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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1286695
- author
- Green, Charlotte J.
; Göransson, Olga
LU
; Kular, Gursant S.
; Leslie, Nick R.
; Gray, Alexander
; Alessi, Dario R.
; Sakamoto, Kei
and Hundal, Harinder S.
- organization
- publishing date
- 2008
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biological Chemistry
- volume
- 283
- issue
- 41
- pages
- 27653 - 27667
- publisher
- American Society for Biochemistry and Molecular Biology
- 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
- 2016-04-01 11:33:01
- date last changed
- 2024-04-08 05:09:14
@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 = {{American Society for Biochemistry and Molecular Biology}},
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}},
doi = {{10.1074/jbc.M802623200}},
volume = {{283}},
year = {{2008}},
}