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P Gual

Publications and source records attributed to P Gual.

14 recordsLinked to original sources
PubMed2001

Differential requirement of the last C-terminal tail of Met receptor for cell transformation and invasiveness.

Biological responses to Hepatocyte Growth Factor are mediated by the tyrosine kinase receptor encoded by the Met oncogene. Under physiological conditions, Met triggers a multi-step genetic program called 'invasive growth' including cell-dissociation, invasion of extracellular matrices and growth. When constitutively activated, Met can induce cell transformation and metastasis. Phosphorylation of two docking tyrosines in the receptor tail is essential for all biological responses. To investigate the role of the C-terminal part of Met, we have generated mutants lacking either the last 26 or 47 amino acids. As expected, mutants lacking the docking sites fail to mediate cell transformation and invasion. Interestingly, while Met Delta26 can mediate invasion, its transforming ability is severely impaired. Moreover, the lack of the last 26 amino acids strongly reduces Met ability to phosphorylate substrates in vitro and in vivo. These data indicate that the last 26 amino acids are required to confer the kinase its full enzymatic activity, which is critical for cell transformation but dispensable for invasive properties. Finally, we also show that up-regulation of Met enzymatic activity by insertion of a point mutation in the kinase domain (M1250T) overcomes the regulatory role played by the last 26 amino acids of the tail. It is concluded that the C-terminal domain of Met is crucial not only for recruitment of transducers but also for regulation of receptor enzymatic activity.

3T3 Cells↗
PubMed2001

Gab1 phosphorylation: a novel mechanism for negative regulation of HGF receptor signaling.

Signal transduction by HGF receptor, the tyrosine kinase encoded by the MET oncogene, switches on a genetic program called 'invasive growth' inducing epithelial cell dissociation, migration, growth, and ultimately leading to differentiation into branched tubular structures. Sustained tyrosine phosphorylation of the downstream adaptor protein Gab1 is required for the HGF response. Here we show that serine/threonine phosphorylation of Gab1 provides a control mechanism for negative regulation. Treatment with okadaic acid, a potent inhibitor of the serine/threonine protein phosphatases PP1 and PP2A, was followed by activation of a number of serine/threonine kinases, hyper-phosphorylation in serine and threonine of Gab1 and severe inhibition of the HGF-induced biological responses. Under these conditions, Gab1 was found to be concomitantly hypo-phosphorylated in tyrosine, and thus endowed with reduced ability to recruit SH2 containing signal transducers such as PI3 kinase. Among the serine-threonine kinases activated by PP1 and PP2A inhibition, we found that PKC-alpha and PKC-beta1 are required for negative regulation of Gab1. These data provide a novel negative mechanism for the HGF receptor signaling pathways and highlight a potentially useful target for inhibitors of invasive growth.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Cross-talk between the proto-oncogenes Met and Ron.

Scatter Factors control a complex genetic program known as 'invasive growth'. HGF (Scatter factor 1) and MSP (Scatter Factor 2) bind to tyrosine kinase receptors encoded by the proto-oncogenes MET and RON. Using the appropriate 'kinase inactive' mutant receptors, we show that ligand-induced activation of Met results in transphosphorylation of Ron, and vice versa. Transphosphorylation is direct, as it occurs in Met or Ron receptors lacking the docking sites for signal transducers. Phosphate groups are transferred to the tyrosine phosphorylation sites responsible both for kinase up-regulation (Met: Y1234/Y1235 and Ron: Y1238/Y1239) and for generation of signal transducer docking sites (Met: Y1349/Y1356 and Ron Y1353/Y1360). The transphosphorylation specifically takes place for the receptor subfamily, as it is not observed between Met or Ron and ErbB1, ErbB2 or TrkA. Cross-linking experiments show that non-covalent Met-Ron complexes are present on the cell surface, before ligand-induced dimerization. Co-expression of a kinase inactive Ron receptor with naturally-occurring oncogenic Met mutants suppresses the transforming phenotype, suggesting a dominant negative role for the inefficient kinase partner. These data show that, while specific for their ligands, scatter factor receptors cross-talk and cooperate in intracellular signaling.

