Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.     

Studies on the physiological function of spermine in the process of progesterone induced toad oocyte maturation

Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation.The ID50 for spermine inhibition via intra -oocyte microinjection on maturation induced by progesterone was 6.8mM(100nl).Spermine could inhibit MPF induced toad oocyte maturation with a much higher ID50.A 55 kD protein was dephosphorylated during the process of progesterone induced oocyte maturation .Spermine selectively promoted the level of phosphorylation of this protein in both progesterone-stimulated and hormone-untreated oocytes.The extent of its dephosphorylation was fairly Correlated with the percentage of GVBD in the hormone stimulated oocytes.The level of endogenous spermine was reduced by 28% between the perod of 0.40 GVBD50 and 0.60 GVBD50,at which 55 kD protein was dephosphorylated.Spermine inhibited progesterone-stimulated protein synthesis in almost the same dose dependent manner as its inhititory effect on the hormone-induced maturation,The endogenous spermine regulated 55 kD protein dephosphorylation which may trigger the increase of protein dephosphorylation which may trigger the increase of protein synthesis and in turn promote the activation of MPF,It is possible that 55 kD protein may be one of the components of messenger ribonucleoprotein(mRNP) particles.     

Tyrosine phosphorylation of p34cdc2 in metaphase II‐arrested pig oocytes results in pronucleus formation without chromosome segregation

At the G2‐M boundary, maturation‐promoting factor (MPF) activation is usually induced in one or both of two ways; tyrosine dephosphorylation of p34^cdc2 or synthesis of cyclin B according to cell type and species. At the end of M‐phase, however, MPF inactivation is normally triggered only by cyclin degradation. We investigated whether tyrosine phosphorylation of p34^cdc2 can inactivate MPF and what kinds of events are induced in pig metaphase II (MII)‐arrested oocytes. First, cyclin B1 in MII‐arrested oocytes is degraded upon fertilization. Second, when MII oocytes were treated with vanadate, an inhibitor of tyrosine phosphatases, they were released from MII arrest, but MPF was inactivated by further tyrosine phosphorylation of p34^cdc2 rather than cyclin B1 degradation. The vanadate‐induced exit from M‐phase is distinct from normal M‐phase exit, which is accompanied by cyclin B1 degradation; the former lacks both sister chromatid separation and second polar body emission. Vanadate itself has no inhibitory effect on chromosome segregation since calcium ionophore induced chromosome segregation in the presence of vanadate. Furthermore, when MII oocytes were treated with olomoucine, an inhibitor of cyclin‐dependent kinases, they exited from MII arrest in a manner similar to vanadate‐treated MII oocytes. Finally, we propose that MPF inactivation by tyrosine phosphorylation of p34^cdc2 enables MII oocytes to form an interphase nucleus, but not to segregate sister chromatid due to the absence of the mechanisms required to trigger sister chromatid separation such as anaphase‐promoting complex (APC)‐mediated proteolysis, on the signaling pathway from intracellular Ca²⁺ increase to MPF inactivation. Mol. Reprod. Dev. 52:107–116, 1999. © 1999 Wiley‐Liss, Inc.     

Effect of insulin on spontaneous and progesterone-induced GVBD on Bufo arenarum denuded oocytes

Progesterone is considered as the physiological steroid hormone that triggers meiosis reinitiation in amphibian oocytes. Nevertheless, isolated oocytes can be induced to undergo germinal vesicle breakdown (GVBD) in a saline medium by means of treatment with various hormones or inducing agents such as other steroid hormones, insulin or an insulin-like growth factor. It has been demonstrated that Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. This study was undertaken to evaluate the participation of the purine and phosphoinositide pathway in the insulin-induced maturation of oocytes competent and incompetent to mature spontaneously, as well as to determine whether the activation of the maturation promoting factor (MPF) involved the activation of cdc25 phosphatase in Bufo arenarum denuded oocytes. Our results indicate that insulin was able to induce GBVD in oocytes incompetent to mature spontaneously and to enhance spontaneous and progesterone-induced maturation. In addition, high intracellular levels of purines such as cAMP or guanosine can reversibly inhibit the progesterone and insulin-induced maturation process in Bufo arenarum as well as spontaneous maturation. Assays of the inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis and its turnover by neomycin and lithium chloride respectively exhibited a different response in insulin- or progesterone-treated oocytes, suggesting that phosphoinositide turnover or hydrolysis of PIP2 is involved in progesterone- but not in insulin-induced maturation. In addition, the inhibitory effect of vanadate suggests that an inactive pre-maturation promoting factor (pre-MPF), activated by dephosphorylation of Thr-14 and Tyr-15 on p34cdc2, is present in Bufo arenarum full-grown oocytes; this step would be common to both spontaneous and hormone-induced maturation. The data presented here strongly suggest that insulin initiates at the cell surface a chain of events leading to GVBD. However, our studies point to the existence of certain differences between the steroid and the peptide hormone pathways, although both involve the decrease in intracellular levels of cAMP, the activation of phosphodiesterase (PDE) and the activation of pre-MPF.     

Influence of insulin-like growth factor-I and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes

The effects of insulin-like growth factor-I (IGF-I) and its interaction with gonadotropins, estradiol, and fetal calf serum (FCS) on in vitro maturation (IVM) of equine oocytes were investigated in this study. We also examined the role of IGF-I in the presence or absence of gonadotropins, estradiol, and FCS in parthenogenic cleavage after oocyte activation with calcium ionophore combined with 6-dimethylaminopurine (6-DMAP), using cleavage rate as a measure of cytoplasmic maturation. Only equine cumulus-oocyte complexes with compact cumulus and homogenous ooplasm (n = 817) were used. In experiment 1, oocytes were cultured in TCM-199 supplemented with BSA, antibiotics, and IGF-I at 0 (control), 50, 100, 200 ng/ml, at 39 degrees C in air with 5% CO(2), 95% humidity for 36 or 48 h. In experiment 2, oocytes were cultured with FSH, LH, estradiol, and FCS with IGF-I at the concentration that promoted the highest nuclear maturation rate in experiment 1. In experiment 3, oocytes from the three experimental groups (IGF-I; hormones; and IGF-I + hormones) were chemically activated by exposure to calcium ionophore followed by culture in 6-DMAP. In experiment 1, IGF-I stimulated equine oocyte maturation in a dose-dependent manner with the highest nuclear maturation rate at a concentration of 200 ng/ml. No significant effect of IGF-I on nuclear maturation was observed in experiment 2. In experiment 3, a significant difference in cleavage rate was observed between the hormone + IGF-I group (15 of 33; 45.4%) compared with IGF-I (10 of 36; 27.8%) and hormone (4 of 31; 12.9%) alone (P < 0.05). These results demonstrated that IGF-I has a positive effect on nuclear maturation rate of equine oocytes in vitro. The addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation, but resulted in a positive effect on cytoplasmic maturation of equine oocytes measured by parthenogenic cleavage.     

Effect of dehydroleucodine on meiosis reinitiation in Bufo arenarum denuded oocytes

In amphibian oocytes meiosis, the transition from G2 to M phase is regulated by the maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34/cdc2 and cyclin B. In immature oocytes there is an inactive complex (pre-MPF), in which cdc2 is phosphorylated on both Thr-161 and Thr-14/Tyr-15 residues. The dephosphorylation of Thr-14/Tyr-15 is necessary for the start of MPF activation and it is induced by the activation of cdc25 phosphatase. Late, to complete the activation, a small amount of active MPF induces an auto-amplification loop of MPF stimulation (MPF amplification). Dehydroleucodine (DhL) is a sesquiterpenic lactone that inhibits mammalian cell proliferation in G2. We asked whether DhL interferes with MPF activation. For this question, the effect of DhL (up to 30 microM) on the resumption of meiosis was evaluated, and visualized by germinal vesicle break down (GVBD), of Bufo arenarum oocytes induced in vitro by either: (i) removing follicle cells; (ii) progesterone stimulation; (iii) VG-content injection; or (iv) injection of mature cytoplasm. The results show that DhL induced GVBD inhibition, in a dose-dependent manner, in spontaneous and progesterone-induced oocyte maturation. Nevertheless, DhL at the doses assayed had no effect on GVBD induced by mature cytoplasm injection, but exerted an inhibitory effect on GVBD induced by GV content. On the basis of these results, we interpreted that DhL does not inhibit MPF amplification and that the target of DhL is any event in the early stages of the cdc25 activation cascade.     

