ORP9 negatively regulates phosphorylation of serine 473 in Akt

Abstract

Oxysterol binding protein-related protein 9 (ORP9) is a member of the ORP gene family implicated in ER/Golgi transport and lipid regulation. Due to alternate promoter start sites, ORP9 exists as a long (ORP9L) and an N-terminal truncated short form (ORP9S). An antibody that recognizes phospho-S473 in the hydrophobic-motif of Akt was found to cross-react with ORP9. Similar to Akt and other AGC kinases, PDK-2 potentially phosphorylates ORP9 at a related motif on S287. RNAi and inducibly overexpressing CHO cells were used to identify the ORP9 S287 kinase, and assess the role of ORP9 in Akt regulation. Initial experiments using 32P labeling, in vitro phosphatase assays and mutagenesis confirmed that ORP9 S287 was phosphorylated. Treatment of CHO cells with LY294002 resulted in decreased ORP9 phosphorylation suggesting involvement of the PI-3 kinase pathway. However, TPAβ increased ORP9 S287 phosphorylation and co-immunoprecipation assays revealed an interaction between ORP9 and PKCβII, indicating that PKCβII is the ORP9 S287 kinase. RNAi of ORP9L in HEK 293 cells increased Akt phospho-S473 4-fold whereas Akt phospho-T308 was unaffected, implying that ORP9 is a negative regulator of Akt activity. In addition, enforced overexpression of ORP9S, but not ORP9L, in CHO cells inhibited proliferation. This may reflect inhibition of Akt phosphorylation at S473 and subsequent activation of downstream targets involved in cell proliferation. We propose that ORP9 negatively regulates the PI-3 kinase/Akt pathway by altering phospho-S473 through interaction with PKCβII, a potential PDK-2.