<a name="home"></a>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

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 NFκB 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 NFκB 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 NFκB. 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.

The PIM1 Kinase Is a Critical Component of a Survival Pathway Activated by Docetaxel and Promotes Survival of Docetaxel-treated Prostate Cancer Cells

“…Our studies indicate that, in RWPE-2 cells, docetaxel activates NFB in a PIM1-dependent manner. Previous reports have shown that the related PIM2 kinase can activate NFB activity (72), although alternative opinions about the PIM1 kinase have been presented (73). PIM2 activates NFB activity through phosphorylation and activation of the COT/ TPL2 kinase, a kinase with known IB kinase-like activity (72).…”

Modulation of peroxisome proliferator–activated receptor γ stability and transcriptional activity in adipocytes by resveratrol

“…Previous studies have shown that resveratrol activates PPARγ in a variety of tissues (17–19), including macrophages (17) when measured using transactivation reporter assays. However, studies of other transcription factors such as Elk-1 and c-Fos (20) or control of T cell receptor β expression (21) have demonstrated discrepancies between luciferase-based transactivation assays and regulation of endogenous targets of transcription factor activity. These studies point to the pitfalls of measuring gene expression outside the usual chromatin structure as is the case with luciferase reporter assays.…”