Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and beta1 integrin to promote cell migration

The WD repeat scaffolding protein RACK1 can mediate integration of the insulin-like growth factor I receptor (IGF-IR) and integrin signaling in transformed cells. To address the mechanism of RACK1 function, we searched for regulatory proteins that associate with RACK1 in an IGF-I-dependent manner. The serine threonine phosphatase protein phosphatase 2A (PP2A) was found associated with RACK1 in serum-starved cells, and it dissociated immediately upon stimulation with IGF-I. This dissociation of PP2A from RACK1 and an IGF-I-mediated decrease in cellular PP2A activity did not occur in cells expressing either the serine 1248 or tyrosine 1250/1251 mutants of the IGF-IR that do not interact with RACK1. Recombinant RACK1 could bind to PP2A in vitro and restore phosphatase activity to PP2A from IGF-I-stimulated cells. Ligation of integrins with fibronectin or Matrigel was sufficient to facilitate IGF-I-mediated dissociation of PP2A from RACK1 and also to recruit beta1 integrin as PP2A dissociated. By using TAT-fused N-terminal and C-terminal deletion mutants of RACK1, we determined that both PP2A and beta1 integrin interact in the C terminus of RACK1 within WD repeats 4 to 7. This suggests that integrin ligation displaces PP2A from RACK1. MCF-7 cells overexpressing RACK1 exhibited enhanced motility, which could be reversed by the PP2A inhibitor okadaic acid. Small interfering RNA-mediated suppression of RACK1 also decreased the migratory capacity of DU145 cells. Taken together, our findings indicate that RACK1 enhances IGF-I-mediated cell migration through its ability to exclusively associate with either beta1 integrin or PP2A in a complex at the IGF-IR.     

Sonic Hedgehog pathway is essential for neuroblastoma cell proliferation and tumor growth

Products of prolidase [E.C. 3.4.13.9] activity, proline or hydroxyproline, contribute to up-regulation of hypoxia-inducible factor-1α (HIF-1α). Prolidase activity is regulated by β(1)-integrin signaling. We studied the effects of echistatin (a well-known disintegrin) and thrombin (a serine protease capable of activation of integrin α(2)β(1) receptor) on prolidase activity and expressions of prolidase, α(2)β(1)-integrin receptor, focal adhesion kinase (FAK), MAP-kinases (ERK(1) and ERK(2)), and nuclear HIF-1α in human colon adenocarcinoma (DLD-1) cells. It has been found that treatment of the cells with thrombin contributes to decrease in the expression of prolidase and simultaneously increase in its phosphorylation, resulting in maintenance of the enzyme activity. The phenomenon was accompanied by thrombin-dependent recovery of depressed autophosphorylation of FAK (pY(397)) under the effect of FAK inhibitor (1,2,4,5-benzenetetramine tetrahydrochloride). Although integrin α(2)β(1) receptor expression was not affected by thrombin, the signaling induced by thrombin up-regulated nuclear HIF-1α expression. It was accompanied by increase in the expression of MAP kinases, ERK1 and ERK2. It suggests that integrin-dependent signaling through p-FAK is up-regulated in DLD-1 cells and it may represent potential target for anti-cancer therapy.     

Contrasting roles for integrin beta 1 and beta 5 cytoplasmic domains in subcellular localization, cell proliferation, and cell migration

To carry out a detailed comparison of the roles of integrin beta 1 and beta 5 cytoplasmic domains, we expressed both wild type beta 1 and chimeric beta 1/5 constructs in CHO cells. In the latter, the cytoplasmic domain of beta 1 was replaced with that of beta 5. The human beta 1 and beta 1/5 constructs appeared at similar levels at the cell surface (mostly as alpha 5 beta 1 heterodimers) and contributed equally to CHO cell adhesion to fibronectin. However, beta 1 but not beta 1/5 localized to focal adhesion-like structures when CHO cells were spread on fibronectin. Furthermore, only the beta 1-CHO cells showed increased proliferation in response to fibronectin plus an integrin-activating anti-beta 1 antibody, and showed increased appearance of 32P-labeled protein (p90) that correlated with proliferation. In sharp contrast, the beta 1/5-CHO cells were notably more migratory than beta 1-CHO cells in a transwell haptotactic migration assay. These results indicate that the beta 1 and beta 5 integrin subunit cytoplasmic domains can translate similar adhesive information into highly contrasting subsequent events. Thus, we have established that "inside-out" and "outside-in" integrin signaling pathways are regulated by fundamentally distinct mechanisms. In addition, we suggest that the same properties of the beta 1 cytoplasmic domain that promote recruitment to visible focal adhesion-like structures may also be conductive to cell proliferation. Conversely, the properties of the beta 5 tail that make it less likely to localize into focal adhesion-like structures may contribute to enhanced cell migration.     

