with tyrosine phosphorylated paxillin were observed in fibroblasts overexpressing CrkL. This study demonstrates that the activation of the insulin-like growth factor-I receptor induces changes in the tyrosine phosphorylation and protein-protein interactions of the Crk proteins in vivo. The different responses of CrkL and CrkII to insulin-like growth factor-I receptor activation suggest distinct roles for these two adapter proteins in signal transduction.

Insulin-like growth factor-I rapidly activates multiple signal transduction pathways in cultured rat cardiac myocytes.

In response to insulin-like growth factor-I (IGF-I), neonatal rat cardiac myocytes exhibit a hypertrophic response. The elucidation of the IGF-I signal transduction system in these cells remains unknown. We show here that cardiac myocytes present a single class of high affinity receptors (12,446 +/- 3,669 binding sites/cell) with a dissociation constant of 0.36 +/- 0.10 nM. Two different beta-subunits of IGF-I receptor were detected, and their autophosphorylation was followed by increases in the phosphotyrosine content of extracellular signal-regulated kinases (ERKs), insulin receptor substrate 1, phospholipase C-gamma1, and phosphatidylinositol 3-kinase. IGF-I transiently activates c-Raf in cultured neonatal cardiac myocytes, whereas A-raf is activated much less than c-Raf. Two peaks of ERK activity (ERK1 and ERK2) were resolved in cardiac myocytes treated with IGF-I by fast protein liquid chromatography, both being stimulated by IGF-I (with EC50 values for the stimulation of ERK1 and ERK2 by IGF-I of 0.10 and 0. 12 nM, respectively). Maximal activation of ERK2 (12-fold) and ERK1 (8.3-fold) activities was attained after a 5-min exposure to IGF-I. Maximal activation of p90 S6 kinase by IGF-I was achieved after 10 min, and then the activity decreased slowly. Interestingly, IGF-I stimulates incorporation of [3H]phenylalanine (1.6-fold) without any effect on [3H]thymidine incorporation. These data suggest that IGF-I activates multiple signal transduction pathways in cardiac myocytes some of which may be relevant to the hypertrophic response of the heart.

Tyrosine residues in the C-terminal domain of the insulin-like growth factor-I receptor mediate mitogenic and tumorigenic signals.

We investigated cellular proliferation, the transforming activity, and activation of known signal transduction pathways in NIH-3T3 cells stably expressing insulin-like growth factor-I receptors (IGF-IRs) with amino acid substitutions in the carboxy(C)-terminal domain. The mutant receptors contained substitutions of both tyrosines 1250 and 1251 with phenylalanine and histidine (amino acids present in the analogous positions in the insulin receptor), as well as phenylalanine 1310 replaced by tyrosine (IsY clones) to resemble the placement of tyrosine residues in the C-terminal domain of the insulin receptor. As a control for the IsY clones, a second mutant receptor was expressed with a substitution of phenylalanine 1310 with tyrosine only (DBY clones). Clones expressing IGF-IRs with the IsY substitutions had a significantly slower rate of growth compared with cells expressing an equivalent number of wild-type IGF-IRs (NWT). In contrast, the DBY clones showed relatively normal growth rates. Cells with wild-type IGF-IR demonstrated a transformed phenotype in soft agar assays. The IsY clones lost the transforming ability of the wild type IGF-IR, whereas DBY clones formed colonies. IGF-I-stimulated autophosphorylation of the IGF-IR and tyrosine phosphorylation of IRS-1 and SHC, known substrates in the IGF-IR signal transduction pathway, were studied. Mutated IGF-IRs (IsY and DBY) did not alter the IGF-I-induced tyrosine phosphorylation of these proteins. Furthermore, the mutated IGF-IRs did not alter Grb2 association with phosphorylated IRS-1 and SHC. IGF-I stimulation of Crk-II phosphorylation, a novel substrate of the IGF-IR, was similar in cells expressing mutated and wild-type IGF-IRs. IGF-I-induced activation of phosphatidylinositol (PI) 3'-kinase was equivalent in cells expressing either mutant or wild-type IGF-IRs. These data suggest that the IGF-IR mediates, at least in part, cellular proliferation and increased transforming ability through its C-terminal domain. The exact postreceptor signaling pathway(s) involved have yet to be fully elucidated.

Sphingosine 1-phosphate stimulates tyrosine phosphorylation of Crk.