3T3 Cells↗
PubMed2000

[Prevalence of eating disorders in a representative sample of female adolescents from Navarra (Spain)].

BACKGROUND: In spite of the social concern about eating disorders (ED), information of its prevalence in Spain is limited. Further studies in communities are needed to estimate the frequency of this problem in representative samples of the population. SUBJECTS AND METHODS: To estimate the prevalence of anorexia nervosa (AN), bulimia nervosa (BN), and partial syndromes of the "non specified eating disorders" (NOSED) type, a representative sample of the female population of Navarre was selected using a random multistage sampling scheme. A sample of 2862 participants aged 12 to 21 was studied. The screening procedure was performed using the Eating Attitudes Test (EAT) (values over 30 as the cutoff point) and each diagnosis was confirmed using a semi-structured interview performed by a psychiatry according to DSM-IV criteria. RESULTS: The overall prevalence of ED in this female population was 4.1% (95% CI: 3.45-4.95). The disorder specific prevalences being NOSED 3.1% (95% CI: 2.5-3.8), BN 0.8 (95% CI: 0.5-1.2) and AN 0.3 (95% CI: 0.1-0.6). CONCLUSIONS: Our results show a slight lower prevalence than what other Spanish authors have reported, although we found higher prevalences of incomplete syndromes and suggest that a high social burden does exist and primary prevention strategies are needed.

Adolescent↗
PubMed2000

Sustained recruitment of phospholipase C-gamma to Gab1 is required for HGF-induced branching tubulogenesis.

A distinctive property of Hepatocyte Growth Factor (HGF) is its ability to induce differentiation of tubular structures from epithelial and endothelial cells (branching tubulogenesis). The HGF receptor directly activates PI3 kinase, Ras and STAT signalling pathways and phosphorylates the adaptator GRB2 Associated Binder-1 (Gab1). Gab1 is also phosphorylated in response to Epidermal Growth Factor (EGF) but is unable to induce tubule formation. Comparison of 32P-peptide maps of Gab1 from EGF- versus HGF-treated cells, demonstrates that the same sites are phosphorylated in vivo. However, while both EGF and HGF induce rapid tyrosine phosphorylation of Gab1 with a peak at 15 min, the phosphorylation persists for over 1 h, only in response to HGF. Nine tyrosines are phosphorylated by both receptors. Three of them (Y307, Y373, Y407) bind phospholipase C-gamma (PLC-gamma). Interestingly, the overexpression of a Gab1 mutant unable to bind PLC-gamma (Gab1 Y307/373/407F) did not alter HGF-stimulated cell scattering, only partially reduced the growth stimulation but completely abolished HGF-mediated tubulogenesis. It is concluded that sustained recruitment of PLCgamma to Gab1 plays an important role in branching tubulogenesis.

Animals↗
PubMed2000

Different point mutations in the met oncogene elicit distinct biological properties.

The MET proto-oncogene, encoding the tyrosine kinase receptor for HGF, controls genetic programs leading to cell growth, invasiveness, and protection from apoptosis. Recently, MET mutations have been identified in hereditary and sporadic forms of papillary renal carcinoma (PRC). Introduction of different naturally occurring mutations into the MET cDNA results in the acquisition of distinct biochemical and biological properties of transfected cells. Some mutations result in a high increase in tyrosine kinase activity and confer transforming ability in focus forming assays. These mutants hyperactivate the Ras signaling pathway. Other mutations are devoid of transforming potential but are effective in inducing protection from apoptosis and sustaining anchorage-independent growth. These Met(PRC) receptors interact more efficiently with the intracellular transducer Pi3Kinase. The reported results show that MET(PRC) mutations can be responsible for malignant transformation through different mechanisms, either by increasing the growth ability of cells or by protecting cells from apoptosis and allowing accumulation of other genetic lesions.-Giordano, S., Maffe, A., Williams, T. A., Artigiani, S., Gual, P., Bardelli, A., Basilico, C., Michieli, P., Comoglio, P. M. Different point mutations in the met oncogene elicit distinct biological properties.

