Importance of C1B domain for lipid messenger-induced targeting of protein kinase C.
The molecular mechanisms by which arachidonic acid (AA) and ceramide elicit translocation of protein kinase C (PKC) were investigated. Ceramide translocated epsilonPKC from the cytoplasm to the Golgi complex, but with a mechanism distinct from that utilized by AA. Using fluorescence recovery after photobleaching, we showed that, upon treatment with AA, epsilonPKC was tightly associated with the Golgi complex; ceramide elicited an accumulation of epsilonPKC which was exchangeable with the cytoplasm. Stimulation with ceramide after AA converted the AA-induced Golgi complex staining to one elicited by ceramide alone; AA had no effect on the ceramide-stimulated localization. Using point mutants and deletions of epsilonPKC, we determined that the epsilonC1B domain was responsible for the ceramide- and AA-induced translocation. Switch chimeras, containing the C1B from epsilonPKC in the context of deltaPKC (delta(epsilonC1B)) and vice versa (epsilon(deltaC1B)), were generated and tested for their translocation in response to ceramide and AA. delta(epsilonC1B) translocated upon treatment with both ceramide and AA; epsilon(deltaC1B) responded only to ceramide. Thus, through the C1B domain, AA and ceramide induce different patterns of epsilonPKC translocation and the C1B domain defines the subtype specific sensitivity of PKCs to lipid second messengers.
Kashiwagi
Kaori
K
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
Shirai
Yasuhito
Y
Kuriyama
Masamitsu
M
Sakai
Norio
N
Saito
Naoaki
N
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002518:Ceramides / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D017354:Point Mutation; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002518:Ceramides; D007527:Isoenzymes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D016718:Arachidonic Acid; C497258:PRKCD protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate