Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection.
We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.
Yan
B
B
Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA.
Wang
H
H
Kon
T
T
Li
C-Y
CY
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D004790:Enzyme Induction; D015870:Gene Expression / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics / Q000502:physiology; D016762:Genes, fos / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D051573:Proto-Oncogene Proteins c-pim-1 / Q000378:metabolism*; D015398:Signal Transduction; D015533:Transcriptional Activation / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D050807:ets-Domain Protein Elk-1 / Q000235:genetics / Q000378:metabolism*
D050807:ets-Domain Protein Elk-1; D051573:Proto-Oncogene Proteins c-pim-1; D020928:Mitogen-Activated Protein Kinases