Modulation of Elk-dependent-transcription by Gene33.
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 NFkappaB 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.
Keeton
Adam B
AB
Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama, 35294-0019.
Messina
Joseph L
JL
D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection; D025521:Tumor Suppressor Proteins
D048868:Adaptor Proteins, Signal Transducing; C484017:ERRFI1 protein, human; D025521:Tumor Suppressor Proteins; D008156:Luciferases