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

Olga Göransson,Dario R Alessi,Alexander Gray,Harinder S Hundal,Nick R Leslie,Kei Sakamoto,Charlotte J Green,Gursant S Kular

doi:10.1074/jbc.m802623200

Olga Göransson, Dario R Alessi + Show 6 more

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https://doi.org/10.1074/jbc.m802623200

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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 PKBalpha 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 (PKB(W80A)) yields an Akti-resistant kinase. Cellular expression of PKB(W80A) 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.

Highlights

Effects of Akti on Insulin Action ulate AS160 and glycogen synthase kinase-3 (GSK3), Protein kinase B (PKB)-directed signaling serves to coordinate the insulin-dependent increase in glucose uptake and storage of glucose as glycogen in tissues such as skeletal muscle
We were surprised that L6 myotubes, which primarily express PKB␣ and PKB␥ [16, 25], did not display any residual PKB phosphorylation that could have been attributed to the gamma (Akt3) isoform after treatment with insulin and 1 ␮M Akti (Fig. 1A)
In line with the observed inhibition of PKB, Akti caused an associated loss in GSK3 phosphorylation and that of AS160; both of which are established physiological downstream targets of PKB (Fig. 1, A and F), whereas tyrosine phosphorylation of IRS1 or phosphorylation/activation of Erk1/Erk2 and that of RSK were unaffected (Fig. 1, A and G)

Summary

Introduction

Effects of Akti on Insulin Action ulate AS160 and GSK3, PKB-directed signaling serves to coordinate the insulin-dependent increase in glucose uptake and storage of glucose as glycogen in tissues such as skeletal muscle. Consistent with the involvement of PI3K in the hormonal regulation of glucose uptake, glycogen synthesis and system A amino acid transport, the ability of insulin to stimulate all three cellular processes was blocked by wortmannin (Fig. 4, A–C).

Results

Conclusion