Biomedical subjects

Y Zick

Publications and source records attributed to Y Zick.

At least 37 records · Page 2Linked to original sources

PubMed1998

Interplay of the proto-oncogene proteins CrkL and CrkII in insulin-like growth factor-I receptor-mediated signal transduction.

The closely related proto-oncogene proteins CrkII and CrkL consist of one SH2 and two SH3 domains and share 60% overall homology with the highest identity within their functional domains. In this study we show that CrkL and CrkII may play overlapping but different roles in insulin-like growth factor (IGF)-I receptor-mediated signal transduction. While both proteins are substrates involved in IGF-I receptor signaling, they apparently demonstrate important different properties and different biological responses. Evidence supporting this hypothesis includes (a) the oncogenic potential of CrkL versus the absence of this potential in CrkII overexpressing cell lines, (b) the inhibition of IGF-I-dependent cell cycle progression by overexpression of CrkII, and (c) the differential regulation of the phosphorylation status of selective proteins in CrkII and CrkL overexpressing cell lines. In addition we demonstrate the specific association of CrkL and CrkII with the newly characterized IRS-4 protein, again in a differential manner. Whereas CrkL strongly interacts with IRS-4 via its SH2 and N-terminal SH3 domains, CrkII interacts only via its SH2 domain, possibly explaining the unstable nature of IRS-4-CrkII association. The results obtained allow us to propose a unique mechanism of CrkL and CrkII tyrosine phosphorylation in response to IGF-I stimulation. Thus these highly homologous proteins apparently possess structural features that allow for the differential association of each protein with different effector molecules, thereby activating different signaling pathways and resulting in unique biological roles of these proteins.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Interaction in vitro of the product of the c-Crk-II proto-oncogene with the insulin-like growth factor I receptor.

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in vitro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in vitro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. Mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in vitro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in vitro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which region of the IGF-I receptor interacts with Crk-II, a peptide association assay was used in vitro. Different domains of the IGF-I receptor were expressed as (His)6-tagged fusion peptides, phosphorylated with activated wheat germ agglutinin-purified IGF-I receptors and tested for association with GST-Crk-II fusion proteins. Using wild-type as well as mutated peptides, we showed that the SH2 domain of Crk-II preferentially binds the peptide encoding the juxtamembrane region of the IGF-I receptor. Phosphorylation of Tyr-950 and Tyr-943 of the receptor is important for this interaction. These findings allow us to propose a model of direct interaction of Crk-II and the IGF-I receptor in vivo. On activation of the IGF-I receptor, Crk-II binds to phosphorylated tyrosine residues, especially in the juxtamembrane region. As a result of this binding, the IGF-I receptor kinase phosphorylates Tyr-221 of Crk-II, resulting in a change in intramolecular folding and binding of the SH2 domain to the phosphorylated Tyr-221, which causes rapid disassociation of the Crk-II-IGF-I receptor complex.

3T3 Cells↗

PubMed1998

The carboxyl-terminal domain of insulin-like growth factor-I receptor interacts with the insulin receptor and activates its protein tyrosine kinase.

Receptors for insulin and insulin-like growth factor-I (IR and IGFIR) consisting of the alpha2beta2 structure are protein tyrosine kinases (PTKs). Carboxyl-terminal (CT) domains of their beta subunits are structurally diverse while the PTK domains share the highest homology. Interactions between CT and PTK domains of IR and IGFIR were studied by means of PTK activity, fluorescence energy transfer or surface plasmon resonance using BIAcore. We present evidence that IGFIR CT directly interacts with both IGFIR and IR. Although binding to both receptors, stimulation of PTK activity only occurs with IR but not IGFIR.

Animals↗

PubMed1997

A molecular basis for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation.

Tumor necrosis factor alpha (TNFalpha) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxyl-terminal region (amino acids 1245-1331) of IR expressed in bacteria as His6 fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNFalpha, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNFalpha for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20-60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNFalpha effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNFalpha on insulin action.

