Identification of NEK9 as a novel substrate of protein kinase B

Protein kinase B (PKB/Akt) regulates diverse cellular processes through phosphorylation of numerous substrate proteins downstream of phosphatidylinositol 3-kinase (PI3K). Although many PKB substrates have been identified, additional substrates remain to be characterized. NEK9 is an essential serine/threonine kinase involved in mitotic progression and contains two putative PKB consensus phosphorylation motifs. However, whether NEK9 is regulated by PKB has remained unknown. In this study, we investigated PKB-dependent phosphorylation of NEK9 using insulin stimulation, pharmacological inhibition, expression of constitutively active PKB, mutational analysis, and in vitro kinase assays. Insulin induced phosphorylation of both recombinant and endogenous NEK9 at putative PKB consensus motifs. This phosphorylation was markedly suppressed by PI3K and PKB inhibitors. Constitutively active PKB induced NEK9 phosphorylation even under serum-starved conditions. Mutational analysis identified Thr333 as the major insulin-induced phosphorylation site in NEK9. In vitro kinase assays demonstrated that PKB directly phosphorylates NEK9 predominantly at Thr333. These findings identify NEK9 as a novel substrate of PKB and suggest that PKB-mediated phosphorylation may contribute to the regulation of NEK9 function.