A novel mechanism for the insulin-like effect of vanadate on glycogen synthase in rat adipocytes

Vanadate activated rat adipocyte glycogen synthase similarly to insulin in a dose- and time-dependent manner. No additional effect was observed when insulin and vanadate were added together. Vanadate also partially counteracted the effect of epinephrine to activate rat adipocyte glycogen phosphorylase similarly to insulin. Inhibition of Na+K+ATPase or stimulation of hydrogen peroxide generation were shown not to be the mechanisms of the insulin-like action of vanadate on glycogen synthase. Vanadate stimulated the phosphorylation of the 95,000-dalton subunit of the insulin receptor on tyrosine residues both in intact adipocytes and in a solubilized insulin receptor fraction. Vanadate also stimulated the phosphorylation of the 95,000-dalton subunit of a highly purified insulin receptor from human placenta. Neither the insulin receptor fraction from rat adipocyte nor the highly purified insulin receptor from human placenta contained any detectable phosphotyrosine phosphatase activity. Potassium fluoride had no stimulatory effect on the phosphorylation of the insulin receptor. Vanadate caused a 10-fold decrease in the Km for ATP, for tyrosine kinase, and enhanced the phosphorylation of histone H2B. These results demonstrate that vanadate enhances the phosphorylation of the insulin receptor by stimulating the kinase reaction in a similar but not identical manner to insulin.     

Maturation/M-phase promoting factor: a regulator of aging in porcine oocytes

Deterioration in the quality of mammalian oocytes during the metaphase-II arrest period is well known as "oocyte aging." Oocytes in which aging has occurred are called aged oocytes, and these oocytes show enhanced activation and higher fragmentation rates after parthenogenetic activation. Previously we showed that porcine aged oocytes had low maturation/M-phase promoting factor (MPF) activity, and we suggested that this low MPF activity contributed at least in part to the aging phenomena. In the present study, we examined the relationship between MPF activity and these aging phenomena by artificially regulating MPF activity in porcine metaphase-II-arrested oocytes. Since we have shown recently that aged porcine oocytes contain abundant phosphorylated inactive MPF, so-called pre-MPF, we used vanadate and caffeine, which affect the phosphorylation status of MPF, to regulate MPF activity. Incubation of 48-h-matured oocytes with vanadate for 1 h increased the phosphorylation of MPF and decreased MPF activity. The parthenogenetic activation and fragmentation rates were significantly increased compared with those of control oocytes. Conversely, treatment of 72-h-cultured aged oocytes with caffeine (last 10 h of culture) decreased the level of pre-MPF and elevated MPF activity. These oocytes revealed significantly lower parthenogenetic activation rates and a lower percentage of fragmentation than did untreated aged oocytes. These results indicate that not only the increased ability for parthenogenetic activation but also the increased fragmentation rate observed in porcine aged oocytes may be attributable in part to the gradual decrease in MPF activity during prolonged culture. Control of MPF phosphorylation with these agents may allow for some degree of manipulation of oocyte aging.     

Cdc2-cyclin B-induced G2 to M transition in perch oocyte is dependent on Cdc25

The G2 to M phase transition in perch oocytes is regulated by maturation promoting factor (MPF), a complex of Cdc2 and cyclin B. In Anabas testudineus, a fresh water perch, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, the maturation inducing hormone (MIH), induced complete germinal vesicle breakdown (GVBD) of oocytes at 21 h. An unusual cyclin, p30 cyclin B, has been identified in oocyte extract using both monoclonal and polyclonal antibodies. Surprisingly, Cdc2 could not be identified, although a Northern blot with Cdc2 cDNA demonstrated expression of the gene. Purification of MPF through an immunoaffinity column followed by SDS-PAGE showed three proteins, Cdc2, cyclin B, and a 20 kDa fragment, indicating earlier failure in immunodetection may be due to the interference by this fragment. In uninduced oocytes, p30 cyclin B was present, and its expression was increased by MIH. MIH increased p30 cyclin B accumulation at 3 h, a high level which was maintained between 9 and 21 h, but an effective increase in GVBD and H1 kinase activation could only be observed between 15 and 21 h. This delay in active MPF formation was found to be related to the activation of Cdc25, phosphorylation of which was detected at 12 h, and a substantial increase occurred during 15-18 h. Sodium orthovanadate, a tyrosine phosphatase inhibitor, inhibited H1 kinase activity and GVBD, suggesting the requirement of Cdc25 activity in MPF activation. Our results show occurrence of pre-MPF in uninduced oocytes and its conversion to active MPF requires dephosphorylation by Cdc25, the existence of which has not yet been shown in fish.     