The proto-oncogene molecule c-Crk plays a role in growth factor-induced activation of Ras. Sphingosine 1-phosphate (SPP), a metabolite of cellular sphingolipids, has previously been shown to play a role in growth factor receptor signaling (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). SPP was found to strongly induce tyrosine phosphorylation of Crk, but not Shc, in NIH-3T3 parental, insulin-like growth factor-I receptor-overexpressing and Crk-overexpressing (3T3-Crk) fibroblasts. Sphingosine, a metabolic precursor of SPP, also produced a slight increase in tyrosine phosphorylation of Crk. In contrast, other sphingolipid metabolites including ceramide did not alter Crk tyrosine phosphorylation. Furthermore, Crk enhanced SPP-induced mitogenesis, as measured by SPP-stimulated [3H]thymidine incorporation in a manner proportional to the level of Crk expression in 3T3-Crk cells. This stimulation appears to be Ras-dependent, whereas SPP stimulation of MAP kinase activity is Ras-independent. These data indicate that SPP activates a tyrosine kinase that phosphorylates Crk and that Crk is a positive effector of SPP-induced mitogenesis.

Mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways are not sufficient for insulin-like growth factor I-induced mitogenesis and tumorigenesis.

Insulin-like growth factor-I (IGF-I) and insulin are known to activate a signaling cascade involving ras --> kappa raf-1 --> mitogen-activated protein (MAP) kinase kinase (MEK) --> p42/p44 MAP kinase (Erk-1 and -2). Recent reports suggest that activation of this ras/MAP kinase pathway is involved in mitogenesis and c-fos transcription but is not required for insulin action on metabolic processes such as glycogen synthesis, lipogenesis, and GLUT-4-mediated glucose transport. Previously we and others have demonstrated that substitution of both tyrosines at positions 1250 and 1251 in the carboxy-terminal region of the human IGF-I receptor has relatively small effects on receptor and endogenous substrate phosphorylation but completely abrogated the ability of these cells to form tumors in nude mice or proliferate in response to IGF-I in culture. Replacement of the tyrosine at position 1316 also did not affect the kinase activity of the receptor with respect to autophosphorylation or phosphorylation of endogenous substrates but did reduce the ability of the receptor to mediate mitogenic or tumorigenic signals. To further characterize the role of these tyrosines in IGF-I receptor function, we have used three distinct approaches to examine the ras/MAP kinase pathway in IGF-I-induced mitogenesis and tumorigenesis in NIH-3T3 cells overexpressing wild-type and mutated IGF-I receptors: 1) tyrosine phosphorylation of the MAP kinases Erk-1 and -2; 2), mobility shifts indicative of MAP kinase phosphorylation; and 3) in vitro MAP kinase activation. We have also examined IGF-I-induced phosphatidylinositol (PI) 3-kinase activation in the same cell lines. By each method we show that the IGF-I-induced MAP kinase phosphorylation/activation and PI 3-kinase activation, are not different between cells overexpressing wild-type IGF-I receptors and cells carrying IGF-I receptors having tyrosine motifs replaced at positions 1250 and 1251. We conclude that mitogenic and tumorigenic signals involving tyrosine residues in the C-terminal domain of the IGF-I-receptor include pathways other than the MAP kinase and PI 3-kinase pathways.

Differential phosphorylation of pp120 by insulin and insulin-like growth factor-1 receptors: role for the C-terminal domain of the beta-subunit.

pp 120, a plasma membrane glycoprotein expressed by hepatocytes, is a substrate of the insulin receptor tyrosine kinase. Since insulin-like growth factor-1 (IGF-1) and insulin receptors are structurally homologous, we investigated whether pp120 is also a substrate of the IGF-1 receptor tyrosine kinase. IGF-1 receptor failed to phosphorylate pp120 in response to IGF-1 in stably transfected NIH 3T3 fibroblasts. However, replacement of the C-terminal domain of the beta-subunit of the IGF-1 receptor with the corresponding fragment in the insulin receptor restored ligand-stimulated pp120 phosphorylation, suggesting that this domain plays a regulatory role in pp120 phosphorylation. Since pp120 is the first identified substrate specific for the insulin vis-à-vis the IGF-1 receptor tyrosine kinase, the pp120 signaling pathway may constitute a novel mechanism for the distinct cellular effects of insulin and IGF-1, the former being principally involved in metabolism, and the latter in mitogenesis.

The proto-oncogene Crk-II enhances apoptosis by a Ras-dependent, Raf-1/MAP kinase-independent pathway.

Human embryonic kidney 293 cells and 293 cells overexpressing different amounts of the adaptor protein Crk-II (ranging from 3- to 10-fold higher levels than the parental cell line) were examined for their ability to undergo apoptosis when maintained in control and serum-free (SF) medium. Parental 293 cells undergo apoptosis only when deprived of serum for prolonged periods of time (24-48 h). On the other hand, 293 cells overexpressing different levels of Crk-II present detectable levels of apoptosis as measured by DNA fragmentation when grown in control medium, with a marked increase when they are deprived of serum for 12-48 h. To determine the pathways involved in Crk-II-induced apoptosis, Crk-II overexpressing cells were transiently transfected with a dominant-negative Ras construct (N17-Ras). Compared to cells transfected with control vectors, the cells overexpressing N17-Ras presented lower levels of apoptosis when maintained in SF-medium. On the other hand, transient transfection of a dominant-negative Raf-1 construct (K375W-Raf-1) did not decrease apoptosis; slightly increasing DNA fragmentation levels were seen. Similar results were obtained when the cells were incubated in the presence of a MEK1 inhibitor. The results presented here suggest that overexpression of Crk-II induces apoptosis via a Ras-dependent, Raf-1/MEK1/ERK-independent pathway.

