Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin

The purified human placental insulin-receptor beta-subunit autophosphorylating activity was found to be inhibited, in a time- and concentration-dependent manner, by the specific thiol-alkylating agents N-ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoic acid). The insulin-receptor kinase was observed to be more sensitive to inhibition by N-ethylmaleimide in the presence [IC50 (concn, giving 50% inhibition) = 25 +/- 3 microM] than in the absence (IC50 = 73 +/- 6 microM) of insulin. Similarly, inhibition by 5,5′-dithiobis-(2-nitrobenzoic acid) occurred with IC50 = 30 +/- 6 microM in the presence and 155 +/- 35 microM in the absence of insulin. Examination of the exogenous-substrate protein kinase activity demonstrated that the differential sensitivity to N-ethylmaleimide was due to direct inhibition of protein kinase activity, as opposed to blockade of the phospho-acceptor properties of the insulin receptor. In contrast, iodoacetamide had essentially no effect on the insulin-receptor beta-subunit autophosphorylating activity and was able to protect partially against the N-ethylmaleimide inhibition in both the presence and the absence of insulin. Consistent with these findings, none of the thiol-specific agents were able to alter significantly insulin binding at concentrations which maximally inhibited the beta-subunit autophosphorylation. Further, in the presence of insulin, the insulin-receptor kinase activity was also observed to be more sensitive to oxidation by H2O2 and FeCl3/ascorbate compared with insulin receptors in the absence of insulin. These results indicate that there is a critical thiol group(s) necessary for the beta-subunit autophosphorylating activity of the insulin-receptor kinase and that in the presence of insulin is more susceptible to exogenously added thiol and oxidizing agents.

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Effect of basic polycations and proteins on purified insulin receptor. Insulin-independent activation of the receptor tyrosine-specific protein kinase by poly(l-lysine)

Biochemical Journal ◽

10.1042/bj2630813 ◽

1989 ◽

Vol 263 (3) ◽

pp. 813-822 ◽

Cited By ~ 32

Author(s):

Y Fujita-Yamaguchi ◽

D B Sacks ◽

J M McDonald ◽

D Sahal ◽

S Kathuria

Keyword(s):

Protein Kinase ◽

Insulin Receptor ◽

Kinase Activity ◽

Specific Protein ◽

Beta Subunit ◽

Protein Kinase Activity ◽

Receptor Kinase ◽

Kinase Activation ◽

Insulin Receptor Kinase ◽

Specific Protein Kinase

Since the studies on tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro suggested that protamine and poly(L-lysine) may activate phosphorylation of the receptor beta subunit [Sacks & McDonald (1988) J. Biol. Chem. 263, 2377-2383], we examined the effects of a variety of basic polycations/proteins and polyamines on insulin receptor kinase activity. The insulin receptor purified from human placental membranes was incubated with each basic polycation/protein or polyamine and assayed for tyrosine-specific protein kinase activity by measuring 32P incorporation into the src-related peptide. At a concentration of 1 microM, poly(L-lysine) and poly(L-ornithine) markedly stimulated kinase activity, whereas poly(L-arginine) and histones H1 and H2B inhibited insulin receptor kinase. In contrast, at a concentration of 1 mM, three polyamines (spermine, spermidine and putrescine) did not alter kinase activity. Poly(L-lysine) and poly(L-ornithine) stimulated the insulin receptor kinase by 5-10-fold at concentrations of 0.1-1 microM. Protamine sulphate also showed a significant stimulatory effect at a concentration of 100 microM. Preincubation of the receptor with poly(L-lysine) or poly(L-ornithine) for 20-60 min resulted in maximal kinase activation. Poly(L-lysine), the most effective activator of the receptor kinase, was used to characterize further the mechanisms of the kinase activation. Poly(L-lysine) activates the insulin receptor kinase by increasing the Vmax. without changing the Km. Poly(L-lysine) markedly stimulates the kinase activity of insulin receptor preparations that have lost both basal kinase activity and the ability to be stimulated by insulin. Insulin and poly(L-lysine) also differed in their ability to stimulate the kinase activity of prephosphorylated receptors. Prephosphorylation of the receptors did not affect the stimulation of the kinase by insulin. In contrast, prephosphorylation of receptors resulted in a markedly enhanced ability of poly(L-lysine) to stimulate kinase activity. These studies suggest that the mechanisms by which poly(L-lysine) and insulin activate the kinase are different. In conjunction with other additional evidence, it is suggested that poly(L-lysine) interacts directly with the beta-subunit of the receptor, thereby activating the receptor kinase.

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Relationship of site-specific beta subunit tyrosine autophosphorylation to insulin activation of the insulin receptor (tyrosine) protein kinase activity.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)68823-4 ◽

1988 ◽

Vol 263 (10) ◽

pp. 4593-4601 ◽

Cited By ~ 14

Author(s):

H E Tornqvist ◽

J Avruch

Keyword(s):

Protein Kinase ◽

Insulin Receptor ◽

Kinase Activity ◽

Beta Subunit ◽

Protein Kinase Activity ◽

Site Specific ◽

Tyrosine Protein Kinase ◽

Relationship Of ◽

Tyrosine Autophosphorylation

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

Biochemical Journal ◽

10.1042/bj2500095 ◽

1988 ◽

Vol 250 (1) ◽

pp. 95-101 ◽

Cited By ~ 75

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.

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Insulin resistance in uremia: insulin receptor kinase activity in liver and muscle from chronic uremic rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1988.254.4.e394 ◽

1988 ◽

Vol 254 (4) ◽

pp. E394-E401 ◽

Cited By ~ 10

Author(s):

F. Cecchin ◽

O. Ittoop ◽

M. K. Sinha ◽

J. F. Caro

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Insulin Receptor ◽

Kinase Activity ◽

Insulin Binding ◽

Beta Subunit ◽

Receptor Kinase ◽

Ad Libitum ◽

Insulin Receptor Kinase ◽

Insulin Receptor Kinase Activity

We have studied the structure and function of the partially purified insulin receptors from liver and skeletal muscle in a rat model of severe chronic uremia. 125I-insulin binding was higher in the liver from uremic rats when compared with ad libitum- and pair-fed controls. Furthermore, the ability of insulin to stimulate the autophosphorylation of the beta-subunit and insulin receptor kinase activity using Glu80, Tyr20 as exogenous phosphoacceptor was increased in the liver of the uremic animals. The structural characteristic of the receptors, as determined by electrophoretic mobilities of affinity labeled alpha-subunit and the phosphorylated beta-subunit, were normal in uremia. 125I-insulin binding and insulin receptor kinase activity were similar in the skeletal muscle from uremic and pair- and ad libitum-fed animals. Thus our data are supportive of the hypothesis that in liver and muscle of chronic uremic rats, insulin resistance is due to a defect(s) distal to the insulin receptor kinase.

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