RESUMO
Amongst the proteins that are subjected to variation during the cell division cycle few are under hormonal regulation. The variation in amount of tyrosine aminotransferase (TAT) in the hepatic tissue is under the control of glucagon, glucocorticoids and insulin. It has been reported that the inducibility of TAT activity by dexamethasone in rat hepatoma (HTC) is limited to the late G1 and the S portions of the cell cycle. Evidence is presented in this report that in the rat hepatoma Fao, insulin (which has the capability to promote both cell growth and hormonal effects via its own receptors) modulates the TAT activity during the cell cycle. The maximal insulin-stimulated induction of TAT activity was observed at the end of the G1 phase and then decreased as cells progressed through their mitotic cycle. The number of insulin binding sites per cell was decreased by only 30% during the same period of time. Furthermore, the extent of receptor autophosphorylation decreased in the same proportion, suggesting that insulin receptors remained functional through the whole cell cycle. In fact, another insulin-stimulated cellular function, neutral amino-acid transport, was not modified as cells progressed into the S phase. Hydroxyurea, which is known to prevent cell progression into the S phase, stabilized the insulin-induced TAT activity at its maximal level for several hours. Reciprocally, removal of hydroxyurea resulted in a concomitant decrease in TAT activity and reinitiation of DNA synthesis.
Assuntos
Ciclo Celular , Insulina/farmacologia , Tirosina Transaminase/metabolismo , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Dexametasona/farmacologia , Indução Enzimática , Hidroxiureia/farmacologia , Cinética , Neoplasias Hepáticas Experimentais , Mitógenos , Fosforilação , Ratos , Receptor de Insulina/metabolismo , Células Tumorais Cultivadas , Tirosina Transaminase/biossínteseRESUMO
Expression of insulin metabolic effects can be obtained by anti-receptor antibodies without activation of the tyrosine kinase activity [O'Brien R. M., Soos M. A. and Siddle K. (1987) EMBO J. 6, 4003-4010; Forsayeth J. R., Caro J. F., Sinha M. K., Maddux B. A. and Goldfine I. D. (1987) Proc. natn. Acad. Sci. U.S.A. 84, 34,448-34,514; Ponzio G., Contreres J. O., Debant A., Baron V., Gautier N., Dolais-Kitabgi J. and Rossi B. (1988) EMBO J. 7, 4111-4117; Hawley D. M., Maddux B. A., Patel R. G., Wong K. Y., Mamula P. W., Firestone G. L., Brunetti A., Verspohl E. and Goldfine I. D. (1989) J. biol. Chem. 264, 2438-2444; Soos M. A., O'Brien R. M., Brindle N. P. J., Stigter J. M., Okamoto A. K., Whittaker J. and Siddle K. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 5217-5221.]. Recently, we have proposed that receptor cross-linking is sufficient in itself to stimulate glycogen synthesis, even if aggregation was performed on receptors mutated on Tyr 1162 and Tyr 1163 and thus devoid of tyrosine kinase activity [Debant A., Ponzio G., Clauser E., Contreres J. O. and Rossi B. (1989) Biochemistry 28, 14-17]. The aim of this study was to gain information on the involvement of receptor clustering in the expression of the different insulin biological effects. To this end, we studied the mimetic effects of wheat-germ agglutinin, which is likely to induce receptor aggregation without interacting with the receptor protein moiety. Wheat-germ agglutinin failed to promote DNA synthesis, whereas the lectin behaved as a potent mimicker of insulin on tyrosine aminotransferase activity and amino-acid transport. However, this stimulatory effect did not parallel the activation of receptor autophosphorylation. Our data reinforce the idea that the expression of the metabolic effects of insulin are not strictly dependent on a general tyrosine kinase activation.
Assuntos
Insulina/metabolismo , Receptor de Insulina/efeitos dos fármacos , Aglutininas do Germe de Trigo/farmacologia , Ácidos Aminoisobutíricos/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , DNA/biossíntese , Fosforilação , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tirosina Transaminase/metabolismoRESUMO
Peroxovanadiums (pVs) are potent protein tyrosine phosphatase (PTP) inhibitors with insulin-mimetic properties in vivo and in vitro. We have established the existence of an insulin receptor kinase (IRK)-associated PTP whose inhibition by pVs correlates closely with IRK tyrosine phosphorylation, activation, and downstream signaling. pVs have also been shown to activate various tyrosine kinases (TKs) that could participate in activation of the insulin-signaling pathway. In the present study we have sought to determine whether pV-induced IRK tyrosine phosphorylation requires the intrinsic kinase activity of the IRK, and whether IRK activation is necessary to realize the early steps in the insulin-signaling cascade. To address this we evaluated the effect of a pure pV compound, bis peroxovanadium 1,10-phenanthroline [bpV(phen)], in HTC rat hepatoma cells overexpressing normal (HTC-IR) or kinase-deficient (HTC-M1030) mutant IRKs. We showed that at a dose of 0.1 mM, but not 1 mM, bpV(phen) induced IRK-dependent events. Thus, 0.1 mM bpV(phen) increased tyrosine phosphorylation and IRK activity in HTC-IR but not HTC-M1030 cells. Tyrosine phosphorylation of insulin signal-transducing molecules was promoted in HTC-IR but not HTC-M1030 cells by bpV(phen). The association of p185 and p60 with the src homology-2 (SH2) domains of Syp and the p85-regulatory subunit of phosphatidylinositol 3'-kinase was induced by bpV(phen) in HTC-IR, but not in HTC-M1030 cells, as was insulin receptor substrate-1-associated phosphatidylinositol 3'-kinase activity. Thus autophosphorylation and activation of the IRK by bpV(phen) is effected by the IRK itself, and the early events in the insulin- signaling cascade follow from this activation event. This establishes a critical role for PTP(s) in the regulation of IRK activity. bpV(phen) could be distinguished from insulin only in its ability to activate ERK1 in HTC-M1030 cells, thus indicating that this event is IRK independent, consistent with our previous hypothesis that bpV(phen) inhibits a PTP involved in the negative regulation of mitogen-activated protein kinases.
