Elk-1 associates with the mitochondrial permeability transition pore complex in neurons

Abstract

The nuclear transcription factor E-26-like protein 1 (Elk-1) is thought to impact neuronal differentiation [Sharrocks, A. D. (2001) Nat. Rev. Mol. Cell Biol. 2, 827-837], cell proliferation [Sharrocks, A. D. (2002) Biochem. Soc. Trans. 30, 1-9], tumorigenesis [Chai, Y. L., Chipitsyna, G., Cui, J., Liao, B., Liu, S., Aysola, K., Yezdani, M., Reddy, E. S. P. & Rao, V. N. (2001) Oncogene 20, 1357-1367], and apoptosis [Shao, N., Chai, Y., Cui, J., Wang, N., Aysola, K., Reddy, E. S. P. & Rao, V. N. (1998) Oncogene 17, 527-532]. In addition to its nuclear localization, Elk-1 is found throughout the cytoplasm, including localization in neuronal dendrites [Sgambato, V., Vanhoutte, P., Pages, C., Rogard, M., Hipskind, R., Besson, M. J. & Caboche, J. (1998) J. Neurosci. 18, 214-226], raising the possibility that Elk-1 may have alternative extranuclear functions in neurons. Using coimmunoprecipitation and reciprocal coimmunoprecipitation from adult rat brain, we found an association between Elk-1 protein and the mitochondrial permeability transition pore complex (PTP), a structure involved in both apoptotic and necrotic cell death. Electron microscopy in adult rat brain sections confirmed this association with mitochondria. Elk-1 was also identified from purified mitochondrial fractions by using Western blotting, and Elk-1 increased its association with mitochondria following proapoptotic stimuli. Consistent with a role for Elk-1 in neuron viability, overexpression of Elk-1 in primary neurons decreased cell viability, whereas Elk-1 siRNA-mediated knockdown increased cell viability. This decrease in viability induced by Elk-1 overexpression was blocked with application of a PTP inhibitor. These results show an association of the nuclear transcription factor Elk-1 with the mitochondrial PTP and suggest an additional extranuclear function for Elk-1 in neurons.

Fig. 1.

Elk-1 associates with mitochondrial PTP proteins. (a) Co-IPs from mouse WB and SN fractions were performed by using Elk-1 antibodies (Santa Cruz Biotechnology), and immunoprecipitates were submitted to the University of Pennsylvania Proteomics Core Facility for peptide sequencing. Listed are mitochondrial proteins identified through peptide sequencing, with check marks representing the antibody and tissue conditions that led to their identification. (b) Shown are co-IPs carried out with an Elk-1 antibody (Santa Cruz Biotechnology) and immunoprecipitates blotted with antibodies to HXK as a positive control, and GAD 65/67 as a negative control. To confirm the co-IP data, reciprocal co-IP was carried out by using antibodies against adenine nucleotide transporter, VDAC, and HXK, and ubiquitous mitochondrial creatine kinase and immunoprecipitates were then analyzed by Western blot using an Elk-1 antibody (Cell Signaling Technology). Shown are the reciprocal co-IPs from SN fractions.

Fig. 2.

Elk-1 associates with mitochondria in adult rat brain section. (a) Immunogold–silver labeling for Elk-1 (arrowhead) is associated with mitochondria (m) from adult rat brain (n = three rats). Images show labeling in a neuronal dendrite. (Scale bar, 500 nm.) (b) Potential interactions of Elk-1 (hexagon) with proteins that form the mitochondrial PTP.

Fig. 3.

Elk-1 increases its association with mitochondria following application of camptothecin and etoposide. (a) Mitochondrial fractions were obtained from primary rat cortical neurons 14–21 days in vitro according to the manufacturer's instructions (Pierce). Shown is a Western blot for mitochondrial lysate with an absence of signal for the nuclear protein NeuN (Abcam) in the mitochondrial fractions, compared with WB fractions. Also shown is the presence of signal for the mitochondrial protein VDAC1 (Santa Cruz Biotechnology) in the mitochondrial fractions. (b) Three hours after treatment with camptothecin or etoposide (15 μM), an increase in Elk-1 signal was detected in the mitochondrial fractions by Western blot. VDAC1 was used as a loading control. Integrated densities measured using image j (National Institutes of Health) are shown below Western blots. Values have been normalized to control to show relative change in density after drug treatment for bands corresponding to Elk-1 and VDAC.

Fig. 4.

Elk-1 overexpression leads to decreased cell viability, whereas Elk-1 siRNA-mediated knockdown leads to increased cell viability. (a) Graph shows whole-cell ATP levels from primary rat hippocampal neurons 48 h following electroporation with either Elk-1 in a pcDNA3.1 vector or pcDNA3.1 vector alone (control). (b) Graph shows whole-cell ATP levels from primary rat hippocampal neurons 48 h following electroporation with either one of two Elk-1 siRNAs (Ambion) or pSilencer Negative Control siRNA (Ambion). ATP levels are normalized to control. Error bars represent standard deviations, and ∗ indicates P < 0.05 when comparing siRNA-mediated knockdown to control or Elk-1 overexpression to control. (c) Graph shows percent of cell death in primary rat hippocampal neurons after transfection with Elk-1 mRNA under control conditions or after preincubation with 25 μM BKA. Note the decrease in cell death after preincubation with BKA.