The insulin-like growth factor-I receptor is required for EWS/FLI-1 transformation of fibroblasts.

Ewing's family of tumors is characterized by a well described reciprocal translocation, t(11;22)(q24;q12), which produces a fusion protein (EWS/FLI-1) that transforms mouse fibroblasts. The EWS/FLI-1 fusion protein has been shown to act as a potent chimeric transcription factor. Overexpression of insulin-like growth factor-I receptor (IGF-IR) has been implicated in many tumor models as playing a role in cell growth and tumorigenesis. In addition, blockade of the IGF-IR inhibits the growth of Ewing's family of tumors cells. Therefore, we first studied whether the presence of the IGF-IR is required for transformation by the EWS/FLI-1 fusion protein. To perform this study, we used two previously described fibroblast cell lines, R- and W, derived from an IGF-IR knockout mouse and a wild-type littermate, respectively. Neither W nor R- cells without the fusion protein formed soft agar colonies. However, W clones expressing the fusion message (WF cells) formed soft agar colonies, whereas R- clones expressing the fusion message (R-F cells) did not form soft agar colonies. Because the IGF-IR is required for EWS/FLI-1 transformation, we chose to investigate whether altered signaling occurs from the IGF-IR when the EWS/FLI-1 fusion is present. WF cells demonstrated a greater degree of ligand-stimulated insulin receptor substrate-1 phosphorylation when compared with W cells, suggesting that expression of the EWS/FLI-1 fusion protein alters the IGF-IR signaling pathway.

Signaling via the insulin-like growth factor-I receptor: does it differ from insulin receptor signaling?

The insulin and insulin-like growth factor (IGF-I) receptors while similar in structure and function serve different physiological functions in vivo. In non-disease states the insulin receptor is primarily involved in metabolic functions whereas the IGF-I receptor mediates growth and differentiation. The separation of these functions is controlled by a number of factors including the tissue distribution of the respective receptors. Modulation of the binding of the ligands insulin or IGF-I and IGF-II to their respective receptors by the local environment of the cell also offers signaling specificity mediated via the receptors. Each ligand bind to its respective receptor with high affinity. This high affinity binding is dictated by the primary sequence of both the ligand and the receptor. Furthermore IGF-binding proteins are specific for IGF-I and IGF-II thereby modulating the binding of the IGFs to the IGF-I receptor. In contrast insulin circulates unbound to any proteins and interacts in the free state with the insulin receptor. It has been postulated that downstream substrates of the activated receptors differ in their specificity for the receptors, thus lending further specificity to the actions mediated by the receptors. While a number of known endogenous substrates such as IRS-1, IRS-2 and She are utilized by both receptors, the structural differences in the beta subunits of the two receptors has lead investigators to suggest that certain substrates may be unique to each receptor. Candidate substrates which show this specificity of action have been and are being described. Full eludication of the specificities of the insulin and IGF-I signaling pathways is of interest of course for a better understanding of intercellular communication. In addition, because the closely related proteins insulin and IGF-I are used clinically, a clear understanding of the pathways activated by these agents is essential if more specific therapeutic modalities are to be developed for use in disease states.

The proto-oncogene product c-Crk associates with insulin receptor substrate-1 and 4PS. Modulation by insulin growth factor-I (IGF) and enhanced IGF-I signaling.

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells. In order to further characterize the role of Crk in the IGF-I signaling pathway, NIH-3T3 and 293 cells were stably transfected with an expression vector containing the Crk cDNA. The various resultant 3T3-Crk clones expressed Crk at approximately 2-15-fold higher levels than parental 3T3 cells. In 3T3-Crk cells, Crk immunoreactivity was detected in insulin receptor substrate-1 (IRS-1) immunoprecipitates. Stimulation with IGF-I resulted in a dissociation of Crk protein from IRS-1. In contrast, the association of the related adaptor protein Grb2 with IRS-1 was enhanced by IGF-I stimulation. Similar results were obtained in stably transfected 293-Crk cells, which express both IRS-1 and the IRS-1-related signaling protein 4PS. In these cells, IRS-1 and 4PS both associated with Crk, and this association was also decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells, as measured by [3H]thymidine incorporation. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling, and may mediate its effects via interaction with IRS-1 and/or 4PS.