| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 95 === PICK1 is a selective anchoring protein for protein kinase C alpha. Previous study showed that over-expression of PICK1 in human leukemia REH cells rendered a higher resistance to etoposide-induced apoptosis through a recruitment of PKC alpha which resulted in maintenance of mitochondrial membrane potential, an enhanced phosphorylation of the anti-apoptotic Bcl-2 protein at Ser70, and inhibition of Bax dimerization. PICK1 contains three major sequence motifs: the PDZ domain, the BAR domain, and the acidic region. To investigate the functional motifs involved in the anti-apoptotic activity, deletion mutants of PICK1 were made and their effects on etoposide-induced cell death were examined. Over-expression of the PDZ domain alone was not sufficient for maintaining mitochondrial membrane potential and the anti-apoptotic activity of PICK1. The PICK1-PDZ also failed to maintain a higher level of phosphor-Bcl2 (Ser70). Since the Lys27 and Asp28 in the PDZ domain have been shown to be essential for interactions with PKCa, these results suggested that either a recruitment of PKC alpha alone was not sufficient for the anti-apoptotic activity of PICK1 or sequences in addition to the PDZ domain were required for recruitment of PKC alpha. In addition, deletion of either the coiled-coil motif in the BAR domain or the acidic region also abolished the pro-survival effect upon etoposide treatment as measured by trypan blue exclusion, caspase activation and mitochondrial membrane potential. These results suggested that the coiled-coil motif and the acidic region were both essential for conferring the pro-survival activity of PIKC1. In addition to etoposide, PICK1 also exhibited a resistance to doxorubicin -induced apoptosis, whereas PICK1 had no apparent effect on either ara-C- or cisplatin-induced apoptosis. These results suggested that the pro-survival activity of PICK1 was selective toward different stresses.
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