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2008 SURI Intern Year: Sophomore Research Project: The Role of PKR in Neuronal Development The double-stranded RNA (dsRNA)-activated protein kinase (PKR), a serine/threonine protein kinase, plays an important role in translational regulation and cell survival, and it is ubiquitously expressed in mammalian cells (Chen et al. 2006). PKR is a component of signal transduction pathways mediating many important cellular functions such as survival, proliferation, differentiation, and stress responses. PKR is activated by a viral infection or double-stranded RNA (Chen et al. 2006). Recent studies have shown that PKR can be activated without dsRNA. Instead PKR can be activated by protein activators, PACT, or its mouse homologue, RAX (Wang et al. 2007). After activation, PKR phosphorylates the α-subunit of eukaryotic translation initiation factor-2 (eIF2α), which leads to the inhibition of translation initiation (Chen et al. 2006). Phosphorylated eIF2α sequesters eIF2B, a rate-limiting component of translation, leading to an inhibition of protein synthesis in the cells (Donze et al. 2004). In some cases, eIF2α phosphorylation leads to cell death (Donze et al. 2004). The role of PKR in neuronal proliferation and differentiation is not known. To investigate the role of PKR, mouse N2a cells will be utilized. The N2a cells will be grown in a 10 percent serum and treated with PIC and PKR inhibitors for 24 , 48, 72, and 96 hours. Then cell proliferation and viability (MTT) will be performed. Also, the morphology of the N2a cells will be examined. The final experiment will be collecting protein samples at 2, 6, 12, and 24 hours after treatment of PIC and PKR inhibitors. In addition, the p-eIF2α will be examined by Western Blots.
Chen G, Ma C, Bower KA, Ke Z, Luo J. Interaction between RAX and PKR modulates the effect of ethanol on protein synthesis and survival of neurons. Journal of Biological Chemistry (2006), 281(23):15909-15915. Donze O, Deng J, Curran J, Sladeck R, Picard D, Sonenberg N. The protein kinase PKR: a molecular clock that sequentially activates survival and death programs. EMBO Journal (2004), 23(3):564-571. Wang X, Fan Z, Wang B, Luo J, Ke Z. Activation of double-stranded RNA-activated protein kinase by mild impairment of oxidative metabolism in neurons. Journal of Neurochemistry (2007), 103(6):2380-2390.
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