Phosphorylation-Dependent Interaction Of Prip With Akt

Phospholipase C (PLC)‐related but catalytically inactive protein (PRIP) is a protein with a domain organization similar to PLC‐δ1. We have reported that PRIP interacts with the catalytic subunits of protein phosphatases, PP1c and PP2Ac, depending on the phosphorylation level of PRIP. We also found that Akt was precipitated along with PRIP by anti‐PRIP antibody from neuronal cells. In this study, we investigated the interaction between PRIP and Akt. Co‐immunoprecipitation assay was performed using COS7 cells expressing glutathione S‐transferase (GST)‐fused PRIP. Both endogenous Akt and PP2Ac were co‐precipitated along with PRIP by GST antibody, and the precipitation was up‐regulated when the cells were stimulated with insulin. GST‐pull down experiments using recombinant GST‐Akt and his‐tagged (His‐) PRIP or the deletion mutant of PRIP (74–298) showed the direct binding of Akt to 74–298 residues in RPIP. Pretreatment of PRIP with Akt in the presence of ATP increased this interaction. Akt phosphorylated PRIP, but not the mutant PRIP whose Ser‐96 was replaced with Ala (S96A). Pre‐treatment of S96A mutant with Akt did not increase the binding to Akt, indicating that phosphorylation of PRIP on Ser‐96 increases the binding of Akt to PRIP. These results indicate that PRIP modulates Akt signaling by the regulation of complex formation between Akt and protein phosphatases.