Identification of serines-967/968 in the juxtamembrane region of the insulin receptor as insulin-stimulated phosphorylation sites

Feng Liu, R. A. Roth

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

A line of Chinese hamster ovary cells overexpressing protein kinase Cα was transfected with cDNAs encoding either the wildtype human insulin receptor or one of two mutant insulin receptors with either Ser-967 and -968 or -974 and -976 in the juxtamembrane region changed to alanine. Both mutant receptors exhibited normal insulin-activated tyrosine kinase activity as assessed by either autophosphorylation or insulin-stimulated increases in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase. The wild-type and mutant insulin receptors were also examined for serine and threonine phosphorylation in response to insulin and activation of protein kinase C. To visualize Ser/Thr-phosphorylation sites of the receptor better in response to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphatase to remove all tyrosine phosphorylation. Phosphopeptides from the three receptors were analysed by high-percentage polyacrylamide/urea gel electrophoresis and two-dimensional t.l.c. The mutant receptor lacking Ser-967 and -968 but not the mutant lacking Ser-974 and -976 was found to be missing phosphorylated peptides in response to insulin and, to a lesser extent, after activation of protein kinase C. However, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase was inhibited to the same extent by activation of protein kinase C in cells expressing the two mutant receptors as in cells expressing the wild-type receptor. These results indicate that these four serine residues in the juxtamembrane region are not major regulatory sites of the intrinsic tyrosine kinase activity of the insulin receptor by protein kinase C, although Ser-967 and/or -968 appear to be phosphorylated in response to insulin.

Original languageEnglish (US)
Pages (from-to)471-477
Number of pages7
JournalBiochemical Journal
Volume298
Issue number2
StatePublished - 1994
Externally publishedYes

Fingerprint

Phosphorylation
Insulin Receptor
Serine
Insulin
Protein Kinase C
Cells
Phosphatidylinositol 3-Kinase
Phosphotyrosine
Chemical activation
Protein-Tyrosine Kinases
Tyrosine
Phosphopeptides
Electrophoresis, Gel, Two-Dimensional
Threonine
Cricetulus
Electrophoresis
Phosphoric Monoester Hydrolases
Alanine
Urea
Ovary

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of serines-967/968 in the juxtamembrane region of the insulin receptor as insulin-stimulated phosphorylation sites. / Liu, Feng; Roth, R. A.

In: Biochemical Journal, Vol. 298, No. 2, 1994, p. 471-477.

Research output: Contribution to journalArticle

@article{4a7edf57947f45aeb11759e34f5f6a68,
title = "Identification of serines-967/968 in the juxtamembrane region of the insulin receptor as insulin-stimulated phosphorylation sites",
abstract = "A line of Chinese hamster ovary cells overexpressing protein kinase Cα was transfected with cDNAs encoding either the wildtype human insulin receptor or one of two mutant insulin receptors with either Ser-967 and -968 or -974 and -976 in the juxtamembrane region changed to alanine. Both mutant receptors exhibited normal insulin-activated tyrosine kinase activity as assessed by either autophosphorylation or insulin-stimulated increases in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase. The wild-type and mutant insulin receptors were also examined for serine and threonine phosphorylation in response to insulin and activation of protein kinase C. To visualize Ser/Thr-phosphorylation sites of the receptor better in response to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphatase to remove all tyrosine phosphorylation. Phosphopeptides from the three receptors were analysed by high-percentage polyacrylamide/urea gel electrophoresis and two-dimensional t.l.c. The mutant receptor lacking Ser-967 and -968 but not the mutant lacking Ser-974 and -976 was found to be missing phosphorylated peptides in response to insulin and, to a lesser extent, after activation of protein kinase C. However, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase was inhibited to the same extent by activation of protein kinase C in cells expressing the two mutant receptors as in cells expressing the wild-type receptor. These results indicate that these four serine residues in the juxtamembrane region are not major regulatory sites of the intrinsic tyrosine kinase activity of the insulin receptor by protein kinase C, although Ser-967 and/or -968 appear to be phosphorylated in response to insulin.",
author = "Feng Liu and Roth, {R. A.}",
year = "1994",
language = "English (US)",
volume = "298",
pages = "471--477",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "2",

}

TY - JOUR

T1 - Identification of serines-967/968 in the juxtamembrane region of the insulin receptor as insulin-stimulated phosphorylation sites

AU - Liu, Feng

AU - Roth, R. A.

PY - 1994

Y1 - 1994

N2 - A line of Chinese hamster ovary cells overexpressing protein kinase Cα was transfected with cDNAs encoding either the wildtype human insulin receptor or one of two mutant insulin receptors with either Ser-967 and -968 or -974 and -976 in the juxtamembrane region changed to alanine. Both mutant receptors exhibited normal insulin-activated tyrosine kinase activity as assessed by either autophosphorylation or insulin-stimulated increases in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase. The wild-type and mutant insulin receptors were also examined for serine and threonine phosphorylation in response to insulin and activation of protein kinase C. To visualize Ser/Thr-phosphorylation sites of the receptor better in response to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphatase to remove all tyrosine phosphorylation. Phosphopeptides from the three receptors were analysed by high-percentage polyacrylamide/urea gel electrophoresis and two-dimensional t.l.c. The mutant receptor lacking Ser-967 and -968 but not the mutant lacking Ser-974 and -976 was found to be missing phosphorylated peptides in response to insulin and, to a lesser extent, after activation of protein kinase C. However, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase was inhibited to the same extent by activation of protein kinase C in cells expressing the two mutant receptors as in cells expressing the wild-type receptor. These results indicate that these four serine residues in the juxtamembrane region are not major regulatory sites of the intrinsic tyrosine kinase activity of the insulin receptor by protein kinase C, although Ser-967 and/or -968 appear to be phosphorylated in response to insulin.

AB - A line of Chinese hamster ovary cells overexpressing protein kinase Cα was transfected with cDNAs encoding either the wildtype human insulin receptor or one of two mutant insulin receptors with either Ser-967 and -968 or -974 and -976 in the juxtamembrane region changed to alanine. Both mutant receptors exhibited normal insulin-activated tyrosine kinase activity as assessed by either autophosphorylation or insulin-stimulated increases in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase. The wild-type and mutant insulin receptors were also examined for serine and threonine phosphorylation in response to insulin and activation of protein kinase C. To visualize Ser/Thr-phosphorylation sites of the receptor better in response to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphatase to remove all tyrosine phosphorylation. Phosphopeptides from the three receptors were analysed by high-percentage polyacrylamide/urea gel electrophoresis and two-dimensional t.l.c. The mutant receptor lacking Ser-967 and -968 but not the mutant lacking Ser-974 and -976 was found to be missing phosphorylated peptides in response to insulin and, to a lesser extent, after activation of protein kinase C. However, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase was inhibited to the same extent by activation of protein kinase C in cells expressing the two mutant receptors as in cells expressing the wild-type receptor. These results indicate that these four serine residues in the juxtamembrane region are not major regulatory sites of the intrinsic tyrosine kinase activity of the insulin receptor by protein kinase C, although Ser-967 and/or -968 appear to be phosphorylated in response to insulin.

UR - http://www.scopus.com/inward/record.url?scp=0028274267&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028274267&partnerID=8YFLogxK

M3 - Article

VL - 298

SP - 471

EP - 477

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 2

ER -