Biomedical subjects

G Pelicci

Publications and source records attributed to G Pelicci.

At least 19 recordsLinked to original sources

PubMed2001

Shc signaling in differentiating neural progenitor cells.

Previously we found that the availability of ShcA adapter is maximal in neural stem cells but that it is absent in mature neurons. Here we report that ShcC, unlike ShcA, is not present in neural stem/progenitor cells, but is expressed after cessation of their division and becomes selectively enriched in mature neurons. Analyses of its activity in differentiating neural stem/progenitor cells revealed that ShcC positively affects their viability and neuronal maturation via recruitment of the PI3K-Akt-Bad pathway and persistent activation of the MAPK pathway. We suggest that the switch from ShcA to ShcC modifies the responsiveness of neural stem/progenitor cells to extracellular stimuli, generating proliferation (with ShcA) or survival/differentiation (with ShcC).

Adaptor Proteins, Signal Transducing↗

PubMed1999

The p66shc adaptor protein controls oxidative stress response and life span in mammals.

Gene mutations in invertebrates have been identified that extend life span and enhance resistance to environmental stresses such as ultraviolet light or reactive oxygen species. In mammals, the mechanisms that regulate stress response are poorly understood and no genes are known to increase individual life span. Here we report that targeted mutation of the mouse p66shc gene induces stress resistance and prolongs life span. p66shc is a splice variant of p52shc/p46shc (ref. 2), a cytoplasmic signal transducer involved in the transmission of mitogenic signals from activated receptors to Ras. We show that: (1) p66shc is serine phosphorylated upon treatment with hydrogen peroxide (H2O2) or irradiation with ultraviolet light; (2) ablation of p66shc enhances cellular resistance to apoptosis induced by H2O2 or ultraviolet light; (3) a serine-phosphorylation defective mutant of p66shc cannot restore the normal stress response in p66shc-/- cells; (4) the p53 and p21 stress response is impaired in p66shc-/- cells; (5) p66shc-/- mice have increased resistance to paraquat and a 30% increase in life span. We propose that p66shc is part of a signal transduction pathway that regulates stress apoptotic responses and life span in mammals.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Polyomavirus large T antigen induces alterations in cytoplasmic signalling pathways involving Shc activation.

It has been extensively demonstrated that growth factors play a key role in the regulation of proliferation. Several lines of evidence support the hypothesis that for the induction of cell cycle progression in the absence of exogenous growth factors, oncogenes must either induce autocrine growth factor secretion or, alternatively, activate their receptors or their receptor substrates. Cells expressing polyomavirus large T antigen (PyLT) display reduced growth factor requirements, but the mechanisms underlying this phenomenon have yet to be explored. We conducted tests to see whether the reduction in growth factor requirements induced by PyLT was related to alterations of growth factor-dependent signals. To this end, we analyzed the phosphorylation status of a universal tyrosine kinase substrate, the transforming Shc adapter protein, in fibroblasts expressing the viral oncogene. We report that the level of Shc phosphorylation does not decrease in PyLT-expressing fibroblasts after growth factor withdrawal and that this PyLT-mediated effect does not require interaction with protein encoded by the retinoblastoma susceptibility gene. We also found that the chronic activation of the adapter protein is correlated with the binding of Shc to Grb-2 and with defects in the downregulation of mitogen-activated protein kinases. In fibroblasts expressing the nuclear oncoprotein, we also observed the formation of a PyLT-Shc complex that might be involved in constitutive phosphorylation of the adapter protein. Viewed comprehensively, these results suggest that the cell cycle progression induced by PyLT may depend not only on the direct inactivation of nuclear antioncogene products but also on the indirect induction, through the alteration of cytoplasmic pathways, of growth factor-dependent nuclear signals.

