Spatio-temporal signaling mechanism of protein kinase C

PKC由10多种亚型组成，多种亚型的存在有力地表明每种亚型在信号转导中具有各自的功能作用。我们已经研究了“何时，何地，哪种PKC亚型被激活，以及如何通过激活来调节细胞功能”，通过使用绿色荧光蛋白（GFP）标记的PKC亚型。我们发现PKC转位在活细胞中是动态的、可逆的，PKC的靶向是亚型特异性、刺激特异性和细胞特异性的，本研究证明：1）花生四烯酸和神经酰胺作用于PKC的CIB结构域，并以亚型特异性的方式将这些PKC亚型转位到高尔基复合体，2）神经酰胺诱导deltaPKC易位至高尔基复合体，并通过酪氨酸磷酸化激活该PKC亚型。3)神经酰胺激活deltaPKC导致细胞凋亡，这与另一种PKC激活剂佛波酯诱导的细胞反应明显不同，4)内源性PKC可以被特异性亚型和物种的siRNA敲低。，5)多种PKC亚型参与小胶质细胞的吞噬作用。最后，我们使用四环素调控系统制备了表达GFP标记的PKC异构体的转基因小鼠。PKC-GFP的表达受时间和空间调控。这些转基因小鼠使我们能够通过生理条件监测PKC在脑切片中的靶向，并且我们观察到活脑中的PKC易位，这与在培养细胞中观察到的不同。

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.