Expression of c-myc gene in human ovary carcinoma cells treated with vanadate

The widely accepted hypothesis of vanadate action on cells postulates that this ion inhibits protein phosphatase(s) that dephosphorylates protein phosphotyrosine residues. This inhibition causes tyrosine hyperphosphorylation of cell proteins followed by changes in physiological action of phosphoproteins resulting in stimulation of cell proliferation, expression of protooncogenes, and transient cell transformation. We have found that treatment of human ovary carcinoma (CaOv) cells with vanadate causes the increase in total protein phosphorylation from 1.5- to 2.0-fold whereas the ratio between phosphoserine, phosphothreonine, and phosphotyrosine content remains unchanged. At the same time, enhancement of c-myc gene expression (not c-fos) was observed. Hence, the increase in the ratio of phosphotyrosine to phosphoserine and phosphothreonine is not an obligatory intermediate stage before vanadate-dependent activation of c-myc expression.     

Reduction of Vanadate by Ascorbic Acid and Noradrenaline in Synaptosomes

The effect of ascorbic acid and noradrenaline on the inhibition of synaptosomal membrane ATPase by vanadate has been studied. Ascorbic acid (2 x 10(-3) M) and noradrenaline (10(-4) M) partly reversed the inhibition by vanadate (10(-6) M); however, when both were administered together the inhibition was completely eliminated. Using electron spin resonance (ESR) spectroscopy, we detected that ascorbic acid (10(-3) M) caused a 42% of reduction of vanadate (10(-4) M). Noradrenaline (10(-4) M) alone also reduced vanadate (10(-4) M) partially. When ascorbic acid and noradrenaline were present together all the vanadate was reduced to vanadyl. The concentration of ascorbic acid present in the brain under physiological conditions is identical to that found effective in our experiments. We suggest that ascorbic acid may protect the ATPase, at least in part, from inhibition by vanadate as a consequence of reducing vanadate to vanadyl. In those tissues where noradrenaline is also present a complete reduction of endogenous vanadium can be presumed.     

Stimulatory (insulin-mimetic) and inhibitory (ouabain-like) action of vanadate on potassium uptake and cellular sodium and potassium in heart cells in culture

(1) The influence of vanadate (Na3VO4) on sodium and potassium uptake as well as on cellular ion contents of sodium and potassium has been studied in heart muscle and non-muscle cells obtained from various species. An ouabain-like inhibition of potassium uptake (up to 50%), combined with a decrease of cellular potassium (up to 20%) has been observed by vanadate (10(-4)-10(-3) M) in heart non-muscle cells obtained from neonatal guinea pigs and chick embryos. In heart muscle and non-muscle cells prepared from neonatal rats, as well as in Girardi human heart cells, a vanadate-induced stimulation of potassium uptake (up to 100%), combined with a rise in cellular potassium (up to 20%) and without significant alteration of cellular sodium, has been found. A slight increase of 22Na+ influx can be measured in rat heart muscle cells and in Girardi human heart cells in the presence of vanadate (10(-4)--10(-3) M). (2) In beating rat heart muscle cells in culture, detrimental effects of serum deprivation--concerning beating properties, potassium uptake and cellular potassium--can at least in part be overcome by addition of vanadate. Furthermore, this compound prevents ouabain-induced signs of toxicity (contractures) in these cells. (3) The stimulatory effects of vanadate on potassium can be mimicked by insulin (1-10 mU/ml). Furthermore, vanadate produces an insulin-like stimulation of 2-deoxy-D-glucose uptake in rat heart muscle and non-muscle cells as well as in Girardi human heart cells. (4) The experimental data demonstrate an ouabain-like inhibition as well as an insulin-mimetic stimulation of potassium-uptake in various heart cells. The reason for this antagonistic mode of action may be due to the different capabilities of the heart cell types to reduce vanadium in the V-valence state of vanadium in the IV-valence state, thereby favouring either ouabain-like inhibition (vanadium V) or insulin-mimetic stimulation (vanadium IV) of potassium transport.     

Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.

The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.     

Effect of vanadate on brain protein phosphorylation

The effect of vanadate on the phosphorylation of synaptosomal membrane proteins prepared from rat cerebral cortex was studied. Vanadate concentrations of 10^–6, 10^–5, and 10^–4 M increased the endogenous phosphorylation activity by 25%, 37%, and 75%, respectively. Increasing the ATP concentration in the assay medium from 50 to 500 M did not influence the above effect. A commercial preparation of the purified protein kinase was stimulated 40% by 10^–3 M vanadate. Calcium-calmodulin dependent activity was stimulated only 20% by 10^–5 M vanadate. The effect was not enhanced by further increasing vanadate concentration. Addition of calcium ions (above 50 M) suppressed the vanadate effect, while an inhibition was observed at high Ca²⁺ concentration (2.5 mM). Below 50 M calcium ions stimulated phosphorylation activity in the absence of vanadate and did not affect the stimulatory action of vanadate. Cyclic AMP-dependent endogenous phosphorylation was also stimulated by vanadate. Activation by cAMP could not be observed at vanadate concentrations above 10^–6 M. Possible mechanisms of the vanadate effect are discussed.     

The insulinomimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells

H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate, or H2O2 alone. Employing Western blotting with anti-P-Tyr antibodies, we have identified in Fao cells at least four proteins (pp180, 150, 114, and 100) whose P-Tyr content is rapidly increased upon treatment of the cells with 3 mM H2O2. Tyrosine phosphorylation of these and additional proteins was markedly potentiated (6-10-fold) when 100 microM sodium orthovanadate was added together with H2O2. The effects of H2O2 and vanadate on protein tyrosine phosphorylation were rapid and specific. The enhanced tyrosine phosphorylation was accompanied by a concomitant inhibition of a cytosolic protein tyrosine phosphatase activity. The latter was inhibited by 50% in 3 mM H2O2-treated cells. The inhibitory effect was augmented in the combined presence of H2O2 and vanadate. Half- and maximal effects of vanadate were obtained at 15 microM and 1 mM, respectively. Vanadate (1 mM) alone, added to the cells, had only a trivial effect on protein tyrosine phosphatase activity. A 45-s challenge with insulin (10(-7) M) of cells pretreated with H2O2 largely mimicked the potentiating effects of vanadate on protein tyrosine phosphorylation but not on protein tyrosine phosphatase activity. Our results suggest the involvement of multiple tyrosine-phosphorylation proteins in mediating the biological effects of H2O2/vanadate. Their enhanced phosphorylation can be attributed at least in part, to the inhibitory effects exerted by H2O2 alone, or in combination with vanadate, on protein tyrosine phosphatase activity. The similarity between proteins phosphorylated in Fao cells in response to H2O2/vanadate or H2O2/insulin, suggests that either treatment stimulates protein tyrosine kinases having similar substrate specificities. The insulin receptor kinase is a likely candidate as its activity is markedly enhanced either by insulin (plus H2O2) or by H2O2/vanadate.     

Myosin light chain phosphorylation inhibits muscle fiber shortening velocity in the presence of vanadate

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30 degrees C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25 degrees C (E. Pate, G. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30 degrees C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 +/- 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10 degrees C. The K(m) for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30 degrees C, is 20 +/- 4 microM for phosphorylated fibers and 192 +/- 40 microM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30 degrees C depends on the phosphate analog, with approximately 12% inhibition in fibers activated in the presence of 5 mM BeF(3) and no inhibition in the presence of 0.25 mM AlF(4). Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue.     

Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase

Both vanadate and hydrogen peroxide (H2O2) are known to have insulin-mimetic effects. We previously reported that the mixture of vanadate plus H2O2 results in the generation of a peroxide(s) of vanadate, which strongly enhances IGF-II binding to rat adipocytes (Kadota et al., 1987b). We now report that pervanadate mimics insulin in isolated rat adipocytes to (1) stimulate lipogenesis, (2) inhibit epinephrine-stimulated lipolysis, and (3) stimulate protein synthesis. The efficacy of pervanadate is comparable to that of insulin. However, it is 10(2)-10(3) times more potent than vanadate alone. Exposure of intact rat adipocytes to pervanadate was found to activate the WGA-purified insulin receptor tyrosine kinase assayed with the exogenous substrate poly(Glu80/Tyr20) in a dose-dependent manner to a maximum of 1464% of control at 10(-3) M compared with a maximum insulin effect of 1046% at 10(-6) M. In contrast, in vitro assayed autophosphorylation of the WGA-purified extract was increased 3-fold after exposure of intact cells to insulin but not significantly increased after pervanadate. Furthermore, high concentrations of pervanadate (10(-5) M) inhibited subsequent in vitro added insulin-stimulated autophosphorylation. In vitro addition of pervanadate to WGA-purified receptors could not stimulate autophosphorylation or exogenous tyrosine kinase activity and did not inhibit insulin-stimulated autophosphorylation. Labeling of intact adipocytes with [32P]orthophosphate followed by exposure to 10(-4) M pervanadate increased insulin receptor beta-subunit phosphorylation (7.9 +/- 3.0)-fold, while 10(-7) M insulin and 10(-4) vanadate increased labeling (5.3 +/- 1.8)- and (1.1 +/- 0.2)-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vanadate stimulates oxygen consumption and tyrosine phosphorylation in electropermeabilized human neutrophils.

To determine the role of protein phosphorylation in neutrophil activation, electropermeabilized cells were treated with vanadate, a phosphatase inhibitor. Micromolar concentrations of vanadate elicited a NADPH-dependent burst of oxygen utilization in permeabilized, but not in intact cells, indicating an intracellular site of action. Stimulation of oxygen consumption by vanadate was reversible, concentration dependent and required the presence of ATP and Mg2+. Generation of a respiratory burst by vanadate was associated with accumulation of phosphorylated proteins. Such accumulation was due, at least in part, to inhibition of phosphoprotein phosphatase activity, as indicated by pulse-chase experiments. No evidence for stimulation of protein kinases by vanadate was found. Phosphoamino acid analysis revealed that a large fraction of the vanadate-induced phosphorylation occurred on tyrosine residues. The pronounced accumulation of tyrosine-phosphorylated proteins was confirmed by immunoblotting with anti-phosphotyrosine antibodies. The data suggest that neutrophils possess one or more constitutively active tyrosine kinases and that phosphoprotein accumulation is normally prevented by vigorous concomitant phosphatase activity. Inhibition of the latter by vanadate leads to phosphoprotein accumulation and is accompanied by stimulation of oxygen consumption.     

ATP-citrate lyase is another enzyme the histidine phosphorylation of which is inhibited by vanadate

We have recently shown that phosphorylation of histidine residue of the alpha-subunit of the succinyl-CoA synthetase is inhibited by both vanadate and vanadyl. To assess the university of this inhibition, we have estimated the effect of vanadate on the phosphorylation of another enzyme ATP-citrate lyase, prepared from rat liver. This enzyme contains histidine as the only amino acid with an acid-labile (P-N) phosphate bond. The 67% inhibition of endogenous phosphorylation by 1 mM vanadate disappeared after cleavage of the acidic P-N bond of histidine with acidic sample solution. The remaining 33 per cent radioactivity was due to labelling of the acid-stable phosphoamino acids (P-serine and P-threonine), the phosphorylation of which was not affected by vanadate. The dose response curve for vanadate inhibition closely resembles that shown previously for inhibition of phosphorylation of histidine in the succinyl-CoA synthetase. The results suggest that the action of vanadate on histidinyl phosphorylation is a more general effect (like its influence on phosphorylation of the protein-bound tyrosine).     

Calcium and cyclic GMP regulation of light-sensitive protein phosphorylation in frog photoreceptor membranes

In frog photoreceptor membranes, light induces a dephosphorylation of two small proteins and a phosphorylation of rhodopsin. The level of phosphorylation of the two small proteins is influenced by cyclic GMP. Measurement of their phosphorylation as a function of cyclic GMP concentration shows fivefold stimulation as cyclic GMP is increased from 10(-5) to 10(-3) M. This includes the concentration range over which light activation of a cyclic GMP phosphodiesterase causes cyclic GMP levels to fall in vivo. Cyclic AMP does not affect the phosphorylations. Calcium ions inhibit the phosphorylation reactions. Calcium inhibits the cyclic GMP-stimulated phosphorylation of the small proteins as its concentration is increased from 10(-6) to 10(-3) M, with maximal inhibition of 70% being observed. Rhodopsin phosphorylation is not stimulated by cyclic nucleotides, but is inhibited by calcium, with 50% inhibition being observed as the Ca++ concentration is increased from 10(-9) to 10(-3) M. A nucleotide binding site appears to regulate rhodopsin phosphorylation. Several properties of the rhodopsin phosphorylation suggest that it does not play a role in a rapid ATP-dependent regulation of the cyclic GMP pathway. Calcium inhibition of protein phosphorylation is a distinctive feature of this system, and it is suggested that Ca++ regulation of protein phosphorylation plays a role in the visual adaptation process. Furthermore, the data provide support for the idea that calcium and cyclic GMP pathways interact in regulating the light-sensitive conductance.     

Effects of vanadate on water and electrolyte transport in rat jejunum

The effect of vanadate (orthovanadate, VO4-) on water and ion transport was studied in rat jejunum. Water transport was tested by single-pass perfusion in vivo and ion fluxes by the Ussing-chamber technique in vitro. The results suggest that vanadate has two actions on ion and water transport: At low concentrations (10(-4) M) it causes Cl-, Na+ and water secretion by stimulation of adenylate cyclase; At higher concentrations (10(-3) and 10(-2) M) it decreases net absorption of Na+ and Cl- by inhibition of (Na+ + K+)-ATPase.     

New signal transduction mechanisms of atrial natriuretic factor: inhibition of phosphorylation of protein kinase C and A 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro. ANF (1x10(-8)M - 1x10(-7)M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.     

Inhibition of cardiac slow action potentials by 8-bromo-cyclic GMP occurs independent of changes in cyclic AMP levels

Cyclic GMP inhibits the slow inward Ca current of cardiac cells. This effect could be due to a cyclic GMP-mediated phosphorylation of the Ca channel (or some protein modifying Ca channel activity), or alternatively, to enhanced degradation of cyclic AMP owing to stimulation of a phosphodiesterase by cyclic GMP. To test the latter possibility, we examined the effect of extracellular 8-bromo-cyclic GMP on cyclic AMP levels in guinea pig papillary muscles, in parallel with electrophysiological experiments. Isoproterenol (10(-6) M) significantly increased the cyclic AMP levels and induced Ca-dependent slow action potentials. Superfusion with 8-bromo-cyclic GMP (10(-3) M) inhibited the slow action potentials induced by isoproterenol. However, muscles superfused with 8-bromo-cyclic GMP had cyclic AMP levels identical to those of muscles superfused with isoproterenol alone. Similarly, 8-bromo-cyclic GMP had no effect on the increase in cyclic AMP levels of muscles treated with forskolin (10(-6) M) or histamine (10(-6) M). We conclude that the inhibitory effect of cyclic GMP on slow Ca channels in guinea pig ventricular cells is not due to a decrease in the cyclic AMP levels. We hypothesize that a cyclic GMP-mediated phosphorylation is the most likely explanation for the Ca channel inhibition observed in this preparation.     

Do vanadium ions exert any specific effect on brain protein phosphrylation

It has been shown previously (1) that vanadate stimulates phosphorylation of the overall protens from the synaptic membranes of rat cerebral cortex. The aim of the present experiments was to investigate whether the action of vanadate and also of vanadyl ions could exert any specific effect on endogenous phosphorylation of proteins from subcellular fractions of the rat brain cortex. Both vanadate and vanadyl ions stimulate phosphorylation of the overall proteins from synaptic membranes and to lesser extent from mitochondria. An attempt was made to estimate the contribution of inhibition of ATPase activity to nonspecific stimulation of phosphate labeling in the synaptic membrane fraction. A band of M_r approx. 37 kDa from synaptic membranes was particularly sensitive to vanadate. In mitochondria both vanadate and vanadyl caused a marked, concentration dependent inhibition of phosphorylation of a band corresponding to M_r approx. 34 kDa. The effect was confined exclusively to the mitochondrial fractions (total, perikaryal and two synaptic types). It was absent in all subcellular fractions tested, including the nuclear one. Phosphorylation of the mitochondrial 34 kDa band is not influenced by cyclic AMP, Ca-calmodulin, shift of pH from 6.6 to 8.1. Alkaline hydrolysis removed almost all phosphatelabeled bands of mitochondria, including that of 34 kDa.     

Comparison of inositol phosphates accumulation induced by different effectors in rat thyroid slices.

Rat thyroid slices were submitted to different effectors and hormones in order to investigate their action on the phosphatidylinositol metabolism. Fluoride and vanadate induced a clear increase of the inositol phosphates with half maximal stimulation at 7 mM and 8 mM respectively. The inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulation induced by vanadate was relatively higher than that observed in the case of fluoride stimulation. Carbachol stimulated also the generation of inositol phosphates with half maximal activation at 2.5 x 10(-6) M. In the same conditions, no significant effect on inositol phosphates production could be detected by the action of TSH or TRH.     

Enhanced sensitivity of insulin-resistant adipocytes to vanadate is associated with oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4).

Vanadate (sodium orthovanadate), an inhibitor of phosphotyrosine phosphatases (PTPs), mimics many of the metabolic actions of insulin in vitro and in vivo. The potential of vanadate to stimulate glucose transport independent of the early steps in insulin signaling prompted us to test its effectiveness in an in vitro model of insulin resistance. In primary rat adipocytes cultured for 18 h in the presence of high glucose (15 mm) and insulin (10(-7) m), sensitivity to insulin-stimulated glucose transport was decreased. In contrast, there was a paradoxical enhanced sensitivity to vanadate of the insulin-resistant cells (EC(50) for control, 325 +/- 7.5 microm; EC(50) for insulin-resistant, 171 +/- 32 microm; p < 0.002). Enhanced sensitivity was also present for vanadate stimulation of insulin receptor kinase activity and autophosphorylation and Akt/protein kinase B Ser-473 phosphorylation consistent with more effective PTP inhibition in the resistant cells. Investigation of this phenomenon revealed that 1) depletion of GSH with buthionine sulfoximine reproduced the enhanced sensitivity to vanadate while preincubation of resistant cells with N-acetylcysteine (NAC) prevented it, 2) intracellular GSH was decreased in resistant cells and normalized by NAC, 3) exposure to high glucose and insulin induced an increase in reactive oxygen species, which was prevented by NAC, 4) EPR (electron paramagnetic resonance) spectroscopy showed a decreased amount of vanadyl (+4) in resistant and buthionine sulfoximine-treated cells, which correlated with decreased GSH and increased vanadate sensitivity, while total vanadium uptake was not altered, and 5) inhibition of recombinant PTP1B in vitro was more sensitive to vanadate (+5) than vanadyl (+4). In conclusion, the paradoxical increased sensitivity to vanadate in hyperglycemia-induced insulin resistant adipocytes is due to oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4). Thus, sensitivity of PTP inhibition and glucose transport to vanadate is regulated by cellular redox state.     

Effects of adenosine 3' : 5'-monophosphate and guanosine 3' : 5'-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle.

