Biomedical subjects

A Chavanieu

Publications and source records attributed to A Chavanieu.

24 records · Page 2Linked to original sources

PubMed1993

Resin immobilized synthetic peptides used to characterize phosphorylation and antigenic properties of insulin receptor autophosphorylation domains.

To develop a common strategy in peptide design for kinase assay, antibody production and affinity purification, we investigated phosphorylation and antigenic properties of peptides immobilized on an aminated polyacrylic resin (Expansin) corresponding to autophosphorylation domains of the insulin receptor tyrosine kinase. Immobilized peptides (1143-1155) and peptide (1314-1330), designated p1151 and p1322, respectively, were good substrates for the insulin receptor with Km of 0.74 and 0.78 mM. By contrast, peptide (952-963), designated p960, was poorly phosphorylated. p1151 showed distinctive behaviour as a substrate, displaying a higher basal phosphorylation, a leftward shift of the insulin dose-response curve (ED50 = 0.7 ng mL-1 insulin compared to 20 ng mL-1 for other substrates) and an inhibition by 90% of receptor autophosphorylation (ID50 = 0.5 mM). Similar substrate behaviour was observed with another tyrosine kinase, the pp60c-src. Antibodies against P1151 and p1322 have comparable reactivity in ELISA, but the antibody against p960 was poor. While purified immunoglobulins (IgG) against both p1151 and p1322 were inhibitors of receptor autophosphorylation and kinase, in immunoprecipitation the IgG against p1151 mainly interacted with the phosphorylated receptor and that against p1322 with non-phosphorylated forms. Functional mapping of the receptor with oligoclonal 1322-antibody revealed inhibition of phosphate transfer to exogenous substrate poly(Glu,Tyr) (4:1) but not towards immobilized p1151. These data provide further support for the distinctive features of endogenous phosphorylation domain 1151. We conclude that immobilized peptides on polyacrylic resin offer a major new potential for use in kinase assays, immunization, immunoabsorbent techniques and purification of well defined oligoclonal antibodies.

Amino Acid Sequence↗

PubMed1992

Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1.

Monoclonal antibodies raised against the 85-kDa subunit (p85) of bovine phosphatidylinositol (PI) 3-kinase were found to recognize uncomplexed p85 or p85 in the active PI 3-kinase. Immunoprecipitation studies of Chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and PI 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. Insulin also induced an association of p85 with the tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and other phosphorylated proteins ranging in size from 100 to 170 kDa but not with the activated insulin receptor. In vitro reconstitution studies were used to show p85 in the active PI 3-kinase associated with the tyrosine-phosphorylated IRS-1 but not with the activated insulin receptor. Competition studies using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of IRS-1 revealed that phosphopeptides containing YMXM motifs inhibited this association with different potencies, whereas nonphosphorylated analogues and a phosphopeptide containing the EYYE motif had no effect. Src homology region 2 domains of p85 expressed as glutathione S-transferase fusion proteins also bound to tyrosine-phosphorylated IRS-1. These results suggest that insulin causes the association of PI 3-kinase with IRS-1 via phosphorylated YMXM motifs of IRS-1 and Src homology region 2 domains of p85.

Amino Acid Sequence↗

PubMed1992

Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain.

We studied the inhibitory effect of non-phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143-1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine-phosphorylated peptides were synthesized using the N-(9-fluorenylmethoxycarbonyl)-O-dibenzylphosphono-L- tyrosine. The triphosphorylated peptide (1151-P3) and the non-phosphorylated peptide (1151-NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose-dependent and additive manner. When the receptor was pre-phosphorylated for 1 min with [gamma-32P]ATP, 1151-P3 decreased autophosphorylation to 60% of maximum, whereas 1151-NP had no further effect. In both non-activated and preactivated receptors, 1151-P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu4, Tyr) was dose-dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 1151-P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151-NP and 1151-P3 competition for the phosphorylation of a resin-immobilized 1151 peptide. While 1151-NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 1151-P3 caused a four-fold increase in the phosphorylation of 1151-NP--resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1314-1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non-phosphorylated receptor forms. Nevertheless, the 1322Ab immunoprecipitated a fully autophosphorylated receptor in the presence of 1151-NP, but not of 1151-P3, thus suggesting a conformational change induced by the non-phosphorylated peptide. In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 1151-peptide tyrosines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151-NP--resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.

Amino Acid Sequence↗

PubMed1991

Cellular mechanism of metformin action.

Activation of the insulin receptor tyrosine kinase and tyrosine phosphorylation of intracellular substrates are important steps in insulin signalling. In order to elucidate the cellular mechanism of action of metformin (NN'dimethylbiguanide) we have focused towards the effects of metformin on the insulin receptor kinase, the phosphorylation cascade and the biological effect of insulin. Since annexins (lipocortins) have been recently recognized as substrates of several tyrosine kinases we have investigated the effect of metformin on phosphorylation of annexins after insulin stimulation or microinjection of pp60c-src kinase in Xenopus laevis oocytes. Insulin induced in oocytes progression through the cell cycle from late G2 to M phase (maturation). Microinjection of pp60c-src kinase or treatment with metformin potentiates both the rate and the level of insulin-induced oocyte maturation. In oocytes prelabeled with 32P orthophosphate metformin potentiates insulin induced phosphorylation of annexins. It is concluded that annexins are substrates of the phosphorylation cascade initiated by insulin which is synergistic to the action of pp60c-src kinase and that this early phosphorylation events correlate well with the enhanced biological effect of insulin during metformin treatment.

Animals↗

PubMed1988

Evaluation by push-pull cannulation of ACTH in the cerebrospinal fluid of intact and hypophysectomized rats.

Adrenocorticotropin (ACTH) was measured in the cerebrospinal fluid (CSF) of male rats implanted with a push-pull cannula in the 3rd ventricle (ICV) and perfused for 3-6 hr. In 5 conscious sham-lesioned controls studied one week after cannulation, CSF ACTH displayed a pulsatile circhoral pattern with a mean concentration of 10.3 +/- 1.1 pg ACTH/15 min, and a peak/trough ratio of about 2. This pattern was not affected by 2 min of ether vapor inhalation or by pentobarbital anesthesia. The high mortality rate following ICV push-pull cannulation in rats hypophysectomized for 2 weeks led us to perform the ICV perfusion just after cannulation in 5 hypophysectomized rats when they were still under pentobarbital anesthesia. Even so the levels and pulsatile pattern of ACTH release into the CSF were unchanged despite the complete disappearance of any detectable amount of ACTH from the plasma. In two of the latter specimens perfused again one week later but without anesthesia, CSF ACTH levels still were indistinguishable from controls. It can thus be concluded that the ACTH detected in the 3rd ventricle CSF is of neuronal origin.

Adrenocorticotropic Hormone↗

PubMedDate not supplied

Peptides quantitative structure-function relationships: an automated mutation strategy to design peptides and pseudopeptides from substitution matrices.

The process by which analogs in peptide chemistry are currently designed does not include any quantitative basis for amino acid substitutions from pharmacological leads. Here, we show that substitution matrices such as PAM 250 can provide quantitative constraints compatible with biological activity. This article describes its use in a strategy of rational amino acid substitution in peptides and proteins: we have computed a chemically derived matrix equivalent to the well-known PAM 250 matrix, reflecting the natural mutability rates of amino acids in protein evolutions but that can be extended to all the noncoded amino acids. Some of these noncoded amino acids are widely used to mimic secondary structure, to constrain backbone conformation, or to evade protease degradation. An automated sequence mutation (ASM) strategy has been defined to generate mutations within constraints. Application of such a substitution matrix to quantitative structure-function relationship studies will be of use in the design of proteins and peptides destined to become pharmaceutical drugs. In particular, issues such as which functionally conserved substitutions are able to satisfy conformational restrictions, oral bioavailability, or formulation demands can be quantitatively addressed.

Amino Acid Substitution↗