Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 38
Filter
1.
Exp Gerontol ; 46(11): 934-45, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21871552

ABSTRACT

In 2004, the integrated European project GEHA (Genetics of Healthy Ageing) was initiated with the aim of identifying genes involved in healthy ageing and longevity. The first step in the project was the recruitment of more than 2500 pairs of siblings aged 90 years or more together with one younger control person from 15 areas in 11 European countries through a coordinated and standardised effort. A biological sample, preferably a blood sample, was collected from each participant, and basic physical and cognitive measures were obtained together with information about health, life style, and family composition. From 2004 to 2008 a total of 2535 families comprising 5319 nonagenarian siblings were identified and included in the project. In addition, 2548 younger control persons aged 50-75 years were recruited. A total of 2249 complete trios with blood samples from at least two old siblings and the younger control were formed and are available for genetic analyses (e.g. linkage studies and genome-wide association studies). Mortality follow-up improves the possibility of identifying families with the most extreme longevity phenotypes. With a mean follow-up time of 3.7 years the number of families with all participating siblings aged 95 years or more has increased by a factor of 5 to 750 families compared to when interviews were conducted. Thus, the GEHA project represents a unique source in the search for genes related to healthy ageing and longevity.


Subject(s)
Aging/genetics , Longevity/genetics , Patient Selection , Research Design , Aged , Aged, 80 and over , Cognition , Europe/epidemiology , Family , Female , Genetic Linkage , Genome-Wide Association Study , Humans , Life Style , Male , Middle Aged , Surveys and Questionnaires
2.
Neurogastroenterol Motil ; 20(3): 206-12, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17919311

ABSTRACT

The adaptor protein Rai (ShcC/N-Shc) is almost exclusively present in the nervous system, although little is documented about its expression in the gut and the enteric nervous system (ENS). As Rai is a physiological substrate of Ret, an important factor for the development of ENS, we have evaluated the expression of Rai in the ENS in various segments of the human gastrointestinal tract. The expression of Rai was assessed by immunohistochemistry in disease-free human gut samples (oesophagus, stomach, small bowel and colon) obtained from subjects undergoing surgical procedures. Rai was not expressed in the epithelia or lymphoid tissue, whereas a moderate level of expression was observed in the endothelial cells of blood vessels and on the outer membrane of smooth muscle cells in both the muscularis mucosae and the muscularis propria. In the ENS, strong positivity was observed only in enteric glial cells, overlapping with GFAP and S100. In conclusion, Rai is expressed in the human gut, especially in the enteric glial cells. We conclude that Rai may provide an additional marker for this cell type.


Subject(s)
Enteric Nervous System/metabolism , Neuropeptides/biosynthesis , Biomarkers , Endothelial Cells/metabolism , Epitopes , Gastrointestinal Tract/metabolism , Glial Fibrillary Acidic Protein/biosynthesis , Glial Fibrillary Acidic Protein/genetics , Humans , Immunohistochemistry , Mutation/physiology , Neuropeptides/genetics , S100 Proteins/biosynthesis , S100 Proteins/genetics , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 3 , Tissue Banks
3.
Mol Cell Neurosci ; 34(4): 592-602, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17303436

ABSTRACT

The vulnerability of motor neurons in transgenic SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), may depend on the failure of these cells to activate survival mechanisms in response to the toxic mutant SOD1. To test this we investigated whether defects in the PI3K/Akt pathway, a survival signal, and of its neuron-specific activator, Rai, were important for motor neuron degeneration in these mice. No substantial changes were found in the levels of Rai, PI3K(p85) or phosphorylated Akt (P-Akt) in the ventral horn of spinal cord of SOD1G93A mice during disease progression. P-Akt immunoreactivity was the same in degenerating and healthy motor neurons. Rai ablation in SOD1G93A mice slightly accelerated the motor dysfunction without affecting their life span. Thus, motor neurons in SOD1G93A mice do not lose the pro-survival PI3K/Akt signal nor increase it in order to suppress the cell death mechanisms.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Motor Neurons/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiology , Spinal Cord/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Blotting, Western , Cell Survival/physiology , Disease Models, Animal , Disease Progression , Female , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Mice , Mice, Transgenic , Microscopy, Confocal , Motor Neurons/pathology , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Spinal Cord/pathology , Superoxide Dismutase/genetics
4.
Nat Neurosci ; 4(6): 579-86, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11369938

ABSTRACT

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).