3T3 Cells↗

PubMed1998

Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization.

eps15R was identified because of its relatedness to eps15, a gene encoding a tyrosine kinase substrate bearing a novel protein-protein interaction domain, called EH. In this paper, we report a biochemical characterization of the eps15R gene product(s). In NIH-3T3 cells, three proteins of 125, 108, and 76 kDa were specifically recognized by anti-eps15R sera. The 125-kDa species is a bona fide product of the eps15R gene, whereas p108 and p76 are most likely products of alternative splicing events. Eps15R protein(s) are tyrosine-phosphorylated following epidermal growth factor receptor activation in NIH-3T3 cells overexpressing the receptor, even at low levels of receptor occupancy, thus behaving as physiological substrates. A role for eps15R in clathrin-mediated endocytosis is suggested by its localization in plasma membrane-coated pits and in vivo association to the coated pits' adapter protein AP-2. Finally, we demonstrate that a sizable fraction of eps15R exists in the cell as a complex with eps15 and that its EH domains exhibit binding specificities that are partially distinct from those of eps15. We propose that eps15 and eps15R are multifunctional binding proteins that serve pleiotropic functions within the cell.

3T3 Cells↗

PubMed1997

Modified phage peptide libraries as a tool to study specificity of phosphorylation and recognition of tyrosine containing peptides.

Tyrosine phosphorylation and protein recognition, mediated by phosphotyrosine containing peptides, play an important role in determining the specific response of a cell, when stimulated by external signals. We have used peptide repertoires displayed by filamentous phage as a tool to study the substrate specificity of the protein tyrosine kinase (PTK) p55(fyn) (Fyn). Peptide libraries were incubated for a short time in the presence of Fyn and phages displaying efficiently phosphorylated peptides were selected by panning over anti-phosphotyrosine antibodies. The characterization of the peptides enriched after three phosphorylation/selection rounds allowed us to define a canonical substrate sequence for the kinase Fyn, E-(phi/T)YGx phi, where phi represents any hydrophobic residue. A peptide conforming to this sequence is a better substrate than a second peptide designed to be in accord with the consensus sequence recognised by the Fyn SH2 domain. When the library phosphorylation reaction is carried out in saturation conditions, practically all the tyrosine containing peptides are phosphorylated, irrespective of their context. These "fully modified" peptide libraries are a valuable tool to study the specificity of phosphotyrosine mediated protein recognition. We have used this new tool to identify a family of peptides that bind the PTB domain of the adapter protein Shc. Comparison of the peptide sequences permits us to confirm the essential role of N at position -3, while P often found at position -2 in natural targets is not absolutely required. Furthermore, our approach permits us to reveal an "extended" consensus indicating that residues that do not seem to influence binding in natural peptides can make productive contacts, at least in linear peptides.

Consensus Sequence↗

PubMed1997

Identification of Shc docking site on Ret tyrosine kinase.

The RET proto-oncogene encodes two isoforms of a receptor type tyrosine kinase which plays a role in neural crest and kidney development. Distinct germ-line mutations of RET have been associated with the inherited cancer syndromes MEN2A, MEN2B and FMTC as well as with the congenital disorder Hirschsprung disease (HSCR), whereas somatic rearrangements (RET/PTCs) have been frequently detected in the papillary thyroid carcinoma. Despite these findings, suggesting a relevant role for RET product in development and neoplastic processes, little is known about the signalling triggered by this receptor. In this study, we have demonstrated that the transducing adaptor molecule Shc is recruited and activated by both Ret isoforms and by the rearranged cytoplasmatic Ret/ptc2 oncoproteins as well as by the membrane bound receptor activated by MEN2A or by MEN2B associated mutations. Moreover, our analysis has identified the Ret tyrosine residue and the Shc domains involved in the interaction. In fact, here we show that both the phosphotyrosine binding domains of Shc, PTB and SH2, interact with Ret/ptc2 in vitro. However, PTB domain binds 20 folds higher amount of Ret/ptc2 than SH2. The putative binding site for either SH2 and PTB domains has been identified as Tyr586 of Ret/ptc2 (Tyr1062 on proto-Ret). In keeping with this finding, by using RET/PTC2-Y586F mutant, we have demonstrated that this tyrosine residue, the last amino acid but one before the divergence of the two Ret isoforms, is the docking site for Shc.

3T3 Cells↗

PubMed1997

Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway.

Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N-terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.

3T3 Cells↗

PubMed1996

A family of Shc related proteins with conserved PTB, CH1 and SH2 regions.

