Phosphorylation of the hepatic insulin receptor

Obberghen, Emmanuel Van; Kowalski, Aline

doi:10.1016/0014-5793(82)80094-x

Cited by 126 publications

(50 citation statements)

References 22 publications

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“…This was first demonstrated in rat hepatoma cells and human IM9 lymphoblasts (Kasuga et al, 1982a) and later in freshly isolated rat hepatocytes (Van Obberghen & Kowalski, 1982). In these experiments, cells were preincubated with [32P]Pi to label cellular ATP, solubilized in detergent, and the glycoproteins purified on wheat-germ-agglutinin-agarose.…”

Section: Vol 235mentioning

confidence: 99%

“…Recently, a promising discovery was made by the demonstration that the insulin receptor is an insulinsensitive protein kinase (Kasuga et al, 1982a;Van Obberghen & Kowalski, 1982;Avruch et al, 1982;Petruzzelli et al, 1982). This novel observation is of interest for our understanding of insulin-regulated processes, since it is now recognized that covalent phosphorylation-dephosphorylation of proteins is a mechanism whereby many cellular functions are regulated by hormones and neurotransmitters (Denton et al, 1981;Cohen, 1982;Houslay, 1981).…”

Section: Introductionmentioning

confidence: 99%

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Protein kinase activity of the insulin receptor

Gammeltoft¹,

Obberghen²

1986

Biochemical Journal

138

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The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Vol 235mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Protein kinase activity of the insulin receptor

Gammeltoft¹,

Obberghen²

1986

Biochemical Journal

138

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“…The insulin receptor is an insulin-dependent tyrosine kinase which displays autophosphorylation activity of the / subunit (Kasuga et al, 1982a,b;Van Obberghen & Kowalski, 1982;Roth & Cassell, 1983). Recently, sequencing of cloned cDNA from mRNA of the human insulin receptor has revealed that the , subunit contains a single transmembrane domain and kinase domain that has similar sequences to other tyrosine kinases (Ullrich et al, 1985;Ebina et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

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

Koshio

Akanuma

Kasuga

1988

Biochemical Journal

108

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95H-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-H202 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the ,3 subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H202-treated H-35 cells and the purified fractions incubated with [y-32P]ATP and Mna+. Phosphorylation of the B3 subunit of insulin receptors obtained from H202-treated cells was 150 % of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H202-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, H202 may increase the insulin receptor kinase activity by inducing phosphorylation of the ,3 subunit of insulin receptor.

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“…For example, the purified insulin receptor expresses protein tyrosine kinase activity (12,13). In addition, ligand-stimulated/ligand-dependent receptor phosphorylation is observed in intact cells (14,15) as well as with partially purified insulin receptors (15,16). The ATP binding site and insulinstimulated phosphorylation site coexist on the 83-subunit of the insulin receptor (17).…”

mentioning

confidence: 99%

Protein kinase activity associated with the purified rat hepatic glucocorticoid receptor.

Miller-Diener¹,

Schmidt²,

Litwack³

1985

Proc. Natl. Acad. Sci. U.S.A.

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The M, 94 Phosphorylation/dephosphorylation reactions may be inherent to the mechanism of steroid hormone action (1-4). Treatment of crude estrogen (5) and glucocorticoid (6) receptors with phosphatase decreases hormone binding. Nonspecific phosphatase inhibitors, such as molybdate and fluoride (7,8), prevent loss of steroid binding. The rate of activation of the glucocorticoid receptor and the appearance of a more positively charged molecular species with increased affinity for nuclei and polyanions are stimulated by addition of calf intestinal alkaline phosphatase to rat liver cytosol (9). It is difficult to determine in crude cytosols whether these effects are due to phosphorylation/dephosphorylation of the receptor itself or of some other cytosolic factor(s). However, in vivo phosphorylation of both the progesterone (10) and glucocorticoid (11) receptors has since been demonstrated. In vitro phosphorylation of several purified and partially purified receptors has now been reported. Receptors also possess enzymatic activity. For example, the purified insulin receptor expresses protein tyrosine kinase activity (12,13 Purification of the Glucocorticoid Receptor. Cytosol and purified molybdate-stabilized glucocorticoid receptor were prepared as described (18). Unactivated receptor, which elutes from DEAE-cellulose at ==250 mM potassium phosphate, can be stored for future use at -70°C after elution from Sephadex G-75 with buffer A containing 10 mM Na2MoO4.Phosphorylation Incubation System. Immediately before use, DEAE-cellulose-purified unactivated glucocorticoid receptor, fresh or frozen, was eluted from Sephadex G-75 with buffer A to remove Na2MoO4 and was concentrated 5-to 10-fold by centrifugation on Centricon-30 filters. Activated receptor was prepared by heating an aliquot at 25°C for 30 min and loaded onto DEAE-cellulose followed by elution with a 5-500 mM KCl

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Phosphorylation of the hepatic insulin receptor

Cited by 126 publications

References 22 publications

Protein kinase activity of the insulin receptor

Protein kinase activity of the insulin receptor

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

Protein kinase activity associated with the purified rat hepatic glucocorticoid receptor.

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