Abstract
The present work was initiated with the aim of identifying nuclear genes whose expression is sensitive to the mitochondrial DNA (mtDNA) status of transformed chicken DU24 cells. We cloned and sequenced cDNAs for the mitogen-activated protein kinase kinase type 2, MEK2, a protein involved in the mitogenic growth factor signal transduction pathway in vertebrates. Sequence comparisons between the chicken protein and its mammalian counterparts indicated that MEK2 proteins are highly conserved among vertebrates. Southern blot analysis of endonuclease-digested genomic DNA from primary chick embryo fibroblasts (CEF) suggested that MEK2 is a single-copy gene in this vertebrate species. The steady-state level of MEK2 transcripts is decreased in DUS3 mtDNA-less (rho0) cells developed by long-term exposure of DU24 rho+ cells to ethidium bromide (EtdBr). Run-on in vitro transcription assays and mRNA stability studies indicated that the decrease in MEK