Association of immature hypophosphorylated protein kinase Cε an Anchoring protein CG-NAP

Abstract

Protein kinase C (PKC) family requires phosphorylation of itself to become competent for responding to second messengers. Much attention has been focused on elucidating the role of phosphorylation in PKC activity; however, it remains unknown where this modification takes place in the cells. This study examines whether anchoring protein is involved in the regulation of PKC phosphorylation. A certain population of PKCε in rat brain extracts as well as that expressed in COS7 cells was associated with an endogenous anchoring protein CG-NAP (centrosome and Golgi localized PKN- associated protein). Pulse chase experiments revealed that the associated PKCe was an immature species at the hypophosphorylated state. In vitro binding studies confirmed that non- or hypophosphorylated PKCe directly bound to CG-NAP via its catalytic domain, whereas sufficiently phosphorylated PKCε did not. PKCε mutant at a potential phosphorylation site of Thr-566 or Ser-729 to Ala, possessing almost no catalytic activity, was associated and co-localized with CG-NAP at Golgi/centrosome area. On the other hand, wild type and a phosphorylation-mimicking mutant at Thr-566 were mainly distributed in cytosol and represented second messenger-dependent catalytic activation. These results suggest that CG-NAP anchors hypophosphorylated PKCε at the Golgi/centrosome area during maturation and serves as a scaffold for the phosphorylation reaction.