Abstract
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.
Highlights
Hematopoietic cells are absolutely dependent on peptide growth factors for survival and proliferation
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
We found that Pim-1 inhibited the Elk-1 and NFκB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays
Summary
Hematopoietic cells are absolutely dependent on peptide growth factors for survival and proliferation. Pim-1 is a serine-threonine kinase in hematopoietic cells whose expression is regulated by such growth factors as GMCSF, IL-3, IL-4, IFN-γ and so on (1-3). Some oncogenes encoding serine-threonine kinases such as raf (18,19), mos (20,21) and tpl-2 (22) are known to exert their survival effect by regulating the mitogen-activated protein kinase (MAPK) pathway. Elk-1 and NFκB are transcription factors whose activities are regulated or partially regulated by MAPK (23,24). If Pim-1 affects the MAPK pathway at any level, this should be reflected on the transcriptional activity of Elk-1 or NFκB, the output of the MAPK pathway. We used the transcriptional reporter assay as a quick screening system for our study
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