Shc proteins are targets of activated tyrosine kinases and have been implicated in the transmission of activation signals to Ras. Upon phosphorylation, Shc proteins form stable complexes with cellular tyrosine-phosphorylated proteins and with the Grb2 adaptor protein. Two Shc isoforms of 52 and 46 kDa have been characterized. They share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal phospho-tyrosine binding domain (PTB). We report her ethe initial characterization of two Shc related human cDNAs: ShcB and ShcC. The ShcB and ShcC cDNAs code for proteins that are highly similar and share the same modular organization as Shc. PTB and SH2 domains of ShcB and ShcC have similar binding specificities in vitro and bind to activated EGFR in a phosphotyrosine-dependent manner. Based on these findings we propose to rename Shc as ShcA. Anti-ShcB and anti-ShcC antibodies recognize specific polypeptides of 52, 47 kDa (ShcB) and 54 kDa (ShcC) in mammalian cells. Since these two genes are predominantly expressed in specific brain tissues, these Shc family members may be involved in cell type-specific signaling, in the nervous system.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.

3T3 Cells↗

PubMed1996

Not all Shc's roads lead to Ras.

The Shc proteins have been implicated in the Ras signaling pathway by virtue of their association with the Grb2 adaptor molecule. Several lines of evidence indicate that this association is indeed involved in Ras activation. More recent experiments in mammalian tissue culture cells suggest that domains unique to Shc isoforms, named CH1 and CH2, might be involved in a new network of protein-protein interactions, and hint at other roles that Shc might play in addition to Ras activation.

Animals↗

PubMed1995

Constitutive phosphorylation of Shc proteins in human tumors.

The Shc gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are ubiquitously expressed and are downstream targets and effectors of activated tyrosine kinases (TK). We investigated tyrosine-phosphorylation of Shc proteins in normal and transformed cells. In tumor cells with known TK gene alterations Shc proteins were constitutively phosphorylated and complexed with the activated TK. No constitutive Shc phosphorylation was found in primary cell cultures and normal tissues. In 14 of 27 tumor cell lines with no reported TK alterations, Shc proteins were constitutively phosphorylated and formed stable complexes with novel tyrosine-phosphorylated polypeptides. Ten distinct Shc-associated phosphoproteins were identified with molecular weights ranging from 30 to 200 kDa. In a subset of carcinoma cell lines, phosphorylated Shc proteins complexed with a p175 phosphoprotein that was identified as the constitutively activated EGFR. In one glioblastoma cell line, a Shc-associated p190 was identified as the activated PDGFR. In 13 of 14 acute leukemia samples phosphorylated Shc proteins were constitutively complexed with a p140 phosphoprotein. Some of the Shc-associated phosphoproteins (EGFR, PDGFR, erbB-2, Met, bcr-abl, H4-ret) bound both the Shc- and Grb2-SH2 domains in vitro; others (p175; p70-p80) only the Shc-SH2 domain and yet others (p140) only the Grb2-SH3 domains. These results indicate that Shc proteins are common substrates of constitutively activated TKs and that the analysis of Shc phosphorylation allow the identification of tumors with constitutive TK activation.

Adaptor Proteins, Signal Transducing↗

PubMed1995

The motogenic and mitogenic responses to HGF are amplified by the Shc adaptor protein.

The receptor of Hepatocyte Growth Factor-Scatter Factor (HGF) is a tyrosine kinase which regulates cell motility and growth. After ligand-induced tyrosine phosphorylation, the HGF receptor associates with the Shc adaptor, via the SH2 domain. Site-directed mutagenesis of the HGF receptor indicates that phosphotyrosines Y1349VHV and Y1356VNV can work as docking sites for Shc. The Kd of this interaction, measured in real time using synthetic phosphopeptides and recombinant Shc on a BIAcore biosensor, is 150 nm for both sites. After stimulation of the HGF receptor, Shc is phosphorylated on Y317VNV, generating an high affinity binding site for Grb2 (Kd = 15 nM). This duplicates the high affinity binding site for Grb2 present on the HGF receptor (Y1356VNV). Thus HGF stimulation can trigger the Ras pathway by recruiting Grb2 both directly through the receptor, and indirectly, through Shc. Overexpression of wild-type Shc, but not of the Y317-->F mutant, enhances cell migration and growth in response to HGF. These data show that Shc is a relevant substrate of the HGF receptor, and works as an 'amplifier' of the motogenic as well as of the mitogenic response.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Inhibition of CD4/p56lck signaling by a dominant negative mutant of the Shc adaptor protein.