Animals↗

PubMed1997

Phosphorylation of erythropoietin receptors in the endoplasmic reticulum by pervanadate-mediated inhibition of tyrosine phosphatases.

Erythropoietin (EPO) is the major hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Ligand binding to the erythropoietin receptor (EPO-R), a member of the cytokine receptor family, triggers Tyr phosphorylation of the surface form of the receptor, presumably mediated by the Janus kinase (JAK) 2. To study whether non-surface EPO-R can be phosphorylated, Ba/F3 cells stably transfected with EPO-R were treated with pervanadate (PV), which is widely used as a potent tool to inhibit cellular protein Tyr phosphatases, thus resulting in enhanced Tyr phosphorylation of cellular proteins. PV treatment caused the EPO-R to undergo Tyr phosphorylation in a time-dependent and dose-dependent manner. PV-mediated Tyr phosphorylation of EPO-R occurred at several intracellular sites including the endoplasmic reticulum (ER), because both endoglycosidase H (endo H)-resistant EPO-R and the ER-retained EPO-R mutant (DeltaWS1 EPO-R) were Tyr phosphorylated in response to PV. Moreover, in metabolic labelling experiments, endo H-sensitive EPO-R was also phosphorylated. The phosphorylated fraction accounted for only 30-50% of the newly synthesized EPO-R, the fraction that normally exits from the ER. Tyr phosphorylation could not be detected on proteolytic fragments of the EPO-R, suggesting that this is a highly regulated process. Unlike the wild-type (wt) EPO-R, which was phosphorylated both on EPO binding and after inhibition of Tyr phosphatases by PV treatment, an EPO-R mutant (W282R EPO-R) that does not activate JAK2 was phosphorylated after PV treatment but not by EPO binding. Both EPO-R and JAK2 were phosphorylated with similar kinetics by PV treatment, suggesting that JAK2, as well as protein Tyr kinases different from JAK2, might mediate PV-dependent EPO-R phosphorylation. Furthermore the Tyr-phosphorylated ER-retained EPO-R mutant DeltaWS1 co-immunoprecipitated with JAK2 kinase, indicating that the EPO-R might interact with JAK2 while in the ER.

Animals↗

PubMed1997

p75, a member of the heat shock protein family, undergoes tyrosine phosphorylation in response to oxidative stress.

The combination of H2O2 and vanadate generates aqueous peroxovanadium (pV) species, which are effective cell-permeable oxidants, and potent inhibitors of protein-tyrosine phosphatases. As a result, treatment of intact cells with pV compounds significantly enhances protein Tyr phosphorylation. Here we demonstrate that treatment of intact rat hepatoma Fao cells with pV markedly enhances Tyr phosphorylation of a 75-kDa protein, termed pp75. Amino-terminal sequencing of pp75 revealed that this protein is a member of the 70-75-kDa heat shock protein family, which includes PBP-74, glucose-related protein (GRP)-75, and mortalin. Tyr phosphorylation of pp75 is selective, because other proteins that belong to the heat shock protein 70 family, such as GRP-72, Bip (GRP-78), and HSC-70 fail to undergo Tyr phosphorylation when cells are treated with pV. Our findings suggest that heat shock proteins such as pp75 may undergo tyrosine phosphorylation when intact cells are subjected to oxidative stress induced by pV compounds.

Amino Acid Sequence↗

PubMed1996

Annexin II is a novel player in insulin signal transduction. Possible association between annexin II phosphorylation and insulin receptor internalization.

Annexin II is a Ca2+-, phospholipid-, and actin- binding protein that was implicated in the regulation of vesicular traffic and endosome fusion. It is a known substrate for protein kinases including the platelet-derived growth factor receptor, src protein-tyrosine kinase, and protein kinase C. In the present study we investigated the possible involvement of annexin II in insulin signal transduction. Phosphorylation of annexin II in response to insulin treatment of intact Chinese hamster ovary (CHO)-T cells was detected by 5 min and reached maximal levels after a 2-3-h incubation with the hormone. However, unlike other receptor substrates, annexin II failed to undergo insulin-induced Tyr phosphorylation under conditions where receptor internalization was inhibited. This was evident in CHO cells, overexpressing the insulin receptor, in which internalization was inhibited either by tyrosine kinase inhibitors or by lowering the temperature to 4 degrees C, and in CHO cells overexpressing various insulin receptor mutants in which normal internalization was impaired. Hence, Tyr phosphorylation of annexin II could be part of the internalization and sorting mechanism of the insulin receptor.