Subject(s)
Adaptor Proteins, Signal Transducing , Cell Differentiation/physiology , Nerve Tissue Proteins/physiology , Neurons/physiology , Stem Cells/physiology , Carrier Proteins/metabolism , Cell Death , Cell Survival , Cells, Cultured , Cloning, Molecular , Epidermal Growth Factor/pharmacology , Fetus , Green Fluorescent Proteins , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Mitogen-Activated Protein Kinases/metabolism , Nerve Growth Factor/pharmacology , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proteins/physiology , Recombinant Fusion Proteins/metabolism , Shc Signaling Adaptor Proteins , Stem Cells/cytology , Telencephalon/cytology , Telencephalon/embryology , Transfection , bcl-Associated Death Protein , src Homology Domains
5.
Nature ; 402(6759): 309-13, 1999 Nov 18.
Article in English | MEDLINE | ID: mdl-10580504

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Longevity/physiology , Oxidative Stress , Proteins/physiology , Animals , Apoptosis , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Cyclins/metabolism , Epidermal Growth Factor/pharmacology , Gene Targeting , Herbicides/pharmacology , Heterozygote , Homozygote , Hydrogen Peroxide/pharmacology , Longevity/drug effects , Longevity/genetics , Longevity/radiation effects , Male , Mice , Paraquat/pharmacology , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Proteins/genetics , Selection, Genetic , Shc Signaling Adaptor Proteins , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1 , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tyrosine/metabolism , Ultraviolet Rays , Up-Regulation
6.
J Virol ; 73(2): 1427-37, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9882348

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Antigens, Polyomavirus Transforming/metabolism , Proteins/metabolism , Signal Transduction , 3T3 Cells , Animals , Antigens, Polyomavirus Transforming/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Cytoplasm/metabolism , Mice , Phosphorylation , Phosphotyrosine/metabolism , Rats , Retinoblastoma Protein/metabolism , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1
7.
J Biol Chem ; 273(5): 3003-12, 1998 Jan 30.
Article in English | MEDLINE | ID: mdl-9446614

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , Calcium-Binding Proteins/metabolism , Coated Pits, Cell-Membrane/physiology , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Repressor Proteins , 3T3 Cells , Adaptor Proteins, Signal Transducing , Animals , Clathrin , DNA-Binding Proteins/metabolism , Endocytosis , Intracellular Signaling Peptides and Proteins , Mice , Protein Binding , Transcription Factor AP-2 , Transcription Factors/metabolism
8.
J Mol Biol ; 269(5): 694-703, 1997 Jun 27.
Article in English | MEDLINE | ID: mdl-9223634

ABSTRACT

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.


Subject(s)
Peptide Library , Peptides/metabolism , Proto-Oncogene Proteins/metabolism , Tyrosine/metabolism , Consensus Sequence , Genetic Vectors , Inoviridae/genetics , Phosphopeptides , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-fyn , Selection, Genetic , Substrate Specificity , src Homology Domains
9.
Oncogene ; 14(7): 773-82, 1997 Feb 20.
Article in English | MEDLINE | ID: mdl-9047384

ABSTRACT

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.


Subject(s)
Drosophila Proteins , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , 3T3 Cells , Animals , Binding Sites , COS Cells , Mice , Mutation , Phosphorylation , Proto-Oncogene Proteins c-ret , Tyrosine/metabolism , src Homology Domains
10.
EMBO J ; 16(4): 706-16, 1997 Feb 17.
Article in English | MEDLINE | ID: mdl-9049300

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , ErbB Receptors/metabolism , Proteins/metabolism , Signal Transduction/physiology , 3T3 Cells , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Transformation, Neoplastic , Cloning, Molecular , DNA, Complementary/genetics , Enzyme Activation , Epidermal Growth Factor/pharmacology , GRB2 Adaptor Protein , Genes, fos/genetics , Humans , Mice , Molecular Sequence Data , Phosphorylation , Promoter Regions, Genetic/genetics , Proteins/genetics , Proteins/physiology , RNA Splicing/physiology , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , Tyrosine/metabolism
11.
Oncogene ; 13(3): 633-41, 1996 Aug 01.
Article in English | MEDLINE | ID: mdl-8760305

