Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.     

Phosphotyrosyl turnover in insulin signaling. Characterization of two membrane-bound pp15 protein tyrosine phosphatases from 3T3-L1 adipocytes.

It was shown previously that 422 (aP2) protein, a 15-kDa fatty acid binding protein, is phosphorylated on Tyr19 both in vitro by the insulin receptor tyrosine kinase and in intact 3T3-L1 adipocytes treated with insulin and phenylarsine oxide (PAO). Phospho-422(aP2) protein (pp15) accumulates in cells treated with insulin and PAO because the arsenical blocks turnover of the phosphoryl group of pp15. These findings suggest that a PAO-sensitive enzyme mediates turnover of the pp15 tyrosine phosphoryl group. We have purified and characterized two membrane protein tyrosine phosphatases (PTPases) from 3T3-L1 adipocytes that catalyze hydrolysis of phospho-Tyr19 of authentic pp15. These enzymes, designated PTPases HA1 and HA2, were purified approximately 20,000-fold and approximately 15,000-fold, respectively, and shown to differ markedly in their sensitivity to both vanadate and phosphotyrosine. Both enzymes are inhibited by PAO and accordingly can be labeled with 4-[125I]iodo-PAO. By this method, it was demonstrated that PTPases HA1 and HA2 have molecular masses of approximately 60 kDa and approximately 38 kDa, respectively. Both enzymes exhibit substrate preference for pp15 when compared with other phosphotyrosine-containing protein substrates. Proteins containing phosphoserine and phosphothreonine do not serve as substrates for the enzymes. The pp15 PTPase HA2 is expressed both in 3T3-L1 preadipocytes and adipocytes, whereas pp15 PTPase HA1 is expressed only in 3T3-L1 adipocytes.     

Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase

Insulin stimulates the tyrosine phosphorylation of a 185-kDa putative cytosolic substrate protein (pp185) in diverse cell types. After intravenous insulin infusion into the live intact rat, pp185 and the 95-kDa insulin receptor beta-subunit were the major proteins that tyrosine phosphorylated in liver, skeletal muscle, and adipose tissue. Both proteins were maximally phosphorylated within 30 s, and both increased in phosphotyrosine content in parallel with increasing insulin dose. However, pp185 tyrosine phosphorylation was transient, with almost complete dephosphorylation within 2-3 min despite continued insulin stimulation. To identify pp185 directly, we purified pp185 from insulin-stimulated rat liver, using a denaturation-based extraction procedure that blocks endogenous protein phosphatases and thus allows a high yield, single step isolation of phosphotyrosyl proteins by anti-phosphotyrosine antibody immunoaffinity absorption. From 50 rat livers, 50-100 pmol of pp185 was isolated. Edman degradation of seven internal tryptic peptide fragments of pp185 yielded novel amino acid sequences, indicating that pp185 is a new protein. Antipeptide antibodies were raised which specifically recognize a single, 185-kDa insulin-stimulated phosphotyrosyl protein in liver, skeletal muscle, adipose tissue, and several cultured cell lines. These results indicate that pp185 is expressed in a variety of insulin-responsive tissues, is the major protein rapidly tyrosine phosphorylated under physiological conditions in the intact animal, and also provide a route for cloning the pp185 gene and elucidating the function of pp185 in insulin signal transduction.     

Phenylarsine oxide stimulates a cytosolic tyrosine kinase activity and glucose transport in mouse fibroblasts

In the present report we further approach the mechanism by which insulin and phenylarsine oxide (PAO), a trivalent arsenical compound, regulate glucose transport in mouse fibroblasts (NIH3T3). First, we show that PAO is a powerful stimulatory agent on glucose transport. Second, at least three series of observations indicate that this action of PAO is not mediated through the insulin receptor: (i) the same effect of PAO is observed in NIH3T3 and in transfected cells expressing 6 x 10(6) insulin receptors, while the effect of insulin is markedly increased in the transfected cells; (ii) PAO does not affect the tyrosine phosphorylation of the insulin receptor; (iii) the tyrosine kinase activity of the insulin receptor toward exogenous substrates is not increased by PAO. Since PAO appears to act on glucose transport by a different mechanism than insulin, we have compared the effect of PAO and insulin on tyrosine phosphorylation of cellular proteins. Using Western blot analysis we did not detect common substrates in PAO- and insulin-treated cells. However, we found in cell extracts from both PAO- and insulin-treated cells a 50-kDa protein that is immunoprecipitated by antiphosphotyrosine antibody. In addition, PAO activates a cytosolic tyrosine kinase capable of poly(Glu/Tyr) phosphorylation. As a whole, our data suggest that the 50-kDa protein found in cells incubated with PAO and insulin could be the convergence point of the insulin and PAO signaling pathways.     

