SAKAI Norio Graduate School of Biomedical Sciences Hiroshima University, Professor, 大学院・医歯薬総合研究科, 教授 (70263407)
SHIRAI Yasuhito Kobe University, Biosignal Research Center, Assistant Professor, バイオシグナル研究センター, 助教授 (60263399)
PKC consists of more than 10 isoforms and the existence of multiple isoforms strongly suggests that each isoform has individual functional role in signal transduction. We have been investigated "When, Where, Which isoform of PKC is activated and How the cellular function is regulated by the activation" by using green fluorescent protein(GFP)-tagged PKC isoforms. We have found that PKC translocation is dynamic and reversible in living cells and that PKC targeting is isoform-specific, stimulus-specific and cell-specific.
In the present study, we have demonstrated that 1) arachidonic acid and ceramide act on CIB domains of epsilon and delta PKC and translocate these PKC isoforms to Golgi complex in isoform-specific manner, 2) ceramide induces translocation of deltaPKC to Golgi complex and activates this PKC isoform through tyrosine-phosphorylation., 3) Activation of deltaPKC by ceramide results in apoptosis which is distinctly different cell response from that induced by phorbol ester, another activator of PKC., 4) endogenous PKC can be knock-down by siRNA in isoform and species specifically., 5) Multiple PKC isoforms are involved in phagocytosis of microglial cells. Finally, we produced transgenic mice expressing GFP-tagged PKC isoforms using tetracyclin-regulated system. The expression of PKC-GFP can be regulated spatially and temporally. These transgenic mice enable us to monitor PKC targeting in brain slices by physiological condition and we observed PKC translocation in living brain which was different from that observed in cultured cells.