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We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1-MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.

Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK). This kinase cascade controls the proliferation and differentiation of different cell types. Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen. This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein). In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras. RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy. RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases. RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase. Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription. RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK module.

POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes. Here we show that whereas homeodomain-containing repressors such as Rpx2 seem to recruit only a co-repressor complex, the activity of Pit-1 is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT, mSin3A/B and histone deacetylases, and a co-activator complex that includes the CREB-binding protein (CBP) and p/CAF. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, the histone acetyltransferase functions of CBP or p/CAF are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors.

Both p21ras and phosphatidylinositol 3-kinase (PI 3-k) are critical elements in signaling pathways mediating insulin/IGF-I induced cell cycle progression. For example, microinjection of antibodies, peptides, or recombinant proteins which block the interaction of the SH2 domains of the PI 3-k p85alpha subunit with tyrosine phosphorylated intracellular targets blocks insulin mediated DNA synthesis. We report here that this inhibitory phenotype is observed whether the injections are made into quiescent cells (the standard approach), or at any time point during G1 phase subsequent to stimulation. This observation is not true, however, for the major substrate of the insulin/IGF-I receptor (IRS-1) despite the well known interaction of p85 with IRS-1. Antibodies to IRS-1 are inhibitory only when injected during the first 15 min of G1 phase, as are antibodies to another major IRS-1 binding protein, the tyrosine phosphatase SHP2. We also have microinjected reagents which target proteins involved in the formation of rasGTP and which mediate some of the downstream effects of ras activation. Reagents which target the formation of rasGTP (Shc and dominant negative ras protein) inhibit DNA synthesis only at points early in G1, as do reagents which target components of the MAP kinase pathway. Injection of antibodies to p21ras itself, or a recombinant Raf-1 protein domain which binds to the effector region of ras in a GTP-dependent manner, results in the inhibition of cell cycle progression throughout G1 phase. The results point to a continuous requirement for both PI 3-k and ras activity until cellular commitment to DNA synthesis, although some of the molecules which are both upstream and downstream of these activities are only required transiently. Our results are also consistent with a Raf-1 independent ras activity late in G1, as well as IRS-1 independent effects of PI 3-kinase.

We report that interferon gamma (IFN-gamma) inhibits transcription of the macrophage scavenger receptor gene by antagonizing the Ras-dependent activities of AP-1 and cooperating ets domain transcription factors, apparently as a result of competition between AP-1/ets factors and activated STAT1 for limiting amounts of CBP and p300. Consistent with this model, STAT1 alpha interacts directly with CBP in cells, and microinjection of anti-CBP and anti-p300 antibodies blocks transcriptional responses to IFN-gamma. Cells lacking STAT1 fail to inhibit AP-1/ets activity, and overexpression of CBP both potentiates IFN-gamma-dependent transcription and relieves AP-1/ets repression. Thus, CBP and p300 integrate both positive and negative effects of IFN-gamma on gene expression by serving as essential coactivators of STAT1 alpha, modulating gene-specific responses to simultaneous activation of two or more signal transduction pathways.

Shc is involved in the activation of Ras in response to many growth factors. Shc contains two phosphotyrosine binding domains, an Src homology 2 (SH2) domain in the carboxyl terminus of the protein and a phosphotyrosine binding (PTB) domain in the amino terminus. Since functional roles for these two domains have not been established, we microinjected glutathione S-transferase fusion proteins of either the Shc PTB or SH2 domains into fibroblasts expressing insulin and epidermal growth factor receptors and measured their effects on DNA synthesis. We found that the Shc PTB was necessary for insulin-induced mitogenic signaling, whereas the SH2 domain was not. In contrast, for epidermal growth factor signaling, the Shc SH2 was functionally more important. These differential modes of signal transduction may be an important factor in determining the specificity of the response of a cell to external stimuli.

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.

We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. We identified a human cDNA that is a splice variant of the human GRB10 homolog GRB-IR, which we term GRB10/IR-SV1 (for GRB10/GRB-IR splice variant 1). The protein encoded by the GRB10/IR-SV1 cDNA contains an SH2 domain and a pleckstrin homology domain. Cloning of a full-length human cDNA revealed a predicted coding sequence that was similar to the mouse GRB10 protein, although GRB10/IR-SV1 contained an 80-amino acid deletion. The GRB10/IR-SV1 cDNA is a splice variant of the GRB-IR cDNA such that GRB10/IR-SV1 contains an intact pleckstrin homology domain and a distinct amino terminus. The interaction of GRB10/IR-SV1 with the insulin receptor and the insulin-like growth factor I (IGF-I) receptor is mediated by the SH2 domain, and we show that glutathione S-transferase-SH2 domain fusion proteins interact specifically in vitro with the insulin receptor derived from mammalian cells. The GRB10/IR-SV1 SH2 domain also interacted with an approximately 135-kDa phosphoprotein from unstimulated cell lysates, an interaction that decreased after insulin stimulation. We present evidence that the GRB10/IR-SV1 protein plays a functional role in insulin and IGF-I signaling by showing that microinjection of an SH2 domain fusion protein inhibited insulin- and IGF-I-stimulated mitogenesis in fibroblasts, yet had no effect on mitogenesis induced by epidermal growth factor. Our findings suggest that GRB10/IR-SV1 may serve to positively link the insulin and IGF-I receptors to an uncharacterized mitogenic signaling pathway.

Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.

We have recently characterized a mutant insulin receptor (HIR delta 978) in which the insulin receptor beta-subunit was truncated at amino acid residue 978. Compared with parental Rat1 cells, the cells expressing the truncated receptor exhibited enhanced sensitivity to insulin's biologic actions. All of these effects are now extended to transcriptional events, since we now show enhanced sensitivity to insulin stimulation of c-fos mRNA expression. These effects were insulin-specific, since insulin-like growth factor-1 stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, and thymidine incorporation into DNA were normal. In addition, the truncated receptor exhibited enhanced sensitivity only in vivo, but not in vitro, since the kinase activity of wheat germ agglutinin-purified receptor preparations was comparable between HIR delta 978 and parental Rat1 insulin receptors. Parental rat endogenous insulin-like growth factor-1 receptors and transfected human insulin receptors form hybrid receptors as well as homologous tetrameric receptors. The normal heterotetrameric receptors possess kinase activity in vivo leading to tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with the p85 regulatory subunit of phosphatidyl inositol 3-kinase. Interestingly, preincubation with human-specific anti-insulin receptor antibody abolished the increased insulin sensitivity in glucose incorporation into glycogen in HIR delta 978 cells. Furthermore, microinjection of anti-IRS-1 antibody into HIR delta 978 cells inhibited insulin stimulation of DNA synthesis. In summary: 1) truncated receptors on the cell surface confer enhanced insulin sensitivity in vivo; 2) the normal heterotetrameric receptors are functionally active and couple to IRS-1 efficiently; and 3) IRS-1 is an important molecule transmitting insulin's biological signals in HIR delta 978 cells.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.

Syp (SH-PTP2) was recently identified as a phosphotyrosine phosphatase containing two SH2 domains within its primary structure. In response to appropriate growth factor stimulation, Syp becomes phosphorylated on tyrosine residues and associates with insulin receptor substrate 1 (IRS-1) and/or the corresponding growth factor receptor via its SH2 domains, leading to increased Syp activity. To assess the importance of Syp in mitogenic signaling, we microinjected mammalian fibroblasts with several reagents designed to interfere with Syp SH2/phosphotyrosine interaction in vivo. Insulin-, insulin-like growth factor-1-, and epidermal growth factor-stimulated DNA synthesis, indicated by bromodeoxyuridine (BrdUrd) incorporation, was dramatically decreased following microinjection of a Syp antibody (Ab) (65-85%) or a Syp GST-SH2 fusion protein (approximately 90%) in comparison with cells microinjected with control IgG or glutathione S-transferase (GST), respectively. In addition, microinjection of an IRS-1-derived phosphonopeptide, which inhibits in vitro binding of Syp-SH2 to IRS-1 with an ED50 value of approximately 23 microM, also decreased BrdUrd incorporation in vivo by approximately 50-75%. Microinjection of the Syp Ab, Syp GST-SH2 fusion protein, or the phosphonopeptide had no effect on serum-stimulated BrdUrd incorporation. In conclusion, disruption of Syp function in living cells inhibited cell cycle progression in response to growth factor stimulation, indicating that Syp is a critical positive regulator of mitogenic signal transduction.

Shc proteins contain a single SH2 domain, lack catalytic activity, and are substrates for activated receptors for insulin, insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF). Treatment with these growth factors induced rapid tyrosine phosphorylation of Shc. We investigated the potential role of Shc in mitogenic signaling. Affinity-purified antibodies were microinjected into living Rat1 fibroblasts overexpressing human insulin receptors. Bromodeoxyuridine incorporation into newly synthesized DNA was subsequently studied to assess the importance of Shc. Cellular microinjection of anti-Shc antibody inhibited BrdU incorporation induced by insulin, IGF-1, and EGF, but did not affect cells stimulated by fetal calf serum. Microinjection of an oncogenic p21ras protein (T24) into quiescent cells produced constitutively active mitogenic signaling, and comicroinjection of T24 with the anti-Shc antibody restored insulin and EGF stimulation of DNA synthesis. Immunoprecipitates of Shc from lysates of insulin-stimulated cells removed 70-80% of guanine nucleotide-releasing factor activity. These results indicate that Shc is an important component in a mitogenic signal transduction pathway that is shared by insulin, IGF-1, and EGF. The functional locus of Shc is either upstream of p21ras or lies on a distinct branch of the pathway leading to cell cycle progression.

Insulin treatment of mammalian cells immediately stimulates the tyrosine phosphorylation of a cellular protein of 185 kDa referred to as pp185 or IRS-1 (insulin receptor substrate 1). The potential role of the IRS-1 protein in insulin signaling has been examined by microinjecting affinity-purified antibodies into living cells. Stably transfected Rat-1 fibroblasts, which overexpress the human insulin receptor, were microinjected and subsequently stimulated with insulin or other growth factors. Progression through the cell cycle was monitored by using a single-cell assay, which employs bromodeoxyuridine labeling of DNA and analysis with immunofluorescence microscopy. Microinjection of anti-IRS-1 antibody completely inhibited incorporation of bromodeoxyuridine into the nuclei of cells stimulated with insulin or insulin-like growth factor I but did not affect cells stimulated with serum or a variety of purified growth factors. These studies indicate that IRS-1 is a critical component of the insulin and insulin-like growth factor I signaling pathways, which lead to DNA synthesis and cell growth.