A novel 68-kDa adipocyte protein phosphorylated on tyrosine in response to insulin and osmotic shock

Osmotic shock can cause insulin resistance in 3T3-L1 adipocytes by inhibiting insulin activation of glucose transport, p70S6 kinase, glycogen synthesis, and lipogenesis. By further investigating the relationship between insulin and hypertonic stress, we have discovered that osmotic shock enhanced by 10-fold the insulin-stimulated tyrosine phosphorylation of a 68-kDa protein. Phosphorylation by insulin was maximal after 1 min and was saturated with 50-100 nm insulin. The effect of sorbitol was completely reversible by 2.5 min. pp68 was a peripheral protein that was localized to the detergent insoluble fraction of the low density microsomes but was not associated with the cytoskeleton. Stimulation of the p42/44 and the p38 MAP kinase pathways by osmotic shock had no effect on pp68 phosphorylation. Treatment of adipocytes with the phosphotyrosine phosphatase inhibitor phenylarsine oxide also enhanced insulin-activated tyrosine phosphorylation of pp68 suggesting that osmotic shock may increase pp68 phosphorylation by inhibiting a phosphotyrosine phosphatase. Dissociation of pp68 from the low density microsomes with RNase A indicated that pp68 binds to RNA. Failure to immunoprecipitate pp68 using antibodies directed against known 60-70-kDa tyrosine-phosphorylated proteins suggest that pp68 may be a novel cellular target that lies downstream of the insulin receptor.     

Interaction between the p21ras GTPase activating protein and the insulin receptor.

We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine residues. However, after insulin treatment in the presence of the phosphotyrosine phosphatase inhibitor phenylarsine oxide (PAO), 5-10% of GAP was found to be associated with the insulin receptor, and, in addition, a fraction of total GAP was phosphorylated on tyrosine. Using in vitro binding we showed that the N-terminal part of GAP containing the src-homology domains 2 and 3 (SH2-SH3-SH2 region) is involved in binding to the autophosphorylated insulin receptor beta-chain. In vitro binding between GAP and the autophosphorylated insulin receptor occurred independently of PAO pretreatment. These results suggest that GAP can transiently interact with the insulin receptor after insulin treatment, and this interaction is arrested after PAO pretreatment.     

Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by a mechanism other than an increase in surface transporters

Phenylarsine oxide (PAO) has been shown to exert a biphasic effect on glucose transport in 3T3-L1 adipocytes. At 10 microM, PAO activates transport threefold, but at higher concentrations an inhibition of transport is observed. In this paper we report a procedure for the subcellular fractionation of these cells which we use to examine the distribution of glucose transporters following PAO challenge. Quantitative immunoblotting showed that the glucose transporter content of the plasma membrane fraction increased with increasing PAO concentrations; a parallel increase in another insulin-responsive protein, the transferrin receptor, also occurred. However, cell-surface labeling procedures for the glucose transporter and transferrin receptor showed that PAO actually decreased the cell-surface concentrations of these proteins; the basis of this discrepancy may be that in the presence of PAO, intracellular vesicles containing these proteins associate with the plasma membrane, but do not fuse with it. The possibility that PAO modulated transport by direct interaction with the glucose transporter was investigated by examining the effects of PAO on transport in both erythrocytes and a reconstituted system of purified erythrocyte transporter in lipid vesicles. PAO was without effect on the rate of transport in these systems. The hypothesis that the stimulatory effect of PAO on transport might be due to the activation of the insulin receptor kinase activity was examined by assessing the phosphotyrosine content of the receptor and other proteins using anti-phosphotyrosine antibodies. PAO alone caused no detectable increase in receptor phosphotyrosine content. However, the combination of PAO and insulin led to the tyrosine phosphorylation of two proteins of Mr 68,000 and 57,000 which were not detected in cells treated with either PAO or insulin, and an increased phosphotyrosine content of proteins of Mr 95,000 and 165,000 when compared to cells treated with insulin alone.     

Pharmacological doses of insulin equalize insulin receptor phosphotyrosine content but not tyrosine kinase activity in plasmalemmal and endosomal membranes.

Following insulin administration to intact rats, the insulin receptor kinase activity of subsequently isolated cell fractions was significantly augmented. Of interest was the observation that the endosomal insulin receptor tyrosine kinase displayed four- to six-fold greater autophosphorylation activity than that of plasma membrane. Surprisingly, the endosomal insulin receptor tyrosine kinase displayed a decrease in beta-subunit phosphotyrosine content compared with that seen in the plasma membrane. These observations prompted the suggestion that insulin receptor tyrosine kinase phosphotyrosine dephosphorylation mediated by an endosome-specific phosphotyrosine phosphatase(s) yields activation of the endosomal insulin receptor tyrosine kinase. In a previous study we examined the effect of subsaturating doses of injected insulin. In this work we evaluated insulin receptor tyrosine kinase activity and phosphotyrosine content in plasma membrane and endosomes after a receptor-saturating pharmacological dose of insulin (150 micrograms/100 g body weight). At this dose the phosphotyrosine content per receptor was reduced compared with that seen earlier at insulin doses of 1.5 and 15 micrograms/100 g body weight. Endosomal insulin receptor tyrosine kinase was greater than that seen at the lower nonsaturating insulin doses. Furthermore, endosomal insulin receptor tyrosine kinase activity exceeded that of the plasma membrane, despite retaining about the same phosphotyrosine content per receptor. These data are consistent with the view that insulin receptor tyrosine kinase activity may be regulated by a particular pattern of phosphotyrosine content on the beta-subunit wherein both activating and inhibitory phosphotyrosine residues play a role.     

Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.     

Tyrosine phosphorylation of alpha-actinin in activated platelets

The integrin alpha(IIb)beta(3) mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human alpha-actinin sequences deposited in the Swiss Protein Database. alpha-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with alpha-actinin and anti-phosphotyrosine antibodies. The distribution of alpha-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained alpha-actinin. The purified alpha-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. alpha-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with alpha-actinin. Taken together, these data demonstrate that a fraction of alpha-actinin is tyrosine-phosphorylated in activated platelets.     

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.     

Antiphosphotyrosine antibodies modulate insulin receptor kinase activity and insulin action

Insulin elicits the autophosphorylation of the beta-subunit of its receptor on tyrosine residues: this effect appears to be the earliest post-binding event involved in insulin action. In the present study we have raised highly specific antibodies to phosphotyrosine residues, and we have taken advantage of these antibodies to further evaluate the role of the insulin receptor tyrosine kinase in the generation of insulin's biological responses. Using a cell-free phosphorylation assay, we show here that these antibodies increase the tyrosine kinase activity of the receptor, and its phosphorylation on tyrosine residues. In contrast, the antibodies do not interfere with dephosphorylation of the insulin receptor. Introduction of the same antibodies in living Fao hepatoma cells enhances the effect of insulin on both glucose transport and aminoacid uptake. As a whole our data indicate that the insulin receptor kinase is involved in the generation of an early (glucose transport) and late (aminoacid uptake) response to insulin. Further, conformational changes in phosphotyrosine containing domains of the insulin receptor appear to modulate insulin's biological effects. Finally, the injection of antibodies in intact cells provides us with a novel and promising tool to search for cellular substrates for the insulin receptor tyrosine kinase.     

Proteomic analysis reveals novel molecules involved in insulin signaling pathway

The binding of insulin to its receptor triggers a signaling cascade regulated by protein complexes via tyrosine phosphorylation events on a multitude of associated proteins. To search novel phosphotyrosine proteins or associated proteins involved in insulin signaling pathway, we employed a method in which Rat1 cells stably expressing the human insulin receptor were stimulated with or without insulin and sub-fractionated prior to enrichment of phosphotyrosine proteins by immunoprecipitation and analysis by LC-MS/MS. Bioinformatic analysis and manual confirmation of peptide phosphorylation site assignments led to identification of 35 phosphotyrosine sites derived from 31 protein groups. Over 50% of these proteins were reported for the first time as tyrosine phosphorylated, including gigaxonin, XIAP and CDK10. In addition, we also found that calcium/calmodulin-dependent protein serine kinase (CASK), a key protein in protein-targeting and vesicle transport in neurons, forms a complex with two unidentified phosphotyrosine proteins pp100 and pp95 in response to insulin-stimulation, though CASK is not itself tyrosine phosphorylated. Furthermore, insulin was able to decrease CASK nuclear location, as well as down-regulate the expression of CASK targeted genes. Our results imply CASK as a novel joint knot connecting CASK-mediated pathways with the insulin signaling. Our data provide a wealth of information potentially paving the way to identify new components in the insulin signaling network.     

