Sale, Elizabeth M., Hodginkson, Conrad P., Jones, Neil P. and Sale, Graham J.
A new strategy for studying protein kinase B and its three isoforms. Role of protein kinase B in phosphorylating glycogen synthase kinase-3, tuberin, WNK1, and ATP citrate lyase
Biochemistry, 45, (1), . (doi:10.1021/bi050287i).
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Protein kinase B appears to play a key role in insulin signaling and in the control of apoptosis, although the precise targets of PKB are incompletely understood. PKB exists as three isoforms (alpha, and gamma) that may have unique as well as common functions within the cell. To facilitate understanding the precise roles of PKB and its isoforms, novel tools of widespread applicability are described.
These tools are antisense oligonucleotide probes that enable the specific and potent knock down of endogenous PKB alpha, beta or gamma isoforms, individually or in various combinations, including concurrent removal of all three isoforms. The probes were applied to dissect the role of PKB in phosphorylating glycogen synthase kinase-3 (GSK-3), a critical mediator in multiple responses, and other potentially key targets.
Triple antisense knock down of PKB alpha, beta, and gamma so that total PKB was < 6% blocked insulin-stimulated phosphorylation of endogenous GSK-3 alpha and GSK-3 beta isoforms by 67% and 45%, respectively, showing that GSK-3 alpha and GSK-3 are controlled by endogenous PKB. Each PKB isoform contributed to GSK-3 alpha and GSK-3 beta phosphorylation, with PKB beta having the predominant role.
Knock down of total PKB incompletely blocked insulin-stimulated phosphorylation of GSK-3 alpha and GSK-3 beta, and a pathway involving atypical PKCs, was shown to contribute to the signal. Triple antisense knock down of PKB alpha, beta, and gamma abrogated the insulin-stimulated phosphorylation of WNK1, ATP citrate lyase, and tuberin. However, antisense-mediated knock down of PKB alpha, beta, and gamma had no effect on insulin-stimulated DNA synthesis in 3T3-L1 adipocytes, indicating that pathways other than PKB mediate this response in these cells. Finally, our PKB antisense strategy provides a method of general usefulness for further dissecting the precise targets and roles of PKB and its isoforms.
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