Abstract
The mechanism by which signals such as those produced by glutamate are transferred to the nucleus may involve direct transport of an activated transcription factor to trigger long-term transcriptional changes. Ionotropic glutamate receptor activation or depolarization activates transcription factor NF-kappaB and leads to translocation of NF-kappaB from the cytoplasm to the nucleus. We investigated the dynamics of NF-kappaB translocation in living neurons by tracing the NF-kappaB subunit RelA (p65) with jellyfish green fluorescent protein. We found that green fluorescent protein-RelA was located in either the nucleus or cytoplasm and neurites, depending on the coexpression of the cognate inhibitor of NF-kappaB, IkappaB-alpha. Stimulation with glutamate, kainate, or potassium chloride resulted in a redistribution of NF-kappaB from neurites to the nucleus. This transport depended on an intact nuclear localization signal on RelA. Thus, in addition to its role as a transcription factor, NF-kappaB may be a signal transducer, transmitting transient glutamatergic signals from distant sites to the nucleus.
Highlights
Ments (6 –10) and rapidly activated independent of protein synthesis [11], making this factor a likely candidate as a synaptic tag [3]
We examined whether activated NF-B RelA was transported from distant sites to the nucleus in living cells using jellyfish green fluorescent protein (GFP) fusion technology to attach a fluorescent tag to the RelA subunit of NF-B
Subcellular Distribution and Biochemical Analysis of NF-B RelA tagged with EGFP—To analyze the distribution of NF-B, a GFP fusion protein containing RelA with its nuclear localization signal was constructed (Fig. 1A)
Summary
Cell Culture—Hippocampal neurons were cultured from embryonic day 17 or 18 rats as described by Banker and Cowan [32] and detailed by de Hoop et al [33]. Using forward primer C (5Ј-GGCCGACGAACTGTTCCCCCTCATCTTCC-3Ј) and reverse primer D (5Ј-GGATCCTTAGGAGCTGATCTGAC-3Ј), a RelA fragment containing the first 361 amino acids including transactivation domain TA3, but without TA1 and TA2 [37], was amplified using a 29-base pair overlap with the 3Ј-end of the EGFP fragment, with the pCMV-RelA expression plasmid used as a template. This strategy was chosen to avoid potential toxicity via the induction of neurotoxic NF-B target genes. Pseudo-color images were created using NIH Image Version 1.61
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