Refining the nuclear localization signal within the Egr transcriptional coregulator NAB2.

NAB1 and 2 are coregulators for early growth response (Egr) transcription factors. The NAB1 nuclear localization signal (NLS) was previously described as a bipartite NLS of sequence R(X2 )K(X11 )KRXK. The sequence is conserved in NAB2 as K(X2 )R(X11 )KKXK; however, whether it functions as the NAB2 NLS has not been tested. We show that the KKXK motif in NAB2 is necessary and sufficient to mediate nuclear localization. Mutation of the KKXK motif to AAXA causes cytoplasmic localization of NAB2, while Lys/Arg-to-Ala mutations of the upstream K(X2 )R motif have no effect. Fusion of the KKXK motif to cytoplasmic protein eIF2Bε causes nuclear localization. Altogether, this study refines our knowledge of the NAB2 NLS, demonstrating that KKXK343-346 is necessary and sufficient for nuclear localization.

Role for Egr1 in the Transcriptional Program Associated with Neuronal Differentiation of PC12 Cells.

PC12 cells are a well-established model to study how differences in signal transduction duration can elicit distinct cell behaviors. Epidermal growth factor (EGF) activates transient ERK signaling in PC12 cells that lasts 30-60 min, which in turn promotes proliferation; nerve growth factor (NGF) activates more sustained ERK signaling that lasts 4-6 h, which in turns induces neuronal differentiation. Data presented here extend a previous study by Mullenbrock et al. (2011) that demonstrated that sustained ERK signaling in response to NGF induces preferential expression of a 69-member gene set compared to transient ERK signaling in response to EGF and that the transcription factors AP-1 and CREB play a major role in the preferential expression of several genes within the set. Here, we examined whether the Egr family of transcription factors also contributes to the preferential expression of the gene set in response to NGF. Our data demonstrate that NGF causes transient induction of all Egr family member transcripts, but a corresponding induction of protein was detected for only Egr1 and 2. Chromatin immunoprecipitation experiments provided clearest evidence that, after induction, Egr1 binds 12 of the 69 genes that are preferentially expressed during sustained ERK signaling. In addition, Egr1 expression and binding upstream of its target genes were both sustained in response to NGF versus EGF within the same timeframe that its targets are preferentially expressed. These data thus provide evidence that Egr1 contributes to the transcriptional program activated by sustained ERK signaling in response to NGF, specifically by contributing to the preferential expression of its target genes identified here.