Vanadate stimulates D-glucose transport into sarcolemmal vesicles from rat skeletal muscles.

Vanadate is known to have various insulin-like actions including activation of D-glucose uptake into the skeletal muscle and adipose tissue. In this study, we examined the effect of orthovanadate on D-glucose uptake into sarcolemmal vesicles prepared from rat hind limb skeletal muscles. In the presence of 10 mM vanadate, the initial rate of D-glucose uptake into sarcolemmal vesicles was enhanced 4-5 times above the basal value. Half-maximal concentration for this effect of vanadate was 3 mM. The D-glucose uptake was also stimulated by metavanadate, but not by selenite, selenate, or molybdate. When vanadate was removed from the vesicles by dilution and centrifugation, D-glucose uptake into the vesicles returned to the basal level, indicating that the effect of vanadate was reversible. Saturation curves showed that the Vmax value for the D-glucose uptake was enhanced more than 4-fold by 10 mM vanadate. Therefore, the activation of D-glucose uptake was due, at least in part, to a large increase in the Vmax value. These results suggest that vanadate increases the intrinsic activity (turnover number) of skeletal muscle glucose transporters in a reversible manner.     

Maturation/M-phase promoting factor regulates aging of porcine oocytes matured in vitro

Control of oocyte aging during manipulation of matured oocytes should have advantages for recently developed reproductive technologies, such as cloning after nuclear transfer. We have shown that the enhanced activation ability and fragmentation of porcine in vitro matured and aged oocytes bore a close relationship to the gradual decrease in maturation/M-phase promoting factor (MPF) activity and that porcine aged oocytes contained plenty of MPF, but it was in an inactive form, pre-MPF, as a result of phosphorylation of its catalytic subunit p34(cdc2) and, therefore, had low MPF activity. We incubated porcine oocytes with vanadate and caffeine, which affected the phosphorylation status and MPF activity, and evaluated their activation abilities and fragmentation frequencies. Incubation of nonaged oocytes with vanadate increased p34(cdc2) phosphorylation and reduced MPF activity to levels similar to those of aged oocytes and increased their parthenogenetic activation and fragmentation rates compared with those of the control oocytes. Conversely, treating aged oocytes with caffeine reduced p34(cdc2) phosphorylation and increased MPF activity. These oocytes showed significantly lower parthenogenetic activation and fragmentation rates than aged mature oocytes. These results suggest that MPF activity is a key mechanism of oocyte aging and controlling MPF activity by altering p34(cdc2) phosphorylation with these chemicals may enable oocyte aging to be manipulated in vitro. We expect those ideas will be applied practically to pig cloning.     

Stimulation of insulin-like growth factor II receptor binding and insulin receptor kinase activity in rat adipocytes. Effects of vanadate and H2O2

Autophosphorylation of the insulin receptor on tyrosine residues and activation of the endogenous insulin receptor kinase is postulated to be a critical step in the mechanism of action of insulin. To investigate this hypothesis, the insulin-mimicking effects of vanadate (sodium orthovanadate) and H2O2 (hydrogen peroxide) alone and in combination were examined in freshly isolated rat adipocytes. Vanadate and H2O2 stimulated the translocation of insulin-like growth factor II (IGF-II) receptors to the plasma membrane of rat adipocytes in a manner analogous to insulin. IGF-II binding was increased by maximally effective doses of vanadate (1 mM), H2O2 (1 mM), and insulin (10 ng/ml) to 172 +/- 10, 138 +/- 12, and 289 +/- 16% of control, respectively. Previously (Kadota, S., Fantus, I. G., Hersh, B., and Posner, B. I. (1986) Biochem. Biophys. Res. Commun. 138, 174-178), we showed that the combination of these concentrations of vanadate plus insulin was not more potent than insulin alone. In this study, similar results were found with H2O2 plus insulin. In contrast, the combination of vanadate plus H2O2 was synergistic, effecting an increase of IGF-II binding to 488 +/- 23% of control. Amiloride inhibited the effects of vanadate, H2O2, and insulin. Adipocyte insulin receptors purified by wheat germ agglutinin chromatography were assayed for tyrosine kinase activity using the synthetic substrate poly(Glu,Tyr) (4:1). Basal activity (no in vitro insulin) was stimulated by exposure of intact cells to vanadate, H2O2, insulin, and vanadate + H2O2 to 147.7 +/- 4.3, 178.2 +/- 43.4, 495.0 +/- 67.1, and 913.2 +/- 92.0% of control, respectively. The stimulation of tyrosine kinase activity by these agents was accounted for by the insulin receptor as the augmented activity was completely immunoprecipitated with insulin receptor antibody. In these studies, the increase in IGF-II binding correlated significantly with the activation of the insulin receptor-tyrosine kinase (r = 0.927, p less than 0.001). These data support the hypothesis that activation of the insulin receptor kinase is linked to insulin action.     

