Association of immature hypophosphorylated protein kinase cepsilon with an anchoring protein CG-NAP.
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 PKC epsilon 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 PKC epsilon was an immature species at the hypophosphorylated state. In vitro binding studies confirmed that non- or hypophosphorylated PKC epsilon directly bound to CG-NAP via its catalytic domain, whereas sufficiently phosphorylated PKC epsilon did not. PKC epsilon 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 PKCepsilon at the Golgi/centrosome area during maturation and serves as a scaffold for the phosphorylation reaction.
Takahashi
M
M
Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
Mukai
H
H
Oishi
K
K
Isagawa
T
T
Ono
Y
Y
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D007527:Isoenzymes / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D015290:Second Messenger Systems
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; C486537:Akap9 protein, rat; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D007527:Isoenzymes; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon