Spatio-temporal signaling mechanism of protein kinase C

PKC由10多种异构体组成，多种异构体的存在强烈表明每种异构体在信号转导中具有单独的功能作用。我们利用绿色荧光蛋白（GFP）标记的PKC异构体，研究了PKC的“何时、何地、哪种异构体被激活以及激活如何调节细胞功能”。我们发现PKC易位在活细胞中是动态和可逆的，PKC靶向是异构体特异性、刺激特异性和细胞特异性的。在本研究中，我们已经证明了1)烯四烯酸和神经酰胺作用于epsilon和deltaPKC的CIB结构域，并以特定的方式将这些PKC异构体转运到高尔基复合体上，2)神经酰胺诱导deltaPKC转运到高尔基复合体上，并通过酪氨酸磷酸化激活PKC异构体。(3)神经酰胺激活deltaPKC导致细胞凋亡，这与PKC的另一种激活剂磷酸酯诱导的细胞凋亡有明显不同。4)内源性PKC可以被siRNA特异性地敲除异构体和物种。5)多种PKC亚型参与小胶质细胞的吞噬作用。最后，我们利用四环素调控系统生产了表达gfp标记PKC亚型的转基因小鼠。PKC-GFP的表达受时空调控。这些转基因小鼠使我们能够在生理条件下监测PKC在脑切片中的靶向性，我们观察到PKC在活脑中的易位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.