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Chitinases/genetics , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA-Binding Proteins/genetics , Plant Proteins , Proteins/genetics , RNA-Binding Proteins/genetics , src Homology Domains/genetics , Amino Acid Sequence , Base Sequence , Chitinases/metabolism , Cloning, Molecular , Conserved Sequence , DNA-Binding Proteins/metabolism , ErbB Receptors/metabolism , Humans , Molecular Sequence Data , Polypyrimidine Tract-Binding Protein , Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Shc Signaling Adaptor Proteins , Signal Transduction/physiology , Src Homology 2 Domain-Containing, Transforming Protein 1 , Tumor Cells, Cultured
12.
Trends Biochem Sci ; 21(7): 257-61, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8755247

ABSTRACT

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.


Subject(s)
ErbB Receptors/metabolism , Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , ras Proteins/metabolism , Animals , Phosphorylation
13.
Mol Cell Biol ; 16(5): 1946-54, 1996 May.
Article in English | MEDLINE | ID: mdl-8628261

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Endoplasmic Reticulum/metabolism , ErbB Receptors/biosynthesis , ErbB Receptors/metabolism , Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , 3T3 Cells , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Endoplasmic Reticulum/ultrastructure , Enzyme Activation , Epidermal Growth Factor/pharmacology , Fluorescent Antibody Technique , Mice , Microscopy, Immunoelectron , Phosphorylation , Protein Biosynthesis , Proteins/analysis , Receptor, ErbB-2/biosynthesis , Receptor, ErbB-2/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , Subcellular Fractions/metabolism , Subcellular Fractions/ultrastructure , Transfection
14.
Oncogene ; 11(5): 899-907, 1995 Sep 07.
Article in English | MEDLINE | ID: mdl-7675449

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Neoplasms/metabolism , Proteins/metabolism , Tyrosine/metabolism , ErbB Receptors/metabolism , GRB2 Adaptor Protein , Humans , Phosphorylation , Protein-Tyrosine Kinases/genetics , Receptors, Platelet-Derived Growth Factor/metabolism , Tumor Cells, Cultured
15.
Oncogene ; 10(8): 1631-8, 1995 Apr 20.
Article in English | MEDLINE | ID: mdl-7731718

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Hepatocyte Growth Factor/pharmacology , Proteins/physiology , Receptor Protein-Tyrosine Kinases/metabolism , Amino Acid Sequence , Animals , Cell Division/drug effects , Cell Movement/drug effects , GRB2 Adaptor Protein , Haplorhini , Humans , Mice , Molecular Sequence Data , Phosphorylation , Proteins/metabolism , Proto-Oncogene Proteins c-met
16.
Oncogene ; 10(6): 1141-7, 1995 Mar 16.
Article in English | MEDLINE | ID: mdl-7700640

ABSTRACT

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.


Subject(s)
CD4 Antigens/metabolism , Mutation , Nuclear Proteins , Protein-Tyrosine Kinases/metabolism , Signal Transduction , Base Sequence , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , DNA, Complementary , DNA-Binding Proteins/metabolism , Genes, Dominant , Humans , Lymphocyte Specific Protein Tyrosine Kinase p56(lck) , Molecular Sequence Data , NFATC Transcription Factors , Phosphorylation , Transcription Factors/metabolism , Tyrosine/metabolism , ras Proteins/metabolism
17.
Oncogene ; 10(5): 907-17, 1995 Mar 02.
Article in English | MEDLINE | ID: mdl-7898932

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Proteins/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Cell Division/drug effects , GRB2 Adaptor Protein , Genes, ras , Humans , Interleukin-3/pharmacology , Interleukin-5/pharmacology , Phosphorylation , Son of Sevenless Proteins , Tumor Cells, Cultured
18.
Oncogene ; 9(12): 3545-55, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7970715

