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Characterization of PDK2 activity against protein kinase B gamma

Hodgkinson, C.P., Sale, E.M. and Sale, G.J. (2002) Characterization of PDK2 activity against protein kinase B gamma Biochemistry, 41, (32), pp. 10351-10359. (doi:10.1021/bi026065r).

Record type: Article


Protein kinase B (PKB), also known as Akt, is a serine/threonine protein kinase controlled by insulin, various growth factors, and phosphatidylinositol 3-kinase. Full activation of the PKB enzyme requires phosphorylation of a threonine in the activation loop and a serine in the C-terminal tail. PDK1 has clearly been shown to phosphorylate the threonine, but the mechanism leading to phosphorylation of the serine, the PDK2 site, is unclear. A yeast two-hybrid screen using full-length human PKBç identified protein kinase C (PKC) ú, an atypical PKC, as an interactor with PKBç, an association requiring the pleckstrin homology domain of PKBç. Endogenous PKBç was shown to associate with endogenous PKCú both in cos-1 cells and in 3T3-L1 adipocytes, demonstrating a physiological interaction. Immunoprecipitates of PKCú, whether endogenous PKCú from insulin-stimulated 3T3-L1 adipocytes or overexpressed PKCú from cos-1 cells, phosphorylated S472 (the C-terminal serine phosphorylation site) of PKBç, in vitro. In vivo, overexpression of PKCú stimulated the phosphorylation of approximately 50% of the PKBç molecules, suggesting a physiologically meaningful effect. However, pure PKCú protein was incapable of phosphorylating S472 of PKBç. Antisense knockout studies and use of a PDK1 inhibitor showed that neither PKB autophosphorylation nor phosphorylation by PDK1 accounted for the S472 phosphorylation in PKCú immunoprecipitates. Staurosporine inhibited the PKCú activity but not the PDK2 activity in PKCú immunoprecipitates. Together these results indicate that an independent PDK2 activity exists that physically associates with PKCú and that PKCú, by binding PKBç, functions to deliver the PDK2 to a required location. PKCú thus functions as an adaptor, associating with a staurosporine-insensitive PDK2 enzyme that catalyzes the phosphorylation of S472 of PKBç. Because both PKCú and PKB have been proposed to be required for mediating a number of crucial insulin responses, formation of an active signaling complex containing PKCú, PKB, and PDK2 is an attractive mechanism for ensuring that all the critical sites on targets such as glycogen synthase kinase-3 are phosphorylated.

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Published date: 13 August 2002


Local EPrints ID: 25073
ISSN: 0006-2960
PURE UUID: 1726e643-d45d-49a4-9bfa-f8009b93fa97

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Date deposited: 24 Apr 2006
Last modified: 17 Jul 2017 16:12

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Author: C.P. Hodgkinson
Author: E.M. Sale
Author: G.J. Sale

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