Androstadienes↗

PubMed1996

Interaction between the insulin receptor and its downstream effectors. Use of individually expressed receptor domains for structure/function analysis.

A structural analysis has been carried out to determine which part of the intracellular domain of the insulin receptor (IR) beta subunit is involved in direct interaction with the receptor substrates IRS-1 and Shc. Toward this end, the juxtamembrane (JM) domain (amino acids 943-984) and the carboxyl-terminal (CT) region (amino acids 1245-1 331) of IR were expressed in bacteria as (His)6-fusion peptides, and their interaction with IRS-1 and Shc was studied. We could demonstrate that the CT region of IR was sufficient to bind Shc, although significant, but much lower binding of Shc to the JM region could be detected as well. Furthermore, in vitro Tyr phosphorylation of the CT region potentiated its interactions with Shc 2-fold. In contrast, the JM region, but not the CT domain of the IR, was sufficient to mediate interactions between the IR and IRS-1. These interactions did not involve the pleckstrin homology (PH) region of IRS-1, since an IRS-1 mutant, in which four "blocks" of the PH domain (Pro5-Pro65) were deleted, interacted with the JM region of IR with the same efficiency as native IRS-1. These results suggest that the IR interacts with its downstream effectors through distinct receptor regions, and that autophosphorylation of Tyr residues located at the CT domain of the IR can modulate these interactions.

Animals↗

PubMed1995

Differential activation of mitogen-activated protein kinase and S6 kinase signaling pathways by 12-O-tetradecanoylphorbol-13-acetate (TPA) and insulin. Evidence for involvement of a TPA-stimulated protein-tyrosine kinase.

AG-18, an inhibitor of protein-tyrosine kinases, was employed to study the role of tyrosine-phosphorylated proteins in insulin- and phorbol ester-induced signaling cascades. When incubated with Chinese hamster ovary cells overexpressing the insulin receptor, AG-18 reversibly inhibited insulin-induced tyrosine phosphorylation of insulin receptor substate-1, with minimal effects either on receptor autophosphorylation or on phosphorylation of Shc64. Under these conditions, AG-18 inhibited insulin-stimulated phosphorylation of the ribosomal protein S6, while no inhibition of insulin-induced activation of mitogen-activated protein kinase (MAPK) kinase or MAPK was detected. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of MAPK kinase and MAPK and phosphorylation of S6 were inhibited by AG-18. This correlated with inhibition of TPA-stimulated tyrosine phosphorylation of several proteins, the most prominent ones being pp114 and pp120. We conclude that Tyr-phosphorylated insulin receptor substrate-1 is the main upstream regulator of insulin-induced S6 phosphorylation by p70s6k, whereas MAPK signaling seems to be activated in these cells primarily through the adaptor molecule Shc. In contrast, TPA-induced S6 phosphorylation is mediated by the MAPK/p90rsk cascade. A key element of this TPA-stimulated signaling pathway is an AG-18-sensitive protein-tyrosine kinase.

Animals↗

PubMed1995

Tyrosine phosphorylation of insulin receptor substrate-1 in vivo depends upon the presence of its pleckstrin homology region.