Vanadate inhibition of brain (Ca+Mg)-ATPase

Vanadate was a potent inhibitor of the membrane-bound (Ca+Mg)-ATPase from rat brain, the concentration required for 50% inhibition under conditions optimal for enzymatic activity being 3 M. Vanadate inhibition increased with the MgCl₂ concentration, half-maximal inhibition occurring at 2 mM MgCl₂, near the MgCl₂ concentration required for half-maximal activation of the ATPase activity. MnCl₂ could substitute for MgCl₂, and at concentrations of 1 mM (Ca+Mn)-ATPase activity was greater than (Ca+Mg)-ATPase activity, although sensitivity to vanadate was less. Vanadate inhibition increased also with the KCl concentration, half-maximal inhibition occurring at 8 mM, again near the concentration required for half-maximal activation of ATPase activity. By contrast, NaCl stimulated (Ca+Mg)-ATPase activity without potentiating vanadate inhibition. These effects of cations on ATPase activity and vanadate inhibition resemble properties of certain transport ATPases and thus suggest mechanistic and functional similarities.     

Vanadate stimulation of IGF binding to rat adipocytes

Preincubation of adipocytes with insulin (10 ng/ml) stimulated binding of IGF-II to maximal levels of 160% above controls. Vanadate also augmented IGF-II binding with an increase of 126% above controls at a concentration of 1 mM. Coincubation of vanadate (1 mM) with a maximal stimulatory dose of insulin (10 ng/ml) produced no additive effect. However, at submaximal doses of insulin (0.1 ng/ml) the effect of vanadate was additive. Amiloride, a potent inhibitor of the insulin receptor kinase, inhibited the effects of both vanadate and insulin. The data are consistent with an effect of vanadate via a similar sequence of steps to that of insulin; perhaps involving activation of the insulin receptor kinase.     

Stimulatory effect of vanadate on hyaluronic acid synthesis in mesothelial cells from rabbit pericardium

Vanadate dose-dependently stimulated the incorporation of [3H]glucosamine into glycosaminoglycan, especially hyaluronic acid, in mesothelial cells from rabbit pericardium. The activity of hyaluronic acid synthase in the mesothelial cells treated with 50 microM vanadate for 0.5-1 h was stimulated to a level about 2 times over that of the control. Neither DNA synthesis nor protein synthesis in the mesothelial cells under the same experimental conditions was affected. The enhancement of the activity of hyaluronic acid synthase in the mesothelial cells treated with vanadate (50 microM) was not inhibited by the addition of cycloheximide (1 microgram/ml). These results suggest that vanadate stimulates the hyaluronic acid synthesis by activation of hyaluronic acid synthase in mesothelial cells from rabbit pericardium.     

Pertussis toxin-insensitive regulation of phosphatidylinositol hydrolysis by vanadate in brain microvessels.

Vanadate, over a concentration range from 0.1 to 0.5 mM, stimulated the incorporation of (32P)-orthophosphate into PI and PA in brain microvessels. At concentrations higher than 0.5 mM, the stimulatory effect of vanadate decreased. Concommitantly, an enhanced DAG production was observed, indicating that vanadate stimulated PI turnover. All these effects were evident at all the times tested. Experiments performed in the presence of pertussis toxin (IAP) indicated that a IAP-sensitive G-protein does not mediate the vanadate stimulated PI effect in brain microvessels.     