Assuntos
Compostos Organometálicos/farmacologia , Fenantrolinas/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Carcinoma Hepatocelular , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Hepáticas , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/efeitos dos fármacos , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/metabolismo , Proteínas Recombinantes de Fusão , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Especificidade por Substrato/efeitos dos fármacos , Células Tumorais Cultivadas , Tirosina/metabolismo , Domínios de Homologia de src/efeitos dos fármacos , Domínios de Homologia de src/fisiologiaRESUMO
The pivotal role that the tyrosine residues in positions 1162 and 1163 play in the control of the insulin action has been clearly established by substitution of these tyrosine residues for phenylalanine [Ellis, L. (1986) Cell 45, 721-732]. We have recently found that this type of mutation, which abolishes the effects of insulin on glucose metabolism, was without any effect on the mitogenic effect of the hormone [Debant, A. (1988) Proc. Natl. Acad. Sci. U.S.A. (in press)]. Here, we provide evidence that a polyclonal antibody, raised against the human insulin receptor, can restore the receptor-mediated stimulation of glycogen synthesis that was abolished by the mutation. Stimulation of the biological effect by the anti-receptor antibody did not necessitate, whatsoever, the activation of the tyrosine kinase activity and/or receptor autophosphorylation. Furthermore, the antibody-induced reversal of the mutation was not observed when we used Fab fragments alone, but addition of anti-(Fab')2 IgG in a second step resulted in a similar effect as that observed with intact IgG. We propose that Tyr 1162 and Tyr 1163 exert their control on the metabolic effects of insulin through the modulation of receptor aggregation.
Assuntos
Insulina/metabolismo , Receptor de Insulina/metabolismo , Animais , Anticorpos , Linhagem Celular , Glicogênio/biossíntese , Imunoglobulina G , Mutação , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/imunologia , TirosinaRESUMO
We provide evidence that both covalent and non-covalent inhibitors of chymotrypsin-like activities inhibit the insulin-induced DNA replication, while the hormonal metabolic effects such as induction of tyrosine aminotransferase activity or increase of amino-acid transport remain unchanged. Besides, the protease inhibitors that we tested were without any effect on both the autocatalytic phosphorylation of insulin receptors and the tyrosine kinase activity towards poly(glutamate/tyrosine). The inhibitory effect of protease inhibitors on DNA synthesis was also visible when fibroblast growth factor (FGF) was used to commit cells in the proliferative cycle. This observation proves that the involvement of a putative protease is not restricted to the insulin mitogenic pathway. Finally, we observed that Fao cells totally escaped the inhibitory action of a covalent inhibitor of chymotrypsin after having been exposed to insulin for 10 h. We propose that a chymotrypsin-like activity is involved in the intracellular signalling leading to the proliferation of rat hepatoma cells up to a non-return point situated in the middle of G1 (6-8 h).
Assuntos
Quimotripsina/antagonistas & inibidores , Replicação do DNA , Endopeptidases/metabolismo , Insulina/farmacologia , Neoplasias Hepáticas Experimentais/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animais , Transporte Biológico , Replicação do DNA/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Citometria de Fluxo , Insulina/metabolismo , Interfase , Cinética , Neoplasias Hepáticas Experimentais/genética , Mitose , Fosforilação , Ratos , Receptor de Insulina/metabolismo , Tosilfenilalanil Clorometil Cetona/farmacologia , Células Tumorais Cultivadas , Tirosina Transaminase/metabolismoRESUMO
Incubating endosomes with ATP decreased binding of 125I-insulin but not 125I-labeled human growth hormone. Increasing ATP concentrations from 0.1 to 1 mM increased beta-subunit tyrosine phosphorylation and insulin receptor kinase (IRK) activity assayed after partial purification. At higher (5 mM) ATP concentrations beta-subunit tyrosine phosphorylation and IRK activity were markedly decreased. This was not observed with nonhydrolyzable analogs of ATP, nor with plasma membrane IRK, nor with endosomal epidermal growth factor receptor kinase autophosphorylation. The inhibition of endosomal IRK tyrosine phosphorylation and activity was completely reversed by bafilomycin A1, indicating a role for endosomal proton pump(s). The inhibition of IRK was not due to serine/threonine phosphorylation nor was it influenced by the inhibition of phosphotyrosyl phosphatase using bisperoxo(1,10-phenanthroline)oxovanadate anion. Prior phosphorylation of the beta-subunit with 1 mM ATP did not prevent the inhibition of IRK activity on incubating with 5 mM ATP. To evaluate conformational change we incubated endosomes with dithiothreitol (DTT) followed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Without DTT the predominant species of IRK observed was alpha2 beta2. With DTT the alpha beta dimer predominated but on co-incubation with 5 mM ATP the alpha2 beta2 form predominated. Thus, ATP-dependent endosomal acidification contributes to the termination of transmembrane signaling by, among other processes, effecting a deactivating conformational change of the IRK.