T-cell antigen receptor stimulation results in phosphorylation of the SH2 containing Shc proteins and recruitment of the Grb2/mSos complex suggesting that Shc proteins are involved in transducing T-cell activating signals to Ras. We have measured the effects of the isolated Shc-SH2 domain and the dominant negative RasN17 protein on activation of the T-cell specific transcription factor NF-AT. The isolated Shc-SH2 domain was designed to compete with endogenous Shc binding to upstream tyrosine phosphorylated proteins and to interfere with coupling to regulators of Ras activation. We have demonstrated that both the Shc-SH2 domain and the RasN17 protein significantly inhibited NF-AT activation by the CD4 coreceptor and the CD4 associated tyrosine kinase p56lck. In contrast, only the RasN17 protein reduced NF-AT activation by the TCR/CD3 complex. Furthermore, tyrosine kinase activity and p56lck protein were found in complexes immunoprecipitated with Shc specific antisera after CD4 triggering but not after CD3 triggering. These results indicate that both CD4 and CD3 signal to Ras and that this signaling is mediated by independent pathways of activation of the Shc adaptor protein.

Base Sequence↗

PubMed1995

Overexpression of Shc proteins potentiates the proliferative response to the granulocyte-macrophage colony-stimulating factor and recruitment of Grb2/SoS and Grb2/p140 complexes to the beta receptor subunit.

The high affinity receptor for GM-CSF consists of a unique alpha subunit and a beta subunit that is shared with receptors for IL-3 and IL-5. Activation of GM-CSF receptor (GMR) triggers two distinct cytoplasmic signalling pathways, JAK2 and Ras, and is sufficient to maintain proliferation of growth factor-dependent cell lines. Shc proteins are phosphorylated upon activation of GMR and may be involved in the transmission of GM-CSF signals to Ras. To define the role of Shc proteins in cells stimulated with GM-CSF, we investigated both the network of interactions that involve Shc after GM-CSF stimulation and the effects of overexpressing Shc proteins on the proliferative response to GM-CSF. Two cytoplasmic complexes, Grb2/Sos and Grb2/p140 bind through the Grb2 SH2 domain to phosphorylated Shc, and are thereby recruited to the beta subunit. Both complexes are stable, even in the absence of ligand, and depend on the direct association of p140 and Sos respectively with the SH3 domains of Grb2. p140 is an uncharacterized protein constitutively phosphorylated on tyrosine and, in its Grb2-bound form, expressed only in hematopoietic cells, the oligomeric complex formed by phosphorylated beta subunit-phosphorylated Shc-Grb2-SoS-p140 is also induced by IL-3 and L-5 stimulation of growth-factor dependent cell lines. Overexpression of wild-type Shc proteins in growth factor-dependent cells increases both MAP kinase activation and proliferation in response to GM-CSF. These effects require the association of Shc with Grb2. Taken together these results indicate that phosphorylation of Shc proteins is a crucial step in the transmission of GM-CSF proliferative stimuli, since it creates a high affinity binding site for the Grb2/SoS complex, whose function is to activate Ras and, for the Grb2/p140 complex, whose function remains unknown.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Chromosome locations of genes encoding human signal transduction adapter proteins, Nck (NCK), Shc (SHC1), and Grb2 (GRB2).