Tyrosine phosphorylation of pp185 by insulin receptor kinase in a cell-free system

Insulin treatment of rat H-35 hepatoma cells causes rapid tyrosine phosphorylation of a high molecular weight protein termed pp185 besides autophosphorylation of the beta-subunit of the insulin receptor (IR) in an intact cell system. To elucidate the molecular basis for tyrosine phosphorylation of pp185, cell-free phosphorylation of pp185 was performed using phosphotyrosine-containing proteins (PYPs) purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibody. After insulin treatment of cells, marked increases of tyrosine phosphorylation of pp185 and IR were observed compared to noninsulin-treated cells. Site-specific antibodies that specifically inactivate IR kinase inhibited tyrosine phosphorylation of pp185 as well as the beta-subunit of IR. PYPs purified from detergent-free cell extracts contained pp185 but little IR; tyrosine phosphorylation of pp185 did not occur. Addition of IR kinase purified from human placenta to these PYPs restored insulin-dependent tyrosine phosphorylation of pp185. These results suggest that tyrosine phosphorylation of pp185 is catalyzed directly by IR kinase in this cell-free system.     

Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.

We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.     

A combination of H2O2 and vanadate concomitantly stimulates protein tyrosine phosphorylation and polyphosphoinositide breakdown in different cell lines

Treatment of four cell lines [rat hepatoma (Fao), murine muscle (BC3H-1), Chinese hamster ovary (CHO), and rat basophilic leukemia (RBL)] with a combination of 3 mM H2O2 and 1 mM sodium orthovanadate markedly stimulates protein tyrosine phosphorylation, which is accompanied by a dramatic increase (5-15-fold) in inositol phosphate (InsP) formation. H2O2/vanadate stimulate best formation of inositol triphosphate while their effects on the mono and di derivatives are more moderate. In the presence of 3 mM H2O2, both protein tyrosine phosphorylation and InsP formation are highly correlated and manifest an identical dose-response relationship for vanadate. Half-maximal and maximal effects are obtained at 30 and 100 microM, respectively. This stimulatory effect of H2O2/vanadate is not mimicked by other oxidants such as spermine, spermidine, KMnO4, and vitamin K3. In RBL cells, the kinetics of inositol triphosphate formation correlate with tyrosine phosphorylation of a 67-kDa protein, while tyrosine phosphorylation of a 55-kDa protein is closely correlated with both inositol monophosphate formation and serotonin secretion from these cells. Taken together, these results suggest a causal relationship between tyrosine phosphorylation triggered in a nonhormonal manner and polyphosphoinositide breakdown. Furthermore, these results implicate protein tyrosine phosphorylation in playing a role in the stimulus-secretion coupling in RBL cells.     

Insulin and insulin-like growth factor 1 stimulate the phosphorylation on tyrosine of a 160 kDa cytosolic protein in 3T3-L1 adipocytes.

Insulin and IGF-1 (insulin-like growth factor 1) rapidly stimulate the phosphorylation on tyrosine of a 160 kDa cytosolic protein (pp160) in intact 3T3-L1 adipocytes. Half-maximal phosphorylation of pp160 is attained with either 4 nM-insulin or 20 nM-IGF-1. A semi-quantitative immunoblotting procedure using anti-phosphotyrosine antibody revealed that the insulin-stimulated 3T3-L1 adipocyte possesses approx. 3 x 10(5) and 0.6 x 10(5) phosphotyrosyl sites, respectively, in pp160 and insulin receptor beta-subunit. Removal of insulin from stimulated cells results in the rapid (within 15 min) loss of phosphate groups from tyrosyl residues in both pp160 and receptor beta-subunit. Whereas pp160 remains maximally phosphorylated on tyrosine for up to 60 min in the presence of 100 nM-insulin, IGF-1 at the same concentration induces only a transient response that is maximally 50% of that observed with insulin. pp160 is not phosphorylated on tyrosine in response to platelet-derived growth factor or epidermal growth factor. Although pp160 appears to be a soluble cytoplasmic protein, in the presence of 1 mM-ZnCl2 it becomes membrane-associated. In view of its apparent cytoplasmic localization and its inability to bind to either wheat-germ agglutinin or concanavalin A, pp160 does not appear to be a typical glycoprotein growth-factor receptor. Our results suggest that pp160 may be a physiologically important cellular substrate of the insulin-receptor tyrosine kinase in the intact 3T3-L1 adipocyte.     