The effects of adenosine 3' : 5'-monophosphate (cyclic AMP), guanosine 3' : 5'-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P). While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10(-5) M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP. Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10(-8) M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10(-8) M, while with cyclic AMP a concentration of 10(-5) M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P. These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

Effect of vanadate on the cellular accumulation of pp15, an apparent product of insulin receptor tyrosine kinase action

The possible involvement of a 15-kDa phosphotyrosyl protein, pp15, in insulin action was investigated by using the insulin-mimetic agent, vanadate. Vanadate, a phosphotyrosine phosphatase inhibitor, was found to mimic insulin in 3T3-L1 adipocytes by three criteria. First, kinetic and concentration-dependence studies verified the insulin-like effect of vanadate in activating 2-deoxyglucose uptake. Insulin had an additive activating effect at a submaximal vanadate concentration, but showed no further activation at a saturating vanadate concentration. The trivalent arsenical, phenylarsine oxide (PAO) which forms complexes with vicinal dithiols, markedly inhibited vanadate-activated hexose transport in agreement with our previous studies in which PAO abolished the insulin-activated component of sugar uptake. Second, in situ phosphorylation experiments showed that vanadate activated tyrosine phosphorylation of the insulin receptor's beta-subunit. Exposure of vanadate-treated cells to PAO further increased the level of beta-subunit phosphorylation. The increased level of phosphorylation in the presence of PAO occurred only on tyrosyl residues. Third, vanadate caused the accumulation of a phosphorylated 15-kDa protein in the presence of PAO, but not in its absence. The characteristics of this protein were identical to those of pp15: 1) both proteins behaved identically by two-dimensional gel electrophoresis, 2) digestion of both proteins with trypsin gave rise to apparently identical phosphopeptides, and 3) both proteins contained phosphotyrosine as the only phosphoamino acid. The results indicate that both vanadate and insulin stimulate the accumulation of pp15 in the presence of PAO. The dithiol,2,3-dimercaptopropanol, but not a monothiol, reversed the effects of PAO on the inhibition of vanadate-induced hexose transport and the accumulation of pp15, thus implicating a vicinal dithiol in these actions of vanadate and insulin. Our results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin receptor tyrosine kinase action, is coupled to signal transmission to the glucose transport system.     

Interleukin-10 activation of the interleukin-10E1 pathway and tissue inhibitor of metalloproteinase-1 expression is enhanced by proteasome inhibitors in primary prostate tumor lines

The interleukin-10 (IL-10) activation of Janus kinase (JAK) family members (JAK1/TYK2) and IL-10E1 is subsequently inactivated by approximately 3-4 h in primary prostate tumor lines. We examined the effect of proteasome inhibition on IL-10 activation of the IL-10E1 pathway following stimulation of HPCA-10a cells. Treatment of HPCA-10a cells with the proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL), led to stable tyrosine phosphorylation of the IL-10 receptor and IL-10E1 following stimulation. Further investigation showed that these stable phosphorylation events were the result of prolonged activation of JAK1 and TYK2 plus IL-10E1. IL-10E1 signaling normally induced the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and LLnL treatment of the HPCA-10a and HPCA-10c cells significantly enhanced IL-10 induction of TIMP-1 levels to block tumor cell invasion in modified Boyden chamber invasion assays. These observations were confirmed using pharmacologic inhibitors by Western blot and ELISAs. In the presence of LLnL, stable phosphorylation of IL-10E1 and induction of TIMP-1 was abrogated if the tyrosine kinase inhibitor, staurosporine, was added. The effect of staurosporine on IL-10E1 phosphorylation and TIMP-1 could be overcome if the phosphatase inhibitor, vanadate, was also added, suggesting that phosphorylated IL-10E1 could be stabilized by phosphatase, but not by proteasome inhibition. These observations are consistent with the hypothesis that proteasome-mediated protein degradation can modulate the activity of the IL-10E1 pathway and TIMP-1 induction by regulating the deactivation of JAK1/TYK2.