Interaction of PKC with Ahnak potentiates Raf phosphorylation

Ahnak is a protein of an exceptionally large size (700kDa) and contains 36 central repeated units (CRUs). We previously reported that CRUs of Ahnak act as scaffolding protein networking PLC‐γ and PKC. Here, we demonstrated that four central repeated units (4CRUs) of Ahnak protein bind and activate PKCα in the PS/DG‐independent manner. Moreover, treatment of PMA to NIH3T3 cells expressing 4CRUs of Ahnak showed an enhanced c‐Raf, MEK, and Erk phosphorylation, compared to parental cells. To evaluate the gain‐of‐function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in Ahnak knockout mouse embryonic cells (MEF). Membrane translocation of PKCα and phosphorylation of Raf in Ahnak null MEF were decrease comparing to wild type MEF. Several lines of evidence suggest that PKCα activity regulated by the association with phosphatase 2A (PP2A). Co‐immunoprecipitation assay with antibodies against PP2A indicate that the association of PKCα with PP2A was broken off in NIH3T3 cells expressing 4CRUs of Ahnak in response to PMA, leading to enhanced PKCα phosphorylation. These data suggest that 4CRUs of Ahnak potentiate PKC‐dependent c‐Raf/MEK/Erk cascade through inhibiting interaction of PKC with PP2A.