Tyrosine phosphorylation of beta3 integrin provides a binding site for Pyk2

Integrins expressed on leukocytes possess the ability to maintain themselves in a non-adhesive state, thus preventing unwarranted adhesion and uncontrolled inflammation. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(V)beta(3). Firm adhesion to the extracellular matrix and directed cellular motility requires the reorganization of the actin cytoskeleton. The ability of beta(3) to recruit signaling and scaffolding molecules to propagate alpha(V)beta(3) -mediated signals is regulated in part by the phosphorylation of the beta(3) cytoplasmic tail. The identities of integrin-associated signaling molecules within alpha(V)beta(3) podosomes and in particular the proximal binding partners of the beta(3) cytoplasmic tail are not completely known. Here we show that alpha(V)beta(3) ligation induces Pyk2-Tyr-402 phosphorylation and its association with the beta(3) cytoplasmic tail in a beta(3)-Tyr-747 phosphorylation-dependent manner. Pyk2 binding to the beta(3) cytoplasmic tail is direct and dependent upon Pyk2-Tyr-402 and beta(3) -Tyr-747 phosphorylations. These data identify Pyk2 as a phosphorylated beta(3) binding partner, providing a potential structural and signaling platform to achieve alpha(V)beta(3) -mediated remodeling of the actin cytoskeleton.     

Adeno-associated virus type 2 contains an integrin alpha5beta1 binding domain essential for viral cell entry

Integrins have been implicated as coreceptors in the infectious pathways of several nonenveloped viruses. For example, adenoviruses are known to interact with alphaV integrins by virtue of a high-affinity arginine-glycine-aspartate (RGD) domain present in the penton bases of the capsids. In the case of adeno-associated virus type 2 (AAV2), which lacks this RGD motif, integrin alphaVbeta5 has been identified as a coreceptor for cellular entry. However, the molecular determinants of AAV2 capsid-integrin interactions and the potential exploitation of alternative integrins as coreceptors by AAV2 have not been established thus far. In this report, we demonstrate that integrin alpha5beta1 serves as an alternative coreceptor for AAV2 infection in human embryonic kidney 293 cells. Such interactions appear to be mediated by a highly conserved domain that contains an asparagine-glycine-arginine (NGR) motif known to bind alpha5beta1 integrin with moderate affinity. The mutation of this domain reduces transduction efficiency by an order of magnitude relative to that of wild-type AAV2 vectors in vitro and in vivo. Further characterization of mutant and wild-type AAV2 capsids through transduction assays in cell lines lacking specific integrins, cell adhesion studies, and cell surface/solid-phase binding assays confirmed the role of the NGR domain in promoting AAV2-integrin interactions. Molecular modeling studies suggest that NGR residues form a surface loop close to the threefold axis of symmetry adjacent to residues previously implicated in binding heparan sulfate, the primary receptor for AAV2. The aforementioned results suggest that the internalization of AAV2 in 293 cells might follow a "click-to-fit" mechanism that involves the cooperative binding of heparan sulfate and alpha5beta1 integrin by the AAV2 capsids.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

Farnesyl pyrophosphate promotes and is essential for the binding of RACK1 with beta-tubulin

Receptors for activated C kinase (RACKs) are a group of protein kinase C (PKC) binding proteins that have been shown to be crucial in the translocation and subsequent functioning of PKC on activation. RACK1 isolated from BALB/3T3 cells transformed with S-ras(Q61K) exhibits receptor activity for PKCgamma as competent as that of RACK1 from BALB/3T3 cells without transformation. However, the ability of RACK1 from transformed cells to bind with beta-tubulin peptide specific for Taxol (PEPtaxol) is defective. Interestingly, when farnesyl pyrophosphate was added at the submicrogram level, the association between RACK1 and PEPtaxol was enhanced significantly in a dosage-dependent manner. A parallel finding for the enhanced effect of farnesyl pyrophosphate on tubulin binding was established with mice RACK1 expressed in vitro. On the other hand, geranylgeranyl pyrophosphate, and retinoic acid failed to modulate the binding between RACK1 and tubulin. The dissociation of RACK1 and tubulin was not effective at damaging the binding between RACK1 and membrane receptor integrin beta1 in transformed cells. These findings indicate that depletion of farnesyl pyrophosphate provides a mechanism to seal PKC signaling on the membrane with immobile RACK1 and to divert cells to aberrant growth, such as transformation.     

An interaction between insulin-like growth factor-binding protein 2 (IGFBP2) and integrin alpha5 is essential for IGFBP2-induced cell mobility

In the study we report here, we tested the hypothesis that insulin-like growth factor-binding protein 2 (IGFBP2) promotes cell mobility through its interaction with integrin alpha5. Our previous microarray studies showed that IGFBP2 activates the expression of integrin alpha5. In addition, IGFBP2 has an Arg-Gly-Asp (RGD) domain, which is a known integrin binding motif. We first confirmed our microarray results by showing that the expression of integrin alpha5 is indeed up-regulated at the protein level in IGFBP2-overexpressing SNB19 glioma cells. Using co-immunoprecipitation, we confirmed that IGFBP2 does interact with integrin alpha5. To confirm that IGFBP2 interacts directly with integrin alpha5 through the RGD domain, we created an RGD --> RGE mutant (D306E) IGFBP2 and stably overexpressed the mutant IGFBP2 in the same cell line. Co-immunoprecipitation then showed that D306E-IGFBP2 had no detectable binding with integrin alpha5. We further observed that IGFBP2-overexpressing cells have extensive cell surface lamellipodia, whereas D306E-IGFBP2-overexpressing cells show abundant cell surface focal adhesions. Consistent with this, phenotype analysis then showed that IGFBP2-overexpressing cells have elevated migration rates compared with vector control; in contrast, the migration rates of the D306E-IGFBP2-overexpressing cells were not elevated and were comparable with that of vector control. Decreased expression of integrin alpha5 by small interference RNA in IGFBP2-overexpressing cells also reduced cell mobility. Therefore, we have concluded that one mechanism by which IGFBP2 activates IGFBP2-induced cell mobility is through its interaction with integrin alpha5 and this interaction is specifically mediated through the RGD domain on IGFBP2.     

The integrin binding site 2 (IBS2) in the talin rod domain is essential for linking integrin beta subunits to the cytoskeleton

Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984-2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258-22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in alpha helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this alpha helix, which disrupted the alpha-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to alphaIIbbeta3 integrin in focal adhesions and to inhibit in vitro this association as shown by an alphaIIbbeta3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1(-/-) cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.     

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on fibrillar as well as soluble collagen. These data show that GPVI plays the central role in platelet-collagen interactions by activating different adhesive receptors, including alpha2beta1 integrin, which strengthens adhesion without being essential.     

Cross-talk of integrin alpha3beta1 and tissue factor in cell migration

In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is beta1 integrin-dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing alphavbeta3 or certain beta1 integrin heterodimers (alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha9beta1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates alpha3beta1-dependent migration on laminin 5. Expression of TF suppresses alpha3beta1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2-dependent phosphorylation of TF. In both cases, release of alpha3beta1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra- and extracellular interactions and phosphorylation regulates TF's function in cell motility.     

Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration

The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin alpha(5)beta(1), and the focal adhesion kinase phosphorylation. E(4)-PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin alpha(5) subunit as well as alpha(2) and alpha(3) subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin alpha(5)beta(1) to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the alpha(5) subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.     

Identification and characterization of the integrin alpha2beta1 binding motif in chondroadherin mediating cell attachment

Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)β(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)β(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)β(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology.     

