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.

scholarly journals Differential Regulation of Angiotensin II-induced Expression of Connective Tissue Growth Factor by Protein Kinase C Isoforms in the Myocardium

2005 ◽  
Vol 280 (16) ◽  
pp. 15719-15726 ◽  
Author(s):  
Zhiheng He ◽  
Kerrie J. Way ◽  
Emi Arikawa ◽  
Eva Chou ◽  
Darren M. Opland ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Growth Factor ◽  
Protein Kinase ◽  
Connective Tissue ◽  
Mrna Expression ◽  
Tissue Growth ◽  
Dominant Negative ◽  
Wild Type ◽  
Kinase C

Protein kinase C (PKC) and angiotensin II (AngII) can regulate cardiac function in pathological conditions such as in diabetes or ischemic heart disease. We have reported that expression of connective tissue growth factor (CTGF) is increased in the myocardium of diabetic mice. Now we showed that the increase in CTGF expression in cardiac tissues of streptozotocin-induced diabetic rats was reversed by captopril and islet cell transplantation. Infusion of AngII in rats increased CTGF mRNA expression by 15-fold, which was completely inhibited by co-infusion with AT1 receptor antagonist, candesartan. Similarly, incubation of cultured cardiomyocytes with AngII increased CTGF mRNA expression by 2-fold, which was blocked by candesartan and a general PKC inhibitor, GF109203X. The role of PKC isoform-dependent action was further studied using adenoviral vector-mediated gene transfer of dominant negative (dn) PKC or wild type PKC isoforms. Expression of dnPKCα, -ϵ, and -ζ isoforms suppressed AngII-induced CTGF expression in cardiomyocytes. In contrast, expression of dominant negative PKCδ significantly increased AngII-induced CTGF expression, whereas expression of wild type PKCδ inhibited this induction. This inhibitory effect was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of PKCδ (δTg mice). Thus, AngII can regulate CTGF expression in cardiomyocytes through a PKC activation-mediated pathway in an isoform-selective manner both in physiological and diabetic states and may contribute to the development of cardiac fibrosis in diabetic cardiomyopathy.

Evidence against a role for protein kinase C in the regulation of the angiotensin II (AT1A) receptor

1996 ◽  
Vol 295 (1) ◽  
pp. 119-122 ◽  
Author(s):  
Walter G. Thomas ◽  
Kenneth M. Baker ◽  
George W. Booz ◽  
Thomas J. Thekkumkara
Keyword(s):  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Kinase C ◽  
At1a Receptor

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 Modulation of mouse estrogen receptor transcription activity by protein kinase C delta

1998 ◽  
Vol 20 (2) ◽  
pp. 245-259 ◽  
Author(s):  
H Lahooti ◽  
T Thorsen ◽  
A Aakvaag
Keyword(s):  
Estrogen Receptor ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Transcriptional Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Receptor Phosphorylation ◽  
Pkc Delta ◽  
Kinase C

The effect of protein kinase C (PKC) delta on the transcriptional activity of the mouse estrogen receptor was investigated. The receptor was expressed transiently in Cos-1 and NIH3T3 cells in the presence of wild-type, dominant negative or constitutively active forms of PKC delta. Transfection experiments demonstrated that PKC delta stimulated both unliganded and liganded estrogen receptor transcriptional activity. This stimulatory effect was not observed using PKC alpha or PKC epsilon. 4-Hydroxytamoxifen and the pure anti-estrogen ICI 164,384 reduced receptor transcriptional activity in the presence of PKC delta. The stimulatory effect of PKC delta on estrogen receptor transcriptional activity was mediated by the N-terminal activation function 1 (AF-1) domain. The reduced stimulatory effect of PKC delta on transcriptional activity of the phosphorylation defective mutant of estrogen receptor suggests that phosphorylation of serine 122 in the AF-1 region may mediate the modulatory effect of PKC delta. Wild-type PKC delta caused a twofold increase in estrogen receptor phosphorylation, while a dominant negative mutant of PKC delta reduced the receptor phosphorylation to five percent of that caused by wild-type PKC delta. Our results suggest that PKC delta participates in the signaling pathways that lead to estrogen receptor phosphorylation and its effect on estrogen receptor transcriptional activation is both cell type and promoter specific.

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