Overexpression of Thymidine Kinase mRNA Eliminates Cell Cycle Regulation of Thymidine Kinase Enzyme Activity

Ernst W. Müllner,Thomas Sauer,Markus Hengstschläger,Wolfgang Mikulits,Erhard Wintersberger

doi:10.1074/jbc.271.2.853

Ernst W. Müllner, Thomas Sauer + Show 3 more

Open Access

https://doi.org/10.1074/jbc.271.2.853

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Abstract

Expression of thymidine kinase (TK) enzyme activity and mRNA is strictly S phase-specific in primary cells. In contrast, DNA tumor virus-transformed cells have enhanced and constitutive levels of TK mRNA during the whole cell cycle. Their TK protein abundance, however, still increases at the G1-S transition and stays high throughout G2 until mitosis. Therefore, post-transcriptional control must account for the decoupling of TK mRNA from protein synthesis in G1. To characterize the underlying mechanism, we studied the consequences of TK mRNA abundance on the cell cycle-dependent regulation of TK activity in nontransformed cells. Constitutive as well as conditional human and mouse TK cDNA vectors were stably transfected into mouse fibroblasts, which were subsequently synchronized by centrifugal elutriation. Low constitutive TK mRNA expression still resulted in a fluctuation of TK activity with a pronounced maximum in S phase. This pattern of cell cycle-dependent TK activity variation reflected the one in primary cell but is caused by post-transcriptional control. Increasing overexpression of TK transcripts after hormonal induction compromised this regulation. At the highest constant mRNA levels, regulation of enzyme activity was totally abolished in each phase of the cell cycle. These data indicate that post-transcriptional regulation of TK is tightly coupled to the amount of mRNA; high concentrations apparently titrate a factor(s) required for repressing TK production during G1 and presumably also G2.

Highlights

During growth stimulation and the cell cycle of normal cell types, thymidine kinase (TK,1 EC 2.7.1.21) enzyme activity is strongly increased, mainly by transcriptional activation of the gene just prior to the onset of DNA synthesis (Stuart et al, 1985; Coppock and Pardee, 1987; Pardee, 1989)
During the cell cycle of polyoma virus-transformed COP-8 mouse fibroblasts, there is almost no fluctuation of TK mRNA and only a moderate 4-fold induction of TK protein as well as enzyme activity at the G1-S boundary, which remain elevated throughout the G2 phase until mitosis (Fig. 1)
We examined molecular mechanisms contributing to post-transcriptional regulation of cytosolic TK enzyme activity during the cell cycle of apparently normal, nontransformed cells

Summary

Introduction

During growth stimulation and the cell cycle of normal cell types, thymidine kinase (TK,1 EC 2.7.1.21) enzyme activity is strongly increased, mainly by transcriptional activation of the gene just prior to the onset of DNA synthesis (Stuart et al, 1985; Coppock and Pardee, 1987; Pardee, 1989). Overexpression of TK mRNA Eliminates TK Activity Regulation transformed cells depends on the expression levels of TK transcripts by a specific regulatory mechanism.

Results

Conclusion