Activation of maturation promoting factor in Bufo arenarum oocytes: injection of mature cytoplasm and germinal vesicle contents

Although progesterone is the established maturation inducer in amphibians, Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. In this species it is possible to obtain oocytes competent and incompetent to undergo spontaneous maturation according to the seasonal period in which animals are captured. Reinitiation of meiosis is regulated by maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34cdc2 and cyclin B. Although the function and molecule of MPF are common among species, the formation and activation mechanisms of MPF differ according to species. This study was undertaken to evaluate the presence of pre-MPF in Bufo arenarum oocytes incompetent to mature spontaneously and the effect of the injection of mature cytoplasm or germinal vesicle contents on the resumption of meiosis. The results of our treatment of Bufo arenarum immature oocytes incompetent to mature spontaneously with sodium metavanadate (NaVO3) and dexamethasone (DEX) indicates that these oocytes have a pre-MPF, which activates and induces germinal vesicle breakdown (GVBD) by dephosphorylation on Thr-14/Tyr-15 by cdc25 phosphatase and without cyclin B synthesis. The injection of cytoplasm containing active MPF is sufficient to activate an amplification loop that requires the activation of cdc25 and protein kinase C, the decrease in cAMP levels, and is independent of protein synthesis. However, the injection of germinal vesicle content also induces GVBD in the immature receptor oocyte, a process dependent on protein synthesis but not on cdc25 phosphatase or PKC activity.     

Differential dephosphorylation of the insulin receptor and its 160-kDa substrate (pp160) in rat adipocytes.

A permeabilized rat adipocyte model was developed which permitted an examination of: 1) insulin receptor autophosphorylation, 2) phosphorylation of a putative insulin receptor substrate of 160 kDa, pp160, and 3) the dephosphorylation reactions associated with each of these phosphoproteins. Rat adipocytes, preincubated with [32P]orthophosphate for 2 h, were exposed to insulin (10(-7) M) at the time of digitonin permeabilization. Phosphorylation of pp160 and autophosphorylation of the insulin receptor increased as a function of Mn2+ concentration in the media with near maximum responses at 10 mM. Maximum response was at least as large as the intact cell response to 10(-7) M insulin. In contrast, magnesium did not increase phosphorylation of pp160 although an increase in receptor autophosphorylation was observed. Autophosphorylation was preserved at digitonin concentrations of 20-100 micrograms/ml, but pp160 phosphorylation was negligible beyond 40 micrograms/ml. Our previous work demonstrated that the insulin receptor was associated with a phosphotyrosine phosphatase activity in permeabilized adipocytes (Mooney, R., and Anderson, D. (1989) J. Biol. Chem. 264, 6850-6857). The current permeabilized adipocyte model made possible an examination of the effects of phosphotyrosine phosphatase inhibitors, including several divalent metal cations (Zn2+, Co2+, and Ni2+), vanadate, and molybdate on both net phosphorylation of pp160 and autophosphorylation of the insulin receptor. Zn2+ at 100 microM, Ni2+ at 1 mM, and Co2+ at 1 or 5 mM increased insulin-dependent phosphorylation of pp160 at least 5-fold and autophosphorylation 2-fold. At higher concentrations of Zn2+ (1 mM) and Ni2+ (5 mM), however, no increase in phosphorylation of pp160 was observed and autophosphorylation was inhibited. Vanadate (1 mM) and molybdate (100 microM) increased insulin-dependent phosphorylation of pp160 by 3-fold when tested separately and 7-fold in combination. Insulin receptor autophosphorylation was increased 50% by each and 3-fold when the agents were combined. Dephosphorylation of pp160 and the insulin receptor was analyzed directly by permeabilizing prelabeled insulin-treated adipocytes in the presence of EDTA (10 mM). Dephosphorylation of pp160 was especially rapid with a t1/2 of approximately 10 s. The t1/2 for the insulin receptor was 37 s. Zn2+ at 1 mM (a concentration that inhibited the insulin receptor kinase) was a strong inhibitor of dephosphorylation, prolonging the rate of pp160 dephosphorylation more than 12-fold and insulin receptor dephosphorylation 3-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation

Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation. Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts. At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of [3H]methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP. This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate. Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates. When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate. In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation. In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs. This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells. Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes. However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.