Quantification and identification of mitochondrial proteins containing vicinal dithiols

Vicinal dithiols may play a role in mitochondrial antioxidant defences and in redox signalling. We quantified protein vicinal dithiols within mammalian mitochondria using the vicinal dithiol-specific reagent phenylarsine oxide (PAO). We found 5-15% of thiols exposed on mitochondrial proteins were vicinal dithiols and that these thiols were particularly sensitive to oxidation by hydrogen peroxide. To visualise these proteins we used PAO to block vicinal dithiols, followed by alkylation of other thiols with N-ethylmaleimide (NEM). The PAO was then removed with 2,3-dimercapto-1-propanesulfonic acid (DMPS) and the exposed vicinal dithiols were labelled with iodoacetamide-biotin. To identify these proteins, we developed a selective proteomic methodology, based on Redox difference in gel electrophoresis (Redox-DIGE). Vicinal dithiol proteins were selectively labelled with a red fluorescent thiol-reactive Cy5 maleimide and mixed with Cy3 maleimide labelled protein in which vicinal dithiols remained untagged. Individual proteins were resolved by 2D gel electrophoresis and fluorescent scanning revealed vicinal dithiol proteins by the increase in Cy5 red fluorescence. These proteins were identified by peptide mass fingerprinting and mass spectrometry. These findings are consistent with roles for mitochondrial vicinal dithiol proteins in antioxidant defence and redox signalling and these methodologies will enable these roles to be explored.     

An altered IGF-I receptor is present in human leukemic cells.

We have characterized and analyzed IGF-I- and insulin-stimulated cell growth, receptor binding, and autophosphorylation in the human leukemic cell line HL-60. IGF-I-stimulated cell growth occurred at low (5 ng/ml) and insulin stimulated only at high (500 ng/ml) concentrations. Binding of 125I-IGF-I to partially purified plasma membrane proteins followed the characteristics of IGF-I receptor binding. 125I-IGF-I binding, as determined by chemical cross-linking, occurred to a 145-kDa protein. IGF-I, as well as insulin, stimulated the autophosphorylation of a 105-kDa band (pp105), but we could not detect a 95-kDa band corresponding to the known molecular mass of the IGF-I and insulin receptor beta-subunits. Phosphorylation of pp105 followed the dose-response characteristics of the IGF-I receptor. The phosphorylation of pp105 occurred at tyrosine and threonine, and the pattern of HPLC tryptic peptide maps showed marked differences when compared with that of a phosphorylated insulin receptor beta-subunit. Enzymatic deglycosylation of pp105 resulted only in a slight reduction of the molecular weight. These data suggest that pp105 is the beta-subunit of an IGF-I receptor variant with a higher molecular weight, similar to that found in fetal tissue. The HL-60 cell may acquire, at least in part, malignant growth characteristics through reexpression of the fetal version of the IGF-I receptor.     

H2O2 potentiates phosphorylation of novel putative substrates for the insulin receptor kinase in intact Fao cells

Western blotting with anti-phosphotyrosine antibodies was employed in order to study insulin-dependent protein tyrosine phosphorylation in intact Fao cells. In insulin-treated cells, a prominent 180-kDa protein underwent tyrosine phosphorylation, which peaked at 45 s and then rapidly declined. Pretreatment of the cells with 1 mM Bt2cAMP or 0.16 microM 12-O-tetradecanoylphorbol-13-acetate inhibited the insulin-dependent phosphorylation of pp 180, while 1 mM vanadate or 3 mM H2O2 markedly potentiated it. These results indicate that phosphorylation of pp 180 is respectively regulated by agents that are known to synergize with or antagonize the action of the insulin receptor kinase. pp 180 is therefore likely to mediate physiological functions of this receptor kinase. Incubation of Fao cells with 3 mM H2O2 for 30 min prior to their treatment with insulin for 45 s allowed the detection of additional, previously undescribed, proteins pp 150, 114, 100, 85, 68, and 56 kDa that underwent insulin-dependent tyrosine phosphorylation. The potentiating effects of H2O2 were time- and dose-dependent and could be reversed by 2 mM dithiothreitol. Proteins phosphorylated in response to H2O2 plus insulin maintained their fully phosphorylated state for at least 20 min. We suggest that these phosphoproteins are potential physiological substrates for the insulin receptor kinase.