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V Baron

Publications and source records attributed to V Baron.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

Dynamin associates with Src-Homology Collagen (Shc) and becomes tyrosine phosphorylated in response to insulin.

The activated insulin receptor phosphorylates docking proteins such as Src-Homology Collagen (Shc) and Insulin Receptor Substrate-1 (IRS-1), which then bind several proteins that contain a Src-Homology 2 (SH2) domain. Both Shc and IRS-1 associate with Growth Factor Receptor-Bound protein 2 (Grb2), an adaptor molecule. The hormone-receptor complex is then rapidly internalized through coated-pits. Dynamin, a 100 kDa protein with GTPase activity, is thought to play a crucial role in receptor-mediated endocytosis. In this study, we show that insulin induces tyrosine phosphorylation of dynamin in cells overexpressing human insulin receptors. Phosphorylation is observed rapidly, i.e. within 1 minute of insulin treatment. Moreover, exposure of cells to the hormone leads to co-immunoprecipitation of dynamin with Shc and with insulin receptor. Since dynamin constitutively associates with Grb2, it could be recruited to the insulin signaling complex through binding of Grb2 to tyrosine-phosphorylated Shc.

Adaptor Proteins, Signal Transducing↗
PubMed1998

[Guidelines for the evaluation of internal quality control of smears for screening of uterine cancer in France in the structures of Pathologic Anatomy and Cytology. French Association for Quality Assurance in Pathologic Anatomy and Cytology (AFAQAP)--Commission for cervical smears].

A national organized mass-screening effective programme is the only way to reduce the risk of cervical cancer, if properly organized and correlated with a system of Quality Assurance. Since 1900, an Association for Quality Assurance was created by the French pathologists, named "AFAQAP". These pathologists thus demonstrated their interest in this kind of action that should be effective if women and clinicians are also implied. The pathologists have concluded the first part of their programme with these French guidelines for internal quality control of pap smears.

Female↗
PubMed1997

Tyr624 and Tyr628 in insulin receptor substrate-2 mediate its association with the insulin receptor.

In addition to the pleckstrin homology domain and the phosphotyrosine binding domain in insulin receptor substrate (IRS)-1 and IRS-2, a region between amino acids 591 and 786 in IRS-2 (IRS-2-(591-786)) binds to the insulin receptor. Based on peptide competition studies, this region interacts with the phosphorylated regulatory loop of the insulin receptor; we designate this region the kinase regulatory loop binding (KRLB) domain. Two tyrosine residues in the KRLB domain at positions 624 and 628 are crucial for this interaction. Phosphorylation of tyrosine residues in the KRLB domain by the insulin receptor inhibits the binding to the receptor. These results reveal a novel mechanism regulating the interaction of the insulin receptor and IRS-2 that may distinguish the signal of IRS-2 from IRS-1.

Amino Acid Sequence↗
PubMed1997

The effect of cyclic adenosine monophosphate on the mitogen-activated protein kinase pathway depends on both the cell type and the type of tyrosine kinase-receptor.

The mitogen-activated protein kinase (MAP kinase) is a key participant in growth factor-stimulated intracellular events such as proliferation and differentiation. We and others have previously described a cross-talk between the MAP kinase pathway and the cAMP pathway. Indeed, in several cell lines and, in particular in fibroblasts, an increase in the level of cAMP produced an inhibition of MAP kinase together with decreased cell proliferation. In contrast, in PC12 cells, cAMP induced an increase in the NGF-induced activation of MAP kinase concomitantly with augmented NGF-induced differentiation. Therefore, it has been proposed that the cellular context is important for the nature of the cAMP effects on growth factor-stimulated MAP kinase activity. Here we show that the type of tyrosine kinase receptor stimulated also participates in the nature of the cAMP effect. Thus, in NIH3T3 fibroblasts expressing NGF receptors (NIH3T3/trk cells) we found that cAMP potentiates NGF-stimulated ERK1 and MEK1 activities, whereas in NIH3T3 fibroblasts expressing insulin receptors (NIH3T3/IR cells) we saw no effect of cAMP on the activation of insulin-stimulated ERK1 and MEK1. In PC12 cells and in Rat1 fibroblasts expressing insulin receptors (PC12/IR and Rat1/IR cells) we observed, respectively, a potentiation and an inhibition of insulin-stimulated ERK1 activity. In addition, cAMP does not seem to modify the basal nor growth factor-stimulated She or IRS-1 tyrosine phosphorylation in the different cell lines studied. Finally, we observed that cAMP inhibited serum- and insulin-induced, but not NGF-induced, cell proliferation in NIH3T3 cells. However, cAMP potentiated insulin-stimulated cell differentiation in PC12/IR cells. These results led us to conclude that the cAMP effect on cell proliferation in NIH3T3 fibroblasts and PC12/IR cells appears to be correlated, in part, with the effect of cAMP on the MAP kinase pathway, but by itself this pathway cannot fully account for these observations.

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

Involvement of Janus kinases in the insulin signaling pathway.

The adaptor molecule growth-factor-receptor-bound protein-2 (Grb2) plays a role in insulin action since it links tyrosine phosphorylated IRS-1 and Shc to the guanine-nucleotide-exchange factor, Sos, which initiates the mitogen-activated-protein (MAP) kinase cascade by producing Ras-GTP. Both IRS-1 and Shc are phosphorylated by the insulin-receptor tyrosine kinase. In the present study, we have investigated whether the tyrosine kinases of the Janus kinase family (JAK) could be involved in insulin signaling by acting on Grb2. In fibroblasts over-expressing insulin receptors we observed that two tyrosine-phosphorylated proteins interact with Grb2 and with a mutant of Grb2, which lacks the Src homology 2 (SH2) domain, indicating that these proteins associate with the SH3 domains of Grb2. Further, we found that both JAK1 and JAK2 constitutively associate with Grb2, through interaction with the SH3 domains of Grb2. Finally, insulin appears to induce the tyrosine phosphorylation of JAK1, but does not modify the tyrosine phosphorylation state of JAK2. In conclusion, our results suggest that the JAK proteins could participate in insulin signal transduction, and could therefore constitute an alternative pathway for mediating some of the pleiotropic responses induced by insulin.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Interaction of the C-terminal acidic domain of the insulin receptor with histone modulates the receptor kinase activity.

In this study, we investigated the role of the insulin receptor domain 1270-1280, an acid-rich sequence located in the receptor C-terminus. Antipeptide IgG raised against this sequence were obtained and used to analyze their effect on receptor function. Antipeptide IgG inhibited receptor autophosphorylation at Tyr1146, Tyr1150 and Tyr1151. These sites are known to be key modulators of the receptor activity. Autophosphorylation at other sites may also have been inhibited. The antipeptide antibody decreased the receptor kinase activity measured with poly(Glu80Tyr20) and a synthetic peptide corresponding to the proreceptor sequence 1142-1158. We provide evidence that the effect of the antibody on substrate phosphorylation may result from the control of the phosphorylation level of the receptor. Concerning the action of the antipeptide IgG on the receptor kinase activity, histone did not behave similarly to poly(Glu80Tyr20). The antibody recognizing sequence 1270-1280 competed with histone for an overlapping binding site. Histone also modulated insulin receptor autophosphorylation, supporting the idea that interference with domain 1270-1280 alters the receptor kinase. Our data suggest that the acidic region including residues 1270-1280 of the insulin receptor C-terminus is involved in the following events: (a) receptor binding with histone, an exogenous substrate of the receptor kinase, and (b) the regulation of receptor autophosphorylation and kinase activity. Based on these observations, we would like to propose that this insulin receptor domain could interact with cellular proteins modulating the receptor kinase.

Animals↗
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↗
PubMed1995

[Mechanism of insulin action].

Insulin is a key hormone regulating glucose homeostasis. Its major target tissues are the liver, the skeletal muscle and the adipose tissue. At the cellular level, insulin activates glucose and amino acids transport, lipid and glycogen metabolism, protein synthesis, and transcription of specific genes. Insulin-induced biological responses are mediated by a specific cell-surface receptor with tyrosine kinase activity. This receptor is a heterotetrameric protein consisting of two extracellular alpha subunits containing the ligand binding site, and two transmembrane beta subunits containing the hormone-sensitive enzymatic activity. The first step following insulin binding consists in receptor autophosphorylation on multiple specific sites and phosphorylation of cellular substrates. We will review the receptor structure, its mechanism of activation, and the autophosphorylation process. Two of the insulin receptor substrates have been identified as IRS-1 and Shc. IRS-1 is phosphorylated at several sites by the insulin receptor, and acts as a docking protein by associating several SH2-containing proteins. One of these proteins is the p85 subunit of P13-kinase which is rapidly stimulated by insulin in adipocytes and skeletal muscle. The phosphorylated IRS-1 also associates Grb2, as does the phosphorylated Shc. This allows recruitment of the preformed complex Grb2-Sos to the plasma membrane. Sos is then capable of stimulating the Ras protein, which in turn activates Raf, the first element of the MAP-kinase cascade. The role of these proteins in insulin signalling will be discussed.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1993

