Abstract 3060: A necessary role of the N-terminal region of JAZ in cell growth inhibition and phosphorylation

JAZ belongs to a new class of evolutionarily conserved C2H2-type zinc finger (ZF) proteins that feature an unusually long linker sequence and preferentially bind to double-stranded RNA. We recently discovered JAZ as a novel positive regulator of p53 transcriptional activity. The mechanism may involve direct binding to p53's C-terminal regulatory domain to activate latent p53. To test a role of JAZ in regulating long-term cell growth, we performed a colony formation assay. Wild-type FLAG- or GFP-tagged JAZ and its deletion mutants that lack its N- or C-terminal region(s) were transfected into NIH mouse fibroblasts that express wild-type p53. Cells were then grown in the growth media in the presence of G418 for two weeks. Results show that transfection of full-length JAZ effectively suppresses colony formation compared to the empty vector or GFP control, supporting a role of wild-type JAZ in activating p53. The JAZ fragments missing the N-terminal region including the first one or two intact zinc finger motifs lose the ability to suppress colony formation, indicating a necessary role for JAZ's N-terminal region in cell growth inhibition. However, the JAZ fragments missing the C-terminal region including the last one or two zinc finger motifs can still suppress colony formation, indicating that JAZ's C-terminal region is not necessary for the suppression. Our previous data showed that both N- and C-terminal regions are necessary for efficient association of JAZ with p53 to stimulate its transcriptional activity. Thus, the inhibitory role of the JAZ fragments missing the C-terminal region does not require JAZ's stimulatory function of p53 transcriptional activity, indicating a novel mechanism. Moreover, we assessed the effect of deletion on FLAG-JAZ's ability to bind poly I[[Unable to Display Character: ∙]]C dsRNA. Results show that poly I[[Unable to Display Character: ∙]]C agarose beads can efficiently “pull down” all the FLAG-JAZ fragments tested, which contain either N- or C-terminal region(s) plus two or more intact ZFs, from the cell lysates. This suggests that while its role is not clear yet, dsRNA-binding is not sufficient for JAZ's role in suppressing colony formation. We also tested the effect of deletion on JAZ's subcellular localization. GFP-JAZ deletion mutants including one that missing the C-terminal region display significantly reduced nuclear localization. JAZ was reported to be a cargo protein for Exportin-5, a nuclear export receptor for cert