Expression of depolarization-induced immediate early gene proteins in PC12 cells

Immediate early genes induced by depolarization are thought to be important in mediating neuronal functional plasticity. We previously identified a group of immediate early genes that are preferentially induced by depolarization and forskolin but not by nerve growth factor or epidermal growth factor in PC12 pheochromocytoma cells. These depolarization-induced genes include synaptotagmin 4; the protein kinases KID-1, PIM-1, and SIK; an orphan transcription factor, Nurr-1; and a transcription corepressor, rTLE-3. All these genes are also induced in the hippocampus in response to kainic-acid induced depolarization. To characterize further the unique functions of these genes in plasticity, we used recombinant proteins to generate and purify antibodies against KID-1 and SIK proteins. Immunoblotting experiments were performed to examine the induced expression of the KID-1 and SIK proteins in PC12 cells. PIM-1 and Nurr-1 protein expression was also examined following stimulation, using commercially available antibodies. There is an increase in synthesis, in PC12 cells, of these four IEG proteins after KCl plus forskolin treatment. Nurr-1 protein peaks between 2 and 4 hr and decreases by 6 hr after the treatment. PIM-1 and KID-1 proteins rise by 1 hr, peak between 2 and 4 hr, and return to their basal levels at 6 hr. SIK protein increases significantly at 2 hr after treatment, peaks between 4 and 6 hr, and returns to the basal level at 8 hr. Immunofluorescence studies demonstrate distinct distribution patterns of each of these depolarization-induced IEG proteins in PC12 cells.