Assuntos
Trifosfato de Adenosina/metabolismo , Endossomos/metabolismo , Insulina/metabolismo , Macrolídeos , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo , Animais , Antibacterianos/farmacologia , Ditiotreitol/farmacologia , Endocitose , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Conformação Proteica , ATPases Translocadoras de Prótons/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Reagentes de Sulfidrila/farmacologia , SuínosRESUMO
In a previous report we described the properties of a rabbit anti-insulin receptor antibody (RAIR-IgG) and its effects on the autophosphorylation and kinase activity of human insulin receptors. The present study was carried out on the hepatoma cell line Fao. We tested the mimetic effects of RAIR-IgG on different biological parameters known to be stimulated by insulin, receptor autophosphorylation and kinase activity. RAIR-IgG stimulated the metabolic effects (glucose and amino acid transport) but, unlike insulin, was unable to promote cell proliferation. These data clearly demonstrated the existence of two distinctly controlled pathways in the mediation of the hormonal response. When we investigated the effects of this antibody at the molecular level we found that in a cell-free system RAIR-IgG weakly stimulated receptor autophosphorylation on non-regulatory sites and failed to stimulate tyrosine kinase activity toward exogenous substrates. Accordingly, RAIR-IgG did not stimulate receptor autophosphorylation in 32P-labelled intact cells. Interestingly, under similar conditions RAIR-IgG elicited ribosomal S6 protein phosphorylation, as did insulin. The possibility that RAIR-IgG activated a cryptic tyrosine kinase activity is discussed.
Assuntos
Insulina/farmacologia , Receptor de Insulina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Divisão Celular/efeitos dos fármacos , Mitógenos , Dados de Sequência Molecular , Fosfopeptídeos/isolamento & purificação , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/imunologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Chinese hamster ovary transfectants that express insulin receptors in which tyrosine residues 1162 and 1163 were replaced by phenylalanine exhibit a total inhibition of the insulin-mediated tyrosine kinase activity toward exogenous substrates [histone, casein, and poly(Glu/Tyr)]; this latter activity is associated with total inhibition of the hypersensitivity reported for insulin in promoting 2-deoxyglucose uptake. We now present evidence that the twin tyrosines also control the insulin-mediated stimulation of glycogen synthesis. Surprisingly, this type of Chinese hamster ovary transfectant is as hypersensitive to insulin for its mitogenic effect as are Chinese hamster ovary cells expressing many intact insulin receptors. Such data suggest that (i) the insulin mitogenic effect routes through a different pathway than insulin uses to activate the transport and metabolism of glucose and (ii) the mitogenic effect of insulin is not controlled by the twin tyrosines. At the molecular level, the solubilized mutated receptor has no insulin-dependent tyrosine kinase activity, whereas this receptor displays measurable insulin-stimulated phosphorylation of its beta subunit in 32P-labeled cells. We therefore propose that the autocatalytic phosphorylating activity of the receptor reports a cryptic tyrosine kinase activity that cannot be visualized by the use of classical exogenous substrates.
Assuntos
Receptor de Insulina/análise , Tirosina/análise , Animais , Linhagem Celular , Cricetinae , Replicação do DNA/efeitos dos fármacos , Desoxiglucose/farmacocinética , Proteínas Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas , SolubilidadeRESUMO
Amino acid sequence of the precursor of the phosphorylated N-glycoprotein (pp63) secreted by rat hepatocytes was deduced from the cDNA sequence. This polypeptide (Mr = 40,586) was rich in both cysteine and proline and contained three potential N-glycosylation sites. A single pp63 mRNA species (approximately 2000 bp), found in normal hepatocytes but not in FaO hepatoma cells, appeared to result from transcription of a single gene. pp63 purified by affinity chromatography inhibited insulin receptor tyrosine kinase and receptor autophosphorylation. Only the phosphorylated form of the protein was active. In additon, pp63 antagonized the growth-promoting action of insulin in FaO cells but did not affect hormone-mediated increase in amino acid transport capacity or tyrosine aminotransferase induction in these cells.