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Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. Membrane translocation in response to peroxovanadate

Wijkander, Jonny; Stenson, Lena LU ; Rahn, Tova; Resjö, Svante LU ; Castan, Isabelle; Manganiello, Vincent; Belfrage, Per LU and Degerman, Eva LU (1997) In Journal of Biological Chemistry 272(34). p.21520-21526
Abstract
Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide... (More)
Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
272
issue
34
pages
21520 - 21526
publisher
ASBMB
external identifiers
  • pmid:9261171
  • scopus:0030820857
ISSN
1083-351X
language
English
LU publication?
yes
id
0fcf3a70-8196-471a-9734-a40f17e71bcc (old id 1111247)
alternative location
http://www.jbc.org/cgi/content/full/272/34/21520
date added to LUP
2008-07-17 10:05:54
date last changed
2017-02-19 03:39:04
@article{0fcf3a70-8196-471a-9734-a40f17e71bcc,
  abstract     = {Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.},
  author       = {Wijkander, Jonny and Stenson, Lena and Rahn, Tova and Resjö, Svante and Castan, Isabelle and Manganiello, Vincent and Belfrage, Per and Degerman, Eva},
  issn         = {1083-351X},
  language     = {eng},
  number       = {34},
  pages        = {21520--21526},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. Membrane translocation in response to peroxovanadate},
  volume       = {272},
  year         = {1997},
}