Endothelin-1 is an essential co-factor for beta2-adrenergic receptor-induced proliferation of human cardiac fibroblasts

We previously demonstrated that chronic stimulation of the beta2-adrenergic receptor (beta2-AR) increases proliferation of cultured human cardiac fibroblasts (CF) via an autocrine mechanism. Here, we investigated the role of endothelin-1 (ET-1) in this process. ETA-receptor antagonism or protein kinase C inhibition abolished the beta2-AR-induced increase in cell proliferation. RT-PCR and ELISA analysis demonstrated that although CF synthesized and secreted ET-1, this occurred independently of beta2-AR stimulation. Furthermore, despite activation of the MAP kinase pathway, ET-1 treatment did not stimulate CF proliferation. Therefore, the role of ET-1 in this process is that of an essential co-factor acting independently of beta2-AR stimulation.     

Glyoxalase 1 is up-regulated in hepatocellular carcinoma and is essential for HCC cell proliferation

Glyoxalase 1 (Glo1), belonging to the glyoxalase system, participates in the detoxification of methylglyoxal (MG), a byproduct of glycolysis. Glo1 is associated with the progression of many human malignancies. However, the role of Glo1 in hepatocellular carcinoma (HCC) is unclear. We have discovered that the expression of Glo1 is up-regulated in HCC tissues compared with adjacent non-tumorous tissues, and knockdown of Glo1 expression by RNA interference significantly inhibited the proliferation of human HCC cell lines. Glo1 knockdown resulted in the accumulation of its cytotoxic substrate, MG. Overall, thus Glo1 might be essential for HCC progression and can be designated as a potential therapeutic target for HCC in the future.     

Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase regulating many cellular processes. PP1alpha and -gamma are closely related isoforms with distinct localization patterns, shown here by time-lapse microscopy of stably expressed fluorescent protein fusions. A pool of PP1gamma is selectively loaded onto chromatin at anaphase. Using stable isotope labeling and proteomics, we identified a novel PP1 binding protein, Repo-Man, which selectively recruits PP1gamma onto mitotic chromatin at anaphase and into the following interphase. This approach revealed both novel and known PP1 binding proteins, quantitating their relative distribution between PP1alpha and -gamma in vivo. When overexpressed, Repo-Man can also recruit PP1alpha to chromatin. Mutating Repo-Man's PP1 binding domain does not disrupt chromatin binding but abolishes recruitment of PP1 onto chromatin. RNA interference-induced knockdown of Repo-Man caused large-scale cell death by apoptosis, as did overexpression of this dominant-negative mutant. The data indicate that Repo-Man forms an essential complex with PP1gamma and is required for the recruitment of PP1 to chromatin.     

The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. We show that Drosophila PP1beta, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1beta. Loss of PP1beta leads to increased levels of phosphorylated nonmuscle MRLC (Sqh) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1beta, and activates PP1beta's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1beta and MYPT genes suggests this role may be conserved.     

RACK1 recruits STAT3 specifically to insulin and insulin-like growth factor 1 receptors for activation, which is important for regulating anchorage-independent growth

Current understanding of the activation of STATs is through binding between the SH2 domain of STATs and phosphotyrosine of tyrosine kinases. Here we demonstrate a novel role of RACK1 as an adaptor for insulin and insulin-like growth factor 1 receptor (IGF-1R)-mediated STAT3 activation specifically. Intracellular association of RACK1 via its N-terminal WD domains 1 to 4 (WD1-4) with insulin receptor (IR)/IGF-1R is augmented upon respective ligand stimulation, whereas association with STAT3 is constitutive. Purified RACK1 or RACK1 WD1-4 associates directly with purified IR, IGF-1R, and STAT3 in vitro. Insulin induces multiprotein complex formation of RACK1, IR, and STAT3. Overexpression or downregulation of RACK1 greatly enhances or decreases, respectively, IR/IGF-1R-mediated activation of STAT3 and its target gene expression. Site-specific mutants of IR and IGF-1R impaired in RACK1 binding are ineffective in mediating recruitment and activation of STAT3 as well as in insulin- or IGF-1-induced protection of cells from anoikis. RACK1-mediated STAT3 activation is important for insulin and IGF-1-induced anchorage-independent growth in certain ovarian cancer cells. We conclude that RACK1 mediates recruitment of STAT3 to IR and IGF-1R specifically for activation, suggesting a general paradigm for the need of an adaptor in mediating activation of STATs by receptor protein tyrosine kinases.