The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity.

Adenosine Triphosphate↗
PubMed1993

Antibodies to the extracellular receptor domain restore the hormone-insensitive kinase and conformation of the mutant insulin receptor valine 382.

A mutation substituting a valine for phenylalanine at residue 382 in the insulin receptor alpha-subunit has been found in two sisters with a genetic form of extreme insulin resistance. This receptor mutation impairs the ability of the hormone to activate autophosphorylation of solubilized receptors and phosphorylation of substrates (Accili, D., Mosthaf, L., Ullrich, A., and Taylor, S. I. (1991) J. Biol. Chem. 266, 434-439). We have previously demonstrated that in native receptors insulin induces a conformational change in the receptor beta-subunit, which is thought to be necessary for receptor activation (Baron, V., Gautier, N., Komoriya, A., Hainaut, P., Scimeca, J. C., Mervic, M., Lavielle, S., Dolais-Kitabgi, J., and Van Obberghen, E. (1990) Biochemistry 29, 4634-4641). Hence, it was thought that a defect in this conformational change might explain the functional defect of the mutant receptor. This appears to be the case, as we demonstrate here that the mutant receptor is locked in its inactive configuration. However, we found two monoclonal antibodies, directed to the extracellular domain, which are capable of restoring the mutant receptor kinase activity. The activation of the mutant receptor was accompanied by restoration of conformational changes in the beta-subunit C terminus. From these data, we draw the two following conclusions. (i) A causal link exists between receptor kinase activation and the occurrence of conformational changes. (ii) Ligands other than insulin, such as antibodies, which perturb the extracellular domain, can function as alternative ways to restore the mutant receptor kinase.

3T3 Cells↗
PubMed1992

The insulin receptor activation process involves localized conformational changes.

The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation.

Animals↗
PubMed1992

Antipeptide antibody to the insulin-like growth factor-I receptor sequence 1232-1246 inhibits the receptor kinase activity.

To approach the question of why insulin-like growth factor-I (IGF-I) and insulin have different physiological actions, we developed antibodies directed against cytoplasmic regions of the IGF-I receptor exhibiting a low degree of homology with the corresponding sequences of the insulin receptor. We found that an antipeptide antibody directed against the beta-subunit carboxyl-terminal sequence (1232-1246) of the IGF-I receptor significantly reduced the in vitro receptor autophosphorylation. The ability of the synthetic peptide corresponding to the IGF-I receptor sequence 1232-1246 to abolish this inhibitory effect reflects the specific nature of the antibody interaction with the targeted domain in the receptor. Antipeptide antibody to IGF-I receptor sequence 1232-1246 also decreased receptor phosphorylation activity toward the exogenous substrate poly(Glu/Tyr). The reduction in poly(Glu/Tyr) phosphorylation was seen even when the antibody was incubated with a receptor previously activated and phosphorylated. Therefore, the inhibitory action on substrate phosphorylation is likely to be unrelated to the antibody reduction of receptor autophosphorylation but rather results from a global decrease in receptor enzymatic activity. The effect of the antipeptide antibody on receptor tyrosine kinase cannot be accounted for by a lowering of the receptor Km for ATP or of its affinity for the substrate poly(Glu/Tyr). Moreover, the interaction of the antibody with the receptor had no repercussion on the ligand binding site as shown by the unaltered IGF-I binding. Taken together our data suggest that the beta-subunit carboxyl-terminal domain of the IGF-I receptor plays a key role in regulating its kinase activity and that the particular sequence recognized by our antipeptide antibody could be involved in negative regulation of receptor functioning.

3T3 Cells↗