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Use of Akt inhibitor and a drug-resistant mutant validates a critical role for protein kinase B/Akt in the insulin-dependent regulation of glucose and system A amino acid uptake

Green, Charlotte J.; Göransson, Olga LU ; Kular, Gursant S.; Leslie, Nick R.; Gray, Alexander; Alessi, Dario R.; Sakamoto, Kei and Hundal, Harinder S. (2008) In Journal of Biological Chemistry 283(41). p.27653-27667
Abstract
Protein kinase B (PKB)/Akt has been strongly implicated in the insulin-dependent stimulation of GLUT4 translocation and glucose transport in skeletal muscle and fat cells. Recently an allosteric inhibitor of PKB (Akti) that selectively targets PKB alpha and -beta was reported, but as yet its precise mechanism of action or ability to suppress key insulin-regulated events such as glucose and amino acid uptake and glycogen synthesis in muscle cells has not been reported. We show here that Akti ablates the insulin-dependent regulation of these processes in L6 myotubes at submicromolar concentrations and that inhibition correlates tightly with loss of PKB activation/phosphorylation. Similar findings were obtained using 3T3-L1 adipocytes. Akti... (More)
Protein kinase B (PKB)/Akt has been strongly implicated in the insulin-dependent stimulation of GLUT4 translocation and glucose transport in skeletal muscle and fat cells. Recently an allosteric inhibitor of PKB (Akti) that selectively targets PKB alpha and -beta was reported, but as yet its precise mechanism of action or ability to suppress key insulin-regulated events such as glucose and amino acid uptake and glycogen synthesis in muscle cells has not been reported. We show here that Akti ablates the insulin-dependent regulation of these processes in L6 myotubes at submicromolar concentrations and that inhibition correlates tightly with loss of PKB activation/phosphorylation. Similar findings were obtained using 3T3-L1 adipocytes. Akti did not inhibit IRS1 tyrosine phosphorylation, phosphatidylinositol 3-kinase signaling, or activation of Erks, ribosomal S6 kinase, or atypical protein kinases C but significantly impaired regulation of downstream PKB targets glycogen synthase kinase-3 and AS160. Akti-mediated inhibition of PKB requires an intact kinase pleckstrin homology domain but does not involve suppression of 3-phosphoinositide binding to this domain. Importantly, we have discovered that Akti inhibition is critically dependent upon a solvent-exposed tryptophan residue (Trp-80) that is present within the pleckstrin homology domain of all three PKB isoforms and whose mutation to an alanine (PKBW80A) yields an Akti-resistant kinase. Cellular expression of PKBW80A antagonized the Akti-mediated inhibition of glucose and amino acid uptake. Our findings support a critical role for PKB in the hormonal regulation of glucose and system A amino acid uptake and indicate that use of Akti and expression of the drug-resistant kinase will be valuable tools in delineating cellular PKB functions. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
283
issue
41
pages
27653 - 27667
publisher
ASBMB
external identifiers
  • wos:000259719200037
  • scopus:55549088220
ISSN
1083-351X
DOI
10.1074/jbc.M802623200
language
English
LU publication?
yes
id
bd2c9f4b-76cd-419b-96b3-f476b1730d12 (old id 1286695)
date added to LUP
2009-01-29 15:17:49
date last changed
2017-09-10 03:30:26
@article{bd2c9f4b-76cd-419b-96b3-f476b1730d12,
  abstract     = {Protein kinase B (PKB)/Akt has been strongly implicated in the insulin-dependent stimulation of GLUT4 translocation and glucose transport in skeletal muscle and fat cells. Recently an allosteric inhibitor of PKB (Akti) that selectively targets PKB alpha and -beta was reported, but as yet its precise mechanism of action or ability to suppress key insulin-regulated events such as glucose and amino acid uptake and glycogen synthesis in muscle cells has not been reported. We show here that Akti ablates the insulin-dependent regulation of these processes in L6 myotubes at submicromolar concentrations and that inhibition correlates tightly with loss of PKB activation/phosphorylation. Similar findings were obtained using 3T3-L1 adipocytes. Akti did not inhibit IRS1 tyrosine phosphorylation, phosphatidylinositol 3-kinase signaling, or activation of Erks, ribosomal S6 kinase, or atypical protein kinases C but significantly impaired regulation of downstream PKB targets glycogen synthase kinase-3 and AS160. Akti-mediated inhibition of PKB requires an intact kinase pleckstrin homology domain but does not involve suppression of 3-phosphoinositide binding to this domain. Importantly, we have discovered that Akti inhibition is critically dependent upon a solvent-exposed tryptophan residue (Trp-80) that is present within the pleckstrin homology domain of all three PKB isoforms and whose mutation to an alanine (PKBW80A) yields an Akti-resistant kinase. Cellular expression of PKBW80A antagonized the Akti-mediated inhibition of glucose and amino acid uptake. Our findings support a critical role for PKB in the hormonal regulation of glucose and system A amino acid uptake and indicate that use of Akti and expression of the drug-resistant kinase will be valuable tools in delineating cellular PKB functions.},
  author       = {Green, Charlotte J. and Göransson, Olga and Kular, Gursant S. and Leslie, Nick R. and Gray, Alexander and Alessi, Dario R. and Sakamoto, Kei and Hundal, Harinder S.},
  issn         = {1083-351X},
  language     = {eng},
  number       = {41},
  pages        = {27653--27667},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Use of Akt inhibitor and a drug-resistant mutant validates a critical role for protein kinase B/Akt in the insulin-dependent regulation of glucose and system A amino acid uptake},
  url          = {http://dx.doi.org/10.1074/jbc.M802623200},
  volume       = {283},
  year         = {2008},
}