To characterize the structural basis for the interactions between the insulin receptor (IR) and its major substrate, insulin receptor substrate-1 (IRS-1), a segment of the NH2-terminal region of IRS-1 (Pro5-Pro65) was deleted. This region contains the first four conserved boxes of a pleckstrin homology (PH) domain, located at the NH2-terminal part of IRS-1. COS-7 cells were then cotransfected with the genes coding for IR and a wild-type (WT) or a mutated form of IRS-1. IRS-1 delta PH underwent significantly reduced insulin-dependent tyrosine phosphorylation compared with WT IRS-1. The reduced in vivo tyrosine phosphorylation of IRS-1 delta PH was accompanied by reduced association between IRS-1 delta PH and its downstream effector p85 regulatory subunit of phosphatidylinositol-3 kinase. In contrast, both WT IRS-1 and IRS-1 delta PH underwent comparable insulin-dependent tyrosine phosphorylation in vitro when incubated with partially purified insulin receptor kinase. These findings suggest that the overall structure of IRS-1 is not altered by deletion of its PH domain and that the PH domain is not the main site for protein-protein interactions between the insulin receptor and IRS-1, at least in vitro. In conclusion, the PH region might facilitate in vivo binding of IRS-1 to membrane phospholipids or other cellular constituents in close proximity to the IR, whereas the actual interactions with the IR are presumably mediated through other domains of the IRS-1 molecule. This could account for the fact that partial deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1, whereas their in vitro interactions remain unaffected.

Amino Acid Sequence↗

PubMed1995

Galectin-8. A new rat lectin, related to galectin-4.

A protein of 35 kDa which has the characteristic properties of galectins (S-type lectins) was cloned from rat liver cDNA expression library. Since names for galectins 1-7 were already assigned, this new protein was named galectin-8. Three lines of evidence demonstrate that galectin-8 is indeed a novel galectin: (i) its deduced amino acid sequence contains two domains with conserved motifs that are implicated in the carbohydrate binding of galectins, (ii) in vitro translation products of galectin-8 cDNA or bacterially expressed recombinant galectin-8 are biologically active and possess sugar binding and hemagglutination activity, and (iii) a protein of the expected size (34 kDa) that binds to lactosyl-Sepharose and reacts with galectin-8-specific antibodies is present in rat liver and comprises approximately 0.025% of the total Triton X-100-soluble hepatic proteins. Overall, galectin-8 is structurally related (34% identity) to galectin-4, a soluble rat galectin with two carbohydrate-binding domains in the same polypeptide chain, joined by a link peptide. Nonetheless, several important features distinguish these two galectins: (i) Northern blot analysis revealed that, unlike galectin-4 that is confined to the intestine and stomach, galectin-8 is expressed in liver, kidney, cardiac muscle, lung, and brain; (ii) unlike galectin-4, but similar to galectins-1 and -2, galectin-8 contains 4 Cys residues; (iii) the link peptide of galectin-8 is unique and bears no similarity to any known protein; (iv) the N-terminal carbohydrate-binding region of galectin-8 contains a unique WG-E-I motif instead of the consensus WG-E-R/K motif implicated as playing an essential role in sugar-binding of all galectins. Together with galectin-4, galectin-8 therefore represents a subfamily of galectins consisting of a tandem repeat of structurally different carbohydrate recognition domains within a single polypeptide chain.

Amino Acid Sequence↗

PubMed1995

Modulation of protein tyrosine phosphorylation in rat insular cortex after conditioned taste aversion training.

Protein tyrosine phosphorylation is a major signal transduction pathway involved in cellular metabolism, growth, and differentiation. Recent data indicate that tyrosine phosphorylation also plays a role in neuronal plasticity. We are using conditioned taste aversion, a fast and robust associative learning paradigm subserved among other brain areas by the insular cortex, to investigate molecular correlates of learning and memory in the rat cortex. In conditioned taste aversion, rats learn to associate a novel taste (e.g., saccharin) with delayed poisoning (e.g., by LiCl injection). Here we report that after conditioned taste aversion training, there is a rapid and marked increase in tyrosine phosphorylation of a set of proteins in the insular cortex but not in other brain areas. A major protein so modulated, of 180 kDa, is abundant in a membrane fraction and remains modulated for more than an hour after training. Exposure of the rats to the novel taste alone results in only a small modulation of the aforementioned proteins whereas administration of the malaise-inducing agent per se has no effect. To the best of our knowledge, this is the first demonstration of modulation of protein tyrosine phosphorylation in the brain after a behavioral experience.

