FIG. 6.

hRev7-mediated Elk-1 activation is mediated through JNK. (A) Depletion of JNK1/2 diminishes Elk-1 phosphorylation induced by hRev7 in vivo. 293T cells were transfected with siRNA duplexes against JNK1/2 (si-JNK) (+) or control siRNA duplexes (−) and additionally Flag-tagged Elk-1 and 0, 0.2, or 1 μg hRev7 expression plasmids. Total protein levels and phosphorylated Elk-1 (P-S383 Elk-1) were determined by immunoblotting (IB). (B) Depletion of JNK1/2 diminishes Elk-1 activation induced by hRev7 in vivo. Luciferase reporter assay (0.25 μg of GAL4-driven TK promoter-reporter) in 293T cells transfected with siRNA duplexes against JNK1/2 (si-JNK) (+) (black bars) or control siRNA duplexes (−) (white bars) and additionally 0, 0.2, or 1 μg hRev7 expression plasmid. Experiments were performed in duplicate and averaged from two independent experiments. (C and D) Same as panels A and B except that the JNK inhibitor SP600125 was added in place of siRNA against JNK1/2 and the phosphorylation status of GAL-Elk-1(223-428) was analyzed instead of full-length Elk-1. (E) Quantitative RT-PCR analysis of egr-1 or c-fos in serum-starved HeLa cells as described in the legend to Fig. 2F. Experiments were performed in the presence of siRNA duplexes against JNK1/2 (siJNK) or control siRNA duplexes against GAPDH. (F) Model showing how hRev7 coordinates the cellular responses to DNA-damaging agents. hRev7 promotes TLS-mediated DNA repair in response to DNA damage induced by agents, such as MMS, and also promotes a transcriptional response through targeting the JNK pathway to transcription factors like Elk-1. The dotted line represents possible cross talk that might occur between the JNK pathway and hRev7 function in TLS. Pol, polymerase.