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), 10351-10359. (doi:10.1021/bi026065r).

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Original Publication URL: http://dx.doi.org/10.1021/bi026065r

Description/Abstract

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.

Item Type: Article
ISSNs: 0006-2960 (print)
Related URLs:
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: University Structure - Pre August 2011 > School of Biological Sciences
ePrint ID: 25073
Date Deposited: 24 Apr 2006
Last Modified: 27 Mar 2014 18:14
URI: http://eprints.soton.ac.uk/id/eprint/25073

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