Functional role of Bcl-2-associated athanogene (BAG) 3 as a regulator of Bak1 protein level in N1E115 cells

抄録

It is known that the Bcl-2-associated athanogene (BAG) 3 is highly expressed in muscle tissues and neuronal tissues, and exhibits cellular protection against apoptosis. Another study reported that BAG3 may regulate protein degradation by controlling the balance between the autophagy system and the ubiquitin-proteasome system. However, the molecular mechanism of BAG3 and its functional role in neuronal cells remains unclear. We analyzed the functional role of BAG3 in N1E115 cells derived from mouse neuroblastoma as a model of neuronal cells. Knockdown of BAG3 in N1E115 cells using si-RNA specifically targeted to BAG3 (si-BAG3) showed marked reduction in the BAG3 protein level in cytoplasm as well as mitochondrial fractions. Concomitant with the reduction in BAG3 protein, treatment with si-BAG3 led to an increase in TUNEL-positive cells and a decrease in cell viability in N1E115 cells. No alteration in the gene expression as well as protein levels of Bcl-2 and Bax were observed in N1E115 cells treated with si-BAG3. Although the cellular Bak1 level was enhanced by treatment with si-BAG3 in N1E115 cells, there was no difference in Bak1 gene expression between si-BAG3-treated and si-Luc-treated N1E115cells. In addition to the loss of function study, we performed a gain of function study using adenoviral vector containing the human BAG3 gene. No alteration in the gene expression as well as protein levels of Bcl-2 and Bax were observed in N1E115 cells treated with adenovirus containing BAG3. Overexpression of BAG3 led to a marked reduction in Bak1 protein in N1E115 cells compared to that overexpressing LacZ although no difference in Bak1 gene expression was observed. Bafilomycin A1, an autophagy inhibitor, can inhibit the modification of Bak1 protein level by BAG3. These results suggest that BAG3 may be critical to protein level in Bak1 via modification of autophagy activity, and that autophagy regulation by BAG3 may play an important role in the apoptosis of neuronal cells.