scholarly journals The insulinomimetic agents H2O2 and vanadate stimulate tyrosine phosphorylation of potential target proteins for the insulin receptor kinase in intact cells

1992 ◽  
Vol 288 (2) ◽  
pp. 631-635 ◽  
Author(s):  
D Heffetz ◽  
W J Rutter ◽  
Y Zick
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Cho Cells ◽  
Receptor Gene ◽  
Chinese Hamster ◽  
Receptor Kinase ◽  
Intact Cells ◽  
Insulin Receptor Gene ◽  
Protein Tyrosine ◽  
Insulin Receptor Kinase

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.

scholarly journals Concanavalin A-induced receptor aggregation stimulates the tyrosine kinase activity of the insulin receptor in intact cells

1990 ◽  
Vol 267 (3) ◽  
pp. 787-794 ◽  
Author(s):  
T Shiba ◽  
K Tobe ◽  
O Koshio ◽  
R Yamamoto ◽  
Y Shibasaki ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Receptor Tyrosine Kinase ◽  
Receptor Kinase ◽  
Intact Cells ◽  
Insulin Receptor Tyrosine Kinase ◽  
Insulin Receptor Kinase ◽  
Rat Hepatoma ◽  
Rat Hepatoma Cells

Concanavalin A (ConA) stimulated the phosphorylation of the beta-subunit of the insulin receptor and an Mr-185,000 protein on serine and tyrosine residues in intact H-35 rat hepatoma cells. This Mr-185,000 protein whose phosphorylation was stimulated by ConA was identical to pp185, a protein reported previously to be a putative endogenous substrate for the insulin receptor tyrosine kinase in rat hepatoma cells. In Chinese hamster ovary (CHO) cells transfected with cDNA of the human insulin receptor, tyrosine-phosphorylation of pp185 was strongly enhanced by ConA compared with the controls, suggesting that the induction of tyrosine-phosphorylation of pp185 was due to stimulation of the insulin receptor kinase by ConA. Moreover, monovalent ConA only slightly induced the tyrosine-phosphorylation of pp185, which was enhanced by the addition of anti-ConA IgG, suggesting that ConA stimulated the insulin receptor kinase mainly by the receptor cross-linking or aggregation in intact cells. These data suggest that the insulin-mimetic action of ConA is related to the autophosphorylation and activation of the insulin receptor tyrosine kinase, as well as the subsequent phosphorylation of pp185 in intact cells.

scholarly journals Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells

1988 ◽  
Vol 250 (1) ◽  
pp. 95-101 ◽  
Author(s):  
O Koshio ◽  
Y Akanuma ◽  
M Kasuga
Keyword(s):  
Insulin Receptor ◽  
Kinase Activity ◽  
Intact Cell ◽  
Insulin Receptors ◽  
Beta Subunit ◽  
Receptor Kinase ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Insulin Receptor Kinase ◽  
Insulin Receptor Kinase Activity

H-35 rat hepatoma cells were labelled with [32P]orthophosphate and their insulin receptors isolated on wheat germ agglutinin (WGA)-agarose and anti-(insulin receptor) serum. The incubation of these cells with 10 mM-H2O2 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the beta subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H2O2-treated H-35 cells and the purified fractions incubated with [gamma-32P]ATP and Mn2+. Phosphorylation of the beta subunit of insulin receptors obtained from H2O2-treated cells was 150% of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H2O2-treated cells, as measured by phosphorylation of src-related synthetic peptide, was increased about 4-fold over control cells. These data suggest that in intact cell systems, H2O2 may increase the insulin receptor kinase activity by inducing phosphorylation of the beta subunit of insulin receptor.

scholarly journals Tyrosine Phosphorylation of pp185 by Insulin Receptor Kinase in a Cell-free System

1989 ◽  
Vol 264 (12) ◽  
pp. 6879-6885 ◽  
Author(s):  
Y Tashiro-Hashimoto ◽  
K Tobe ◽  
O Koshio ◽  
T Izumi ◽  
F Takaku ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Receptor Kinase ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Insulin Receptor Kinase

scholarly journals Interaction between cAMP-dependent and insulin-dependent signal pathways in tyrosine phosphorylation in primary cultures of rat hepatocytes

1997 ◽  
Vol 324 (2) ◽  
pp. 379-388 ◽  
Author(s):  
Yoshiaki ITO ◽  
Yasunobu UCHIJIMA ◽  
Miyako ARIGA ◽  
Taiichiro SEKI ◽  
Asako TAKENAKA ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Β Subunit ◽  
Receptor Kinase ◽  
Dibutyryl Camp ◽  
Insulin Dependent ◽  
Insulin Receptor Kinase

The present studies were undertaken to determine whether the interaction between cAMP-dependent and insulin-dependent pathways in primary cultures of rat hepatocytes affects biological functions and tyrosine phosphorylation. Quiescent hepatocytes were pretreated with dibutyryl cAMP or cAMP-generating agents such as glucagon, and then treated or not with insulin. Preincubation for 6 h with dibutyryl cAMP or glucagon enhanced the effect of insulin on DNA synthesis, but not the effect of insulin on amino acid transport or glycogen and protein synthesis. Tyrosine phosphorylation of intracellular proteins was determined by immunoblot analysis using an anti-phosphotyrosine antibody. Maximum tyrosine phosphorylation of a 195 kDa protein, which may be a substrate of insulin receptor kinase, of 175–180 kDa proteins, including insulin receptor substrate (IRS)-1, and of 90–95 kDa proteins, including the insulin receptor β-subunit, was reached within 30 s of incubation with insulin. Pretreatment for about 3 h with dibutyryl cAMP or cAMP-generating agents clearly increased insulin-dependent tyrosine phosphorylation of the 195 kDa protein, but not IRS-1, IRS-2 or the insulin receptor β-subunit. Because dibutyryl cAMP and cAMP-generating agents did not increase insulin receptor number or its kinase activity, the effect of cAMP on this potentiation of tyrosine phosphorylation is assumed to be exerted at a step distal to insulin receptor kinase activation. The potentiation by cAMP pretreatment of insulin-stimulated tyrosine phosphorylation may in part be secondary to inhibition of phosphotyrosine phosphatase activity, because cAMP pretreatment blunted the effect of Na3VO4 on the net tyrosine phosphorylation of the 195 kDa protein as compared with cells pretreated with no additive. In summary, the interactions between cAMP-dependent and insulin-dependent pathways that lead to augmentation of DNA synthesis appear to parallel the changes in tyrosine phosphorylation. Further studies will be required to determine whether there is a causal relationship between these phenomena.

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