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

1994 ◽  
Vol 298 (2) ◽  
pp. 471-477 ◽  
Author(s):  
F Liu ◽  
R A Roth
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Insulin Receptors ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Juxtamembrane Region ◽  
Kinase C ◽  
Mutant Receptors

A line of Chinese hamster ovary cells overexpressing protein kinase C alpha was transfected with cDNAs encoding either the wild-type 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.

scholarly journals Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region.

1991 ◽  
Vol 115 (6) ◽  
pp. 1535-1545 ◽  
Author(s):  
J M Backer ◽  
S E Shoelson ◽  
E Haring ◽  
M F White
Keyword(s):  
Insulin Receptor ◽  
Cho Cells ◽  
Insulin Receptors ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Coated Pits ◽  
Intracellular Potassium ◽  
Juxtamembrane Region ◽  
High Insulin ◽  
Mutant Receptors

The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IRA1018), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR delta 960). The rate of [125I]insulin internalization in CHO/IR cells at 37 degrees C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA1018 and CHO/IR delta 960 cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [125I]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [125I]insulin uptake by CHO/IR delta 960 and CHO/IRA1018 cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system.

scholarly journals Protein kinase C isoforms β 1 and β 2 inhibit the tyrosine kinase activity of the insulin receptor

Diabetologia ◽  
1997 ◽  
Vol 40 (7) ◽  
pp. 863-866 ◽  
Author(s):  
B. Bossenmaier ◽  
L. Mosthaf ◽  
H. Mischak ◽  
A. Ullrich ◽  
H. U. Häring
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Tyrosine Kinase Activity ◽  
Kinase C ◽  
Protein Kinase C Isoforms

scholarly journals Identification of protein kinase C phosphorylation sites in the angiotensin II (AT1A) receptor

1999 ◽  
Vol 343 (3) ◽  
pp. 637-644 ◽  
Author(s):  
Hongwei QIAN ◽  
Luisa PIPOLO ◽  
Walter G. THOMAS
Keyword(s):  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Single Mutation ◽  
Wild Type ◽  
Receptor Phosphorylation ◽  
Kinase C ◽  
C Terminus ◽  
At1a Receptor ◽  
Mutant Receptors

Protein kinase C (PKC) phosphorylates the C-terminus of the type 1 angiotensin II receptor (AT1), although the exact site(s) of phosphorylation are unidentified. In the present study, we examined the phosphorylation of epitope-tagged wild-type AT1A receptors, transiently expressed in Chinese hamster ovary K1 cells, in response to angiotensin II (AngII) and following selective activation and inhibition of PKC. This phosphorylation was compared with mutant receptors where C-terminal serine residues (Ser331, Ser338 and Ser348) within three putative PKC consensus sites were replaced with alanine, either individually or in combination. Stimulation by AngII or the phorbol ester PMA to activate PKC induced an increase in phosphorylation of the wild-type AT1A receptor, which was prevented by truncation of the receptor C-terminus to remove the last 34 amino acids, including Ser331, Ser338 and Ser348. Whereas single alanine mutation (Ser331Ala, Ser338Ala and Ser348Ala) resulted in decreased receptor phosphorylation, no single mutant completely inhibited either AngII- or PMA-induced phosphorylation. Combined mutation of the three PKC consensus sites caused an ≈ 70% reduction in PMA-mediated phosphorylation. The ≈ 60% reduction in AngII (1 μM)-induced phosphorylation of this triple mutant and the partial inhibition of wild-type receptor phosphorylation by bisindolylmaleimide, a specific PKC inhibitor, suggest a significant contribution of PKC to agonist-stimulated regulation. The ratio of PKC to total receptor phosphorylation was greatest at low doses of AngII (1 nM), consistent with the idea that PKC phosphorylates and regulates receptor function at low levels of stimulation, whereas phosphorylation by other kinases is more prevalent at high levels of agonist stimulation. To determine if a single PKC site is favoured when the contribution of PKC varies, the phosphorylation of wild-type and mutant receptors was examined over a range of AngII concentrations (0, 1, 10 and 100 nM). At all AngII concentrations, single mutation of Ser331, Ser338 or Ser348 was incapable of completely preventing receptor phosphorylation, suggesting no clear preference for PKC consensus-site utilization. Together, these results indicate a redundancy in PKC phosphorylation of the AT1A receptor, whereby all three consensus sites are utilized to some degree following homologous (AngII) and heterologous (PMA) stimulation. The contribution of PKC phosphorylation to receptor regulation is unclear, but multiple PKC phosphorylation of the AT1A receptor may allow independent and/or complementary events to occur at the three separate sites of the C-terminus.

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