Animals↗

PubMed1994

Elevated protein tyrosine phosphatase activity and increased membrane viscosity are associated with impaired activation of the insulin receptor kinase in old rats.

Insulin resistance is very common in the elderly, and may be associated with glucose intolerance or frank diabetes. In previous studies we demonstrated that insulin resistance in old Wistar rats is associated with decreased autophosphorylation and activation of the hepatic insulin receptor kinase (IRK) in vivo. We now show that this defect can be reproduced in vitro, where the extent of insulin-induced activation of IRK in liver membranes of old rats was decreased by approximately 50% compared with young controls. The defect could be largely abolished after solubilization of the membranes with Triton X-100. We also show that: (a) the viscosity of membranes from the old rats was significantly (P < 0.001, n = 4) higher (by 15%) compared with young controls; (b) incubation of plasma membranes from old animals with lecithin liposomes, which lowered their cholesterol levels, partially abolished the defect in IRK activation; and (c) Triton extracts of liver membranes prepared from old rats did not interfere with the activation of IRK derived from young controls. Additionally, non-membrane components did contribute to the development of this defect. We observed a significant (approximately 30%) (P < 0.001, n = 18) elevation of cytosolic protein tyrosine phosphatase (PTP) activity directed against the beta subunit of the insulin receptor in livers of old rats. No such elevation of PTP activity could be demonstrated with synthetic substrates. Our findings are consistent with a model in which increased membrane viscosity as well as enhancement of a cytosolic PTP activity both markedly inhibit the activation in vivo of the hepatic IRK in old animals.

Aging↗

PubMed1994

A novel human insulin receptor gene mutation uniquely inhibits insulin binding without impairing posttranslational processing.

The precise nature of the insulin-binding site of the insulin receptor (IR) has not been determined, although the importance of several regions of the alpha-subunit in insulin binding has been demonstrated. A naturally occurring mutation in a patient with severe insulin resistance that changes the Ser323 codon in the alpha-subunit of the IR to a leucine codon is associated with markedly impaired insulin binding to cells from the patient and to transfected cells expressing the mutant receptor. However, unlike other IR alpha-subunit mutations associated with decreased insulin binding, this mutation does not lead to a defect in posttranslational processing or cell-surface expression of IRs. Thus, the defect in insulin binding associated with the Leu323 mutant IR is a direct result of an alteration in the insulin-binding site. No natural IR mutation described thus far is associated with both decreased insulin binding and normal cell-surface expression of the mutant receptor. This study demonstrates the critical role that Ser323 of the IR alpha-subunit plays in insulin binding, either by forming part of the binding site or by stabilizing its conformation.

3T3 Cells↗

PubMed1993

Hepatic tyrosine-phosphorylated proteins identified and localized following in vivo inhibition of protein tyrosine phosphatases: effects of H2O2 and vanadate administration into rat livers.

Injection of a combination of H2O2 and vanadate (H/V) into the portal vein of rat livers resulted in inhibition of protein tyrosine phosphatase activity and led to a dramatic enhanced in vivo protein tyrosine phosphorylation. Some of the phosphorylated proteins were identified as the beta-subunit of the insulin receptor, the insulin receptor substrate 1 (pp185), PLC-gamma (pp145), and a 100 kDa PLC-gamma-associated protein. Immunofluorescense and immune electron microscopy of frozen liver sections with anti-P-Tyr antibodies revealed that most of the tyrosine-phosphorylated proteins are localized in close proximity to the plasma membrane in intercellular adherence junctions and tight junction regions. This close in vivo association between membranal protein tyrosine kinases, their target proteins, and cytoskeletal elements could enable formation of 'signaling complexes' which may play a role in transmembrane signal transduction. By affinity chromatography over immobilized anti-P-Tyr antibodies, a large number of these tyrosine-phosphorylated proteins were partially purified.