Apoptosis↗
PubMed1999

MET(PRC) mutations in the Ron receptor result in upregulation of tyrosine kinase activity and acquisition of oncogenic potential.

Ron and Met are structurally related receptor tyrosine kinases that elicit a complex biological response leading to invasive growth. Naturally occurring point mutations activate the Met kinase in papillary renal carcinomas (MET(PRC) mutations). By site-directed mutagenesis, we generated homologous amino acid substitutions in the Ron kinase domain and analyzed the biochemical and biological properties of the mutant receptors. Among the mutations studied, D(1232)H and M(1254)T displayed transforming activity in NIH3T3 cells, inducing focus formation and anchorage-independent growth. The D(1232)H and M(1254)T substitutions resulted in increased Ron autophosphorylation both in vivo and in vitro and constitutive binding to intracellular signal transducers. Both mutations yielded a dramatic increase in catalytic efficiency, indicating a direct correlation between kinase activity and oncogenic potential. Molecular modeling of the Ron D(1232)H mutation suggests that this single amino acid substitution favors the transition of the kinase from the inactive to the active state. These data demonstrate that point mutations can confer transforming activity to the Ron receptor and show that RON is a potential oncogene.

3T3 Cells↗
PubMed1998

Interaction of Janus kinases JAK-1 and JAK-2 with the insulin receptor and the insulin-like growth factor-1 receptor.

Insulin and insulin-like growth factor-1 (IGF-1) treatment of cells overexpressing the insulin receptor or the IGF-1 receptor promotes phosphorylation and activation of Janus kinases JAK-1 and JAK-2 but not of TYK-2. With insulin, we observed maximal phosphorylation of JAK-1 within 2 min (5.2 +/- 0.6-fold) and maximal phosphorylation of JAK-2 within 10 min (2.4 +/- 0.6-fold). In cells incubated with IGF-1, we found maximal phosphorylation of JAK-2 within 2 min (1.9 +/- 0.2-fold) and of JAK-1 within 5 min (4.5 +/- 0.4-fold). The JAKs from insulin- or IGF-1-stimulated cells were activated, as shown by their autophosphorylation in vitro. Moreover, they were able to phosphorylate in vitro native insulin receptor substrate (IRS)-1 and a fragment of IRS-2 (GST-IRS-2591-786). Comparison of 32P-peptide maps of IRS-1 phosphorylated in vitro by the insulin receptor vs. JAK-1 showed the occurrence of different phosphopeptides, suggesting that different sites are likely to be phosphorylated by the two kinases. Finally, coprecipitation of receptors and JAK-1 was seen, and phosphorylation of both receptors was found to be necessary for receptor binding to JAK-1. Two domains of JAK- 1 are involved in the formation of the complex between receptor and JAK-1, i.e. the N-terminal portion containing JH7 and JH6 domains, and the C-terminal kinase domain (JH1 domain). Taking our data together, we conclude that: 1) insulin and IGF-1 lead to phosphorylation and activation of JAK-1 and JAK-2 in intact cells; 2) phosphorylation of IRS-I by JAK-1 seems to occur on sites different from those phosphorylated by the insulin receptor; 3) JAK-1 interacts directly with phosphorylated insulin and IGF-1 receptors; and 4) the JH7-JH6 and JH1 domains of JAK-1 are responsible for the interaction with insulin and IGF-1 receptors.

3T3 Cells↗
PubMed1996

Role of the insulin receptor C-terminal acidic domain in the modulation of the receptor kinase by polybasic effectors.

