Identification of 14-3-3zeta as a protein kinase B/Akt substrate.
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 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.
Powell
David W
DW
Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky 40202-1718, USA.
Rane
Madhavi J
MJ
Chen
Qingdan
Q
Singh
Saurabh
S
McLeish
Kenneth R
KR
D048948:14-3-3 Proteins; D020816:Amino Acid Motifs; D002460:Cell Line; D019281:Dimerization; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000378:metabolism
D048948:14-3-3 Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D005982:Glutathione Transferase; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt