• Anti-apoptotic Signaling of the Insulin-like Growth Factor-I Receptor through Mitochondrial Translocation of c-Raf and Nedd4

      Peruzzi, Francesca; Prisco, Marco; Morrione, Andrea; Valentinis, Barbara; Baserga, Renato; Morrione|0000-0002-2319-7884 (2001-07-13)
      The type 1 insulin-like growth factor receptor (IGF-IR) sends a strong anti-apoptotic signal by at least three different pathways. By using mutants of the IGF-IR, we showed that one of the pathways depends on residues of the IGF-IR (serines 1280–1283) that interact with 14.3.3 proteins. The result is the activation of Raf-1 and the mitochondrial translocation of both Raf-1 and Nedd4, a target of caspases. A mutant IGF-IR in which the serines at positions 1280–1283 have been mutated to alanine does not protect from apoptosis and fails to translocate Nedd4 or Raf-1 to the mitochondria. This failure is accompanied by a loss of cytochrome c from the mitochondria. The 14.3.3/Raf-1/Nedd4 pathway is operative in the presence or absence of the insulin receptor substrate-1.
    • Growth and Differentiation Signals by the Insulin-like Growth Factor 1 Receptor in Hemopoietic Cells Are Mediated through Different Pathways

      Valentinis, Barbara; Romano, Gaetano; Peruzzi, Francesca; Morrione, Andrea; Prisco, Marco; Soddu, Silvia; Cristofanelli, Barbara; Sacchi, Ada; Baserga, Renato; Morrione|0000-0002-2319-7884 (1999-04-30)
      The type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in the growth of cells both in vivo and in vitro. The IGF-IR is also capable of inducing differentiation in a number of cell types, raising the question of how the same receptor can send two seemingly contradictory signals, one for growth and one for differentiation. Using 32D cells, which are murine hemopoietic cells, we show that the activated IGF-IR can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one of the major substrates of the IGF-IR, the insulin receptor substrate-1 inhibits IGF-I-mediated differentiation of 32D cells. In the absence of insulin receptor substrate-1, functional impairment of another major substrate of the IGF-IR, the Shc proteins, is associated with a decrease in the extent of differentiation. Although the end points of the respective pathways remain to be defined, these results show for the first time that IGF-I-mediated growth or differentiation of hemopoietic cells may depend on a balance between two of its substrates.
    • Insulin Receptor Substrate-1, p70S6K, and Cell Size in Transformation and Differentiation of Hemopoietic Cells

      Valentinis, Barbara; Navarro, Magali; Zanocco-Marani, Tommaso; Edmonds, Pamela; McCormick, Jason; Morrione, Andrea; Sacchi, Ada; Romano, Gaetano; Reiss, Krzysztof; Baserga, Renato; Morrione|0000-0002-2319-7884 (2000-08-18)
      After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70S6K, which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70S6K and an increase in cell size. Phosphorylationin vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70S6K seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70S6K in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70S6K activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.
    • mGrb10 Interacts with Nedd4

      Morrione, Andrea; Plant, Pamela; Valentinis, Barbara; Staub, Olivier; Kumar, Sharad; Rotin, Daniela; Baserga, Renato; Morrione|0000-0002-2319-7884 (1999-08-20)
      We have utilized the yeast two-hybrid system to identify proteins interacting with mouse Grb10, an adapter protein known to interact with both the insulin and the insulin-like growth factor-I receptors. We have isolated a mouse cDNA clone containing the C2 domain of mouse Nedd4, a ubiquitin protein ligase (E3) that also contains a hect (homologous to the E6-APcarboxyl-terminus) domain and three WW domains. The interaction with Grb10 in the two-hybrid system was confirmed using the full-length Nedd4, and it was abolished by deleting the last 148 amino acids of Grb10, a region that includes the SH2 domain and the newly identified BPS domain. The interaction between Grb10 and Nedd4 was also reproduced in vivo in mouse embryo fibroblasts, where endogenous Nedd4 co-immunoprecipitated constitutively with both the endogenous and an overexpressed Grb10. This interaction was Ca2+-independent. Grb10 interacting with Nedd4 was not ubiquitinated in vivo, raising the possibility that this interaction may be used to target other proteins, like tyrosine kinase receptors, for ubiquitination.
    • Regulation of Id2 Gene Expression by the Insulin-like Growth Factor I Receptor Requires Signaling by Phosphatidylinositol 3-Kinase

      Belletti, Barbara; Prisco, Marco; Morrione, Andrea; Valentinis, Barbara; Navarro, Magali; Baserga, Renato; Morrione|0000-0002-2319-7884 (2001-04-27)
      The Id proteins play an important role in proliferation, differentiation, and tumor development. We report here that Id gene expression can be regulated by the insulin-like growth factor I receptor (IGF-IR), a receptor that also participates in the regulation of cellular proliferation and differentiation. Specifically, we found that the IGF-IR activated by its ligand was a strong inducer of Id2 gene expression in 32D murine hemopoietic cells. This activation was not simply the result of cellular proliferation, as Id2 gene expression was higher in 32D cells stimulated by IGF-I than in cells exponentially growing in interleukin-3. The up-regulation of Id2 gene expression was largely dependent on the presence of insulin receptor substrate-1, a major substrate of the IGF-IR and a potent activator of the phosphatidylinositol 3-kinase (PI3K) pathway. The role of PI3K activity in the up-regulation of Id2 gene expression by the IGF-IR was confirmed by different methods and in different cell types. In 32D cells, the up-regulation of Id2 gene expression by the PI3K pathway correlated with interleukin-3 independence and inhibition of differentiation.
    • The Role of mGrb10α in Insulin-like Growth Factor I-mediated Growth

      Morrione, Andrea; Valentinis, Barbara; Resnicoff, Mariana; Xu, Shi-Qiong; Baserga, Renato; Morrione|0000-0002-2319-7884 (1997-10-17)
      Several isoforms of Grb10 are known to interact with either the insulin receptor or the insulin-like growth factor I (IGF-I) receptor, or both. Inasmuch as the data in the literature on the function of Grb10 are not always concordant, we have investigated the role of one of these isoforms, mGrb10α, in cell proliferation. For this purpose, a plasmid expressing mGrb10α was stably transfected into p6 cells and other mouse embryo fibroblast cell lines. An overexpressed mGrb10α inhibits IGF-I-mediated growth, causes a delay in the S and G2 phases of the cell cycle, and partially reverses the transformed phenotype. In contrast, it has no effect on insulin stimulation of cell proliferation. These studies indicate that this isoform of Grb10 has an inhibitory effect on IGF-I signaling of cell proliferation.