Basic polymers such as polylysine have been found to activate insulin receptor autophosphorylation and kinase activity toward substrates. It was suggested that acidic receptor domains may be involved in the interaction of the receptor with these basic effectors. In a previous study, we have shown that the receptor acid-rich C-terminal sequence, including residues 1270-1280, is involved in the regulation of the receptor kinase activity. Moreover, this domain may be the site of interaction with histone, which is a modulator of the receptor kinase. In this study, we investigated whether the insulin receptor domain comprising amino acids 1270-1280 is involved in the interaction with polybasic effectors. We used anti-peptide serum directed to this sequence, and basic activators such as polylysine, polyarginine and protamine sulfate. Our antibodies inhibit polylysine-induced receptor autophosphorylation, whereas they have no effect on receptor phosphorylation stimulated by concanavalin A which is a non-basic activator of the insulin receptor. Polylysine-induced receptor aggregation was blocked by the antibodies (Fab fragments or whole Ig), indicating that competition occurs between the antibody and polylysine at the level of their binding site to the receptor. Finally, we observed a direct interaction of the 125I-peptide corresponding to receptor sequence 1270-1280 with the basic polymers in dot-blot experiments. Interestingly, the peptide did not bind spermine, a basic molecule which is not an activator of the insulin receptor kinase. Our data indicate that the insulin receptor C-terminal acidic domain including residues 1270-1280 is involved in the interaction of polylysine and other polybasic molecules with the receptor. Since this receptor region has been implicated in the regulation of the receptor kinase activity, we propose that interaction of basic effectors with this domain may be responsible for their activating properties.

Antibodies↗
PubMed1996

Insulin receptor-induced phosphorylation of cellular and synthetic substrates is regulated by the receptor beta-subunit C-terminus.

The transmembrane beta-subunits of the insulin receptor possess hormone-sensitive tyrosine kinase activity. To study the role of the C-terminus domain, a rabbit antipeptide antibody directed to the 1294-1317 domain was produced. The antipeptide antibody inhibited the receptor-induced phosphorylation of poly (Glu, Tyr) and synthetic peptides corresponding to the receptor autophosphorylation sites. In contrast, the same antibody did not inhibit receptor autophosphorylation. The kinetic parameters of the poly(Glu, Tyr) phosphorylation reaction indicated that the antibody interfered with the receptor enzymatic site. Concerning the insulin receptor cellular substrates, the anti-(1294-1317) antibody inhibited Src homology/collagen and IRS-1 phosphorylation. The extent of inhibition was 52% for Src homology/collagen phosphorylation and 30% for IRS-1 phosphorylation. From our data, we conclude that a similar regulation of insulin receptor-induced phosphorylation of artificial and cellular insulin receptor substrates can be generated at the level of the receptor beta-subunit C-terminus.

Amino Acid Sequence↗
PubMed1995

A conformational change in the beta-subunit of the insulin-like growth factor I receptor identified by antipeptide antibodies.

Insulin-like growth factor I (IGF-I) binding to its receptor results in receptor autophosphorylation and phosphorylation of several cellular substrates. The mechanism by which binding of the ligand to the extracellular receptor domain activates the intracellular kinase remains to be defined. Using polyclonal antibodies against four regions of the IGF-I receptor, we searched for putative conformational changes occurring in purified receptors. We studied the ability of the antipeptide antibodies to immunoprecipitate the native, ligand-occupied, or autophosphorylated IGF-I receptor. We found that the antipeptide antibody directed to the sequence 985-998 of the kinase domain immunoprecipitated the phosphorylated receptor, but not the native or the ligand-occupied receptor. By contrast, the antibody against the sequence 950-957 of the juxtamembrane domain immunoprecipitated the three receptor forms. The difference between phosphorylated receptors and unphosphorylated receptors was not observed in Western blot experiments, indicating that the conformational modification of the receptors is not detected upon unfolding. These data demonstrate that the IGF-I receptor undergoes an autophosphorylation-induced conformational change detectable in the kinase domain. Our work provides evidence that conformational changes induced by autophosphorylation may be a common activation mechanism for tyrosine kinase receptors.

3T3 Cells↗
PubMed1989

[Anorexia nervosa: study method and sleep analysis].

By studying anorexia nervosa with an Integrated Inventory and the quality and the quantity of sleep applying Hauri's scale for the analysis of dream contents, the sleeping habits of 50 anorexic patients who were under treatment have been studied. The results show that sleep in these patients is similar and sometimes better in quantity and quality than those in the control group. Their dreams are characterized by an almost total absence of sexual, aggressive and alimentary contents, and that reality, active participation, unpleasant feelings and sensory-perceptive elements are predominant.

Adolescent↗