Animals↗

PubMed1992

The insulinomimetic agents H2O2 and vanadate stimulate tyrosine phosphorylation of potential target proteins for the insulin receptor kinase 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. We have previously shown that a combination of H2O2 and vanadate, when added to intact cells, rapidly stimulates protein tyrosine phosphorylation, owing to the inhibitory effects of these agents on intracellular protein tyrosine phosphatases (PTPases). Employing Western blotting with anti-phosphotyrosine antibodies, we have now identified in Chinese-hamster ovary (CHO) cells transfected with a wild-type insulin-receptor gene (CHO.T cells) several proteins (e.g. pp180, 125, 100, 60 and 52) whose phosphotyrosine content is rapidly increased upon treatment of the cells with a combination of insulin and 3 mM-H2O2. Tyrosine phosphorylation of these and additional proteins was further potentiated when 100 microM-sodium orthovanadate was added together with H2O2. The effects of insulin, insulin/H2O2, and H2O2/vanadate on tyrosine phosphorylation were markedly decreased in CHO cells transfected with an insulin-receptor gene where the twin tyrosines 1162 and 1163 were replaced with phenylalanine (CHO.YF-3 cells). Similarly, most of these proteins failed to undergo enhanced tyrosine phosphorylation in parental CHO cells incubated in the presence of insulin or the insulinomimetic agents. Our findings suggest that inhibition of PTPase activity by H2O2/vanadate augments the autophosphorylation of tyrosines 1162 and 1163 of the insulin receptor kinase, leading to its activation in an insulin-independent manner. As a result, tyrosine phosphorylation of potential targets for this enzyme takes place. Failure of H2O2/vanadate to induce phosphorylation of these proteins in receptor mutants lacking these twin tyrosine residues supports this hypothesis.

Animals↗

PubMed1992

Selective inhibition of protein tyrosine phosphatase activities by H2O2 and vanadate in vitro.

Acute (10-30 min) treatment of intact rat hepatoma (Fao) cells with H2O2, inhibits in vivo protein tyrosine phosphatase activity. Vanadate markedly potentiates this effect although it has only trivial effects of its own. Here we show that H2O2 inhibits a protein tyrosine phosphatase activity, but not a p-nitro phenyl phosphate hydrolysing activity, in cytosolic extracts of these cells. This effect is completely reversed by 10 mM dithiothreitol. Other oxidants have similar inhibitory effects. Vanadate inhibits the protein tyrosine phosphatase activity in vitro, and its effects are additive with those of H2O2. These findings suggest that H2O2 and vanadate interact with the protein tyrosine phosphatases at two independent sites. They also suggest that in intact cells H2O2 has a direct inhibitory effect on protein tyrosine phosphatase activity and an indirect effect of facilitating the entry of vanadate.

Animals↗

PubMed1992

Insulin and insulinomimetic agents induce activation of phosphatidylinositol 3'-kinase upon its association with pp185 (IRS-1) in intact rat livers.

The major cytosolic substrate of the insulin receptor is a 185-kDa phosphoprotein (IRS-1) that contains multiple putative attachment sites for the p85 alpha regulatory subunit of phosphatidylinositol 3'-kinase (PI3K). To examine the possible interaction of pp185 with p85 alpha in vivo, we injected insulin or insulinomimetic agents (a combination of H2O2 and vanadate (H/V)) into the portal vein of anesthetized rats. IN this model system, H/V treatment and, to a lesser extent, injection of insulin resulted in rapid and sustained tyrosine phosphorylation of multiple cellular proteins, including pp185/IRS-1. The latter was found to undergo specific association with the p85 alpha regulatory subunit of PI3K but not with two other proteins that contain src homology domains. As p85 alpha was not detectably phosphorylated on tyrosine residues and did not appear to interact directly with the insulin receptor, we conclude that tyrosine phosphorylation of pp185 promotes its association with p85 alpha and the catalytic subunit of PI3K. The recruitment of the holoenzyme may also involve its enzymatic activation and thus constitute an important step in the transduction of insulin signals.

Animals↗