Abnormalities due to chromosomal aberration or point mutation in gene products of growth factor receptors or in ras gene products, which lie on the same signaling pathway, can cause disease in animals and humans. Thus, it can be important to determine chromosomal map positions of genes encoding "adapter" proteins, which are involved in transducing signals from receptor tyrosine kinases to downstream signal recipients such as ras, because adaptor protein genes could also, logically, serve as targets of mutation, rearrangement, or other aberration in disease. Therefore, DNAs from panels of rodent-human hybrids carrying defined complements of human chromosomes were assayed for the presence of the cognate genes for NCK, SHC, and GRB2, three SH2 or SH2/SH3 (Src homology 2 and 3) domain-containing adapter proteins. Additionally, NCK and SHC genes were more narrowly localized by chromosomal in situ hybridization. The NCK locus is at chromosome region 3q21, a region involved in neoplasia-associated changes; the SHC cognate locus, SHC1, is at 1q21, and the GRB2 locus is at 17q22-qter telomeric to the HOXB and NGFR loci. Both SHC1 and GRB2 are in chromosome regions that may be duplicated in some tumor types.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Involvement of Src-homology/collagen (SHC) proteins in signaling through the insulin receptor and the insulin-like-growth-factor-I-receptor.

Src homology/collagen (SHC) proteins are thought to participate in signaling through both receptor tyrosine kinases, such as the insulin receptor and the EGF (epidermal growth factor) receptor, and cytoplasmic tyrosine kinases, such as v-src and v-fps. Here we approached the insulin-induced and the insulin-like-growth-factor-I-induced (IGF-I-induced) phosphorylation of SHC proteins, and the possible role of these proteins in insulin and IGF-I signaling. First, we showed that SHC proteins are phosphorylated on tyrosine residues upon insulin and IGF-I treatment of fibroblasts transfected with a SHC cDNA construct. More important, ligand-activated insulin and IGF-I receptors phosphorylate SHC proteins in vitro, indicating that SHC proteins could be direct substrates for insulin and IGF-I receptors. Further, insulin or IGF-I treatment of SHC-transfected fibroblasts leads to immunoprecipitation of SHC proteins with insulin-receptor substrate 1 (IRS-1). We next looked at the possible effect of SHC proteins on biological responses in SHC-transfected fibroblasts. We found that the expression of exogenous SHC proteins results in an increased basal MEK (MAPK/ERK-activating kinase) activity. Further, neither the basal nor the insulin-induced or IGF-I-induced PtdIns-3-kinase activity were modified by expression of exogenous SHC proteins. These results illustrate that SHC proteins are implicated in the MAP (mitogen-activated protein)-kinase pathway, but not in that of PtdIns-3-kinase. Finally, we show that SHC-transfected cells, unlike control cells, are able to advance into the early phases of the cell cycle, and are more sensitive to the growth-promoting effect of insulin. In conclusion, SHC proteins are substrates for insulin and IGF-I receptors, and would appear to function as early post-receptor signaling components.

3T3 Cells↗

PubMed1994

Transformation by polyoma virus middle T-antigen involves the binding and tyrosine phosphorylation of Shc.

Polyoma virus middle T-antigen converts normal fibroblasts to a fully transformed, tumorigenic phenotype. It achieves this, at least in part, by binding and activating one of the non-receptor tyrosine kinases, pp60c-src, pp62c-yes or pp59c-fyn (reviewed in refs 2 and 3). As a result, middle T-antigen itself is phosphorylated on tyrosine residues, one of which (Tyr 315) acts as a binding site for the SH2 domains of phosphatidylinositol-3'OH kinase 85K subunit. Here we show that another tyrosine phosphorylation site in middle T-antigen (Tyr 250; refs 4, 5) acts as a binding region for the SH2 domain of the transforming protein Shc. This results in Shc also becoming tyrosine-phosphorylated and binding to the SH2 domain of Grb2 (ref. 10). This probably stimulates p21ras activity through the mammalian homologue of the Drosophila guanine-nucleotide-exchange factor Sos (reviewed in ref. 11). We suggest that middle T-antigen transforms cells by acting as a functional homologue of an activated tyrosine kinase-associated growth-factor receptor.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Cancer genetics.

The accumulation of multiple genetic changes underlies the process of tumorigenesis, and both dominantly acting oncogenes and inactivated tumor suppressor genes co-exist in the same tumor. Individual mutations are thought to independently contribute to the kaleidoscopic transformed phenotype. Several examples have now been found of mutations in genes that, through different mechanisms, act on central control points either to ensure genome stability or to regulate the common pathways that signal cell proliferation, survival and differentiation. Mutations at these loci may have multiple, and apparently unrelated, phenotypic consequences.

Animals↗