Identification of 14-3-3ζ as a Protein Kinase B/Akt Substrate*

By D. Powell, M. Rane, Qingdan Chen, Saurabh Singh, K. Mcleish. Journal of Biological Chemistry, 2002

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Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3ζ, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3ζ and endogenous 14-3-3ζ from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3ζ in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3ζ. Based on a motif search of 14-3-3ζ, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3ζ with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3ζ dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3ζ both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.

Subfields: Molecular Biology

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