Abstract
Gene33 is a cytoplasmic protein expressed in many cell types, including those of renal and hepatic origin. Its expression is regulated by a large number of mitogenic and stressful stimuli, both in cultured cells and in vivo. Gene33 protein possesses binding domains for ErbB receptors, 14‐3‐3 proteins, SH‐3 domains, and GTP bound Cdc42, suggesting that it may play a role in signal transduction. Indeed, these regions of Gene33 have been reported to modulate signaling through the ERK, JNK, and NFκB pathways. In the present work, epitope‐tagged full‐length and truncation mutants, as well as wild‐type Gene33, were overexpressed in 293 cells. The expression of these proteins was compared to the level of endogenous Gene33 by Western blot using a newly developed polyclonal antibody. As proxies for activity of the ERK and JNK pathways, Elk‐ and c‐Jun‐dependent transcription were measured by a luciferase reporter gene. Moderate expression levels of full‐length Gene33 caused a twofold increase in Elk‐dependent transcription, while at higher levels, c‐Jun‐dependent transcription was partially inhibited. The C‐terminal half of Gene33 significantly increased both Elk‐ and c‐Jun‐dependent transcription when expressed at approximately threefold above control levels. This effect on Elk‐dependent transcription was lost at higher levels of Gene33 expression. In contrast, higher levels of the C‐terminal half of Gene33 caused a progressively greater effect on c‐Jun‐dependent transcription. These findings suggest that Gene33 may increase ERK activity, and that the C‐terminal half of Gene33 may act less specifically in the absence of the N‐terminal half, inducing JNK activity. © 2005 Wiley‐Liss, Inc.