ABSTRACT

The FLT4, FLT1 and KDR/FLK1 genes encode structurally similar endothelial cell receptor tyrosine kinases. Recently it has been shown that the FLT1 and KDR/FLK-1 proteins function as high-affinity receptors for vascular endothelial growth factor (VEGF). Here we show that FLT4 does not act as a receptor for VEGF, as VEGF did not show specific binding to the FLT4 tyrosine kinase or induce its autophosphorylation. Also, FLT4 did not interact with KDR in response to VEGF. However, when fused with the ligand binding domain of the colony stimulating factor-1 receptor (CSF-1R), the FLT4 tyrosine kinase was specifically activated by CSF-1. The activated FLT4 tyrosine kinase domain was found to interact with the Src homology 2 domains of the SHC and GRB2 adaptor proteins in vitro and with SHC in cells. CSF-1 stimulation of the CSF-1R/FLT4 receptor chimera induced thymidine incorporation in serum-starved NIH3T3 fibroblasts, but not in porcine aortic or murine lung capillary endothelial cells, although tyrosyl phosphorylation of the receptor and SHC occurred in these cells as well. These results suggest that the endothelial cell FLT4 receptor tyrosine kinase transmits signals for an as yet unidentified growth factor.


Subject(s)
Protein Processing, Post-Translational , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cell Surface/metabolism , Receptors, Growth Factor/metabolism , Signal Transduction , 3T3 Cells , Animals , Base Sequence , Cell Line , Enzyme Activation , Hydrolysis , Mice , Mitogens , Molecular Sequence Data , Oligodeoxyribonucleotides , Peptide Biosynthesis , Phosphorylation , Protein Binding , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Receptors, Cell Surface/biosynthesis , Receptors, Vascular Endothelial Growth Factor , Recombinant Fusion Proteins/metabolism , Transfection , Vascular Endothelial Growth Factor Receptor-3
19.
Oncogene ; 9(10): 2827-36, 1994 Oct.
Article in English | MEDLINE | ID: mdl-8084588

ABSTRACT

The mammalian SHC gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are phosphorylated on tyrosine by a variety of receptor and cytoplasmic tyrosine kinases. Phosphorylated Shc proteins form a complex with the SH2-SH3 containing Grb2 protein which is implicated in the regulation of Ras, suggesting that Shc is involved in the intracellular transmission of growth signals from activated tyrosine kinases to Ras. Overexpression of Shc proteins in cultured fibroblasts induces a transformed phenotype. We now report that, in vitro, the high affinity binding of Grb2 to Shc proteins requires phosphorylation of Shc at Tyr317, which lies within the high affinity binding motif for the Grb2 SH2 domain, pYVNV, where Asn at the +2 position is crucial for complex formation. In vivo, Tyr317 is the major, but not the only, site for Shc phosphorylation, and is the sole Shc high affinity binding site for Grb2. Mutant Shc proteins with substitution of the Tyr317 by Phe lose the capacity to be highly phosphorylated on tyrosine upon growth factor receptor activation, to bind Grb2 and to induce neoplastic transformation. In contrast, Shc proteins that have an extensive aminoterminal deletion, but retain the Tyr317 site and the SH2 domain conserve the capacity to be phosphorylated, to bind to Grb2 and to induce cell transformation. These data indicate that the formation of the Shc-Grb2 complex is a crucial event in the transformation induced by overexpression of Shc and support the notion that Shc proteins can deliver activation signals to RAS.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Cell Transformation, Neoplastic , Proteins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Binding Sites , Cell Line , GRB2 Adaptor Protein , Mice , Molecular Sequence Data , Mutation , Phosphorylation , Proteins/genetics , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , Tyrosine/metabolism
20.
Genomics ; 22(2): 281-7, 1994 Jul 15.
Article in English | MEDLINE | ID: mdl-7806213

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Chromosomes, Human, Pair 17 , Chromosomes, Human, Pair 1 , Chromosomes, Human, Pair 3 , Proteins/genetics , Signal Transduction/genetics , Animals , Chromosome Mapping , GRB2 Adaptor Protein , Humans , Hybrid Cells , Mice/genetics , Oncogene Proteins/genetics , Rodentia , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1
SELECTION OF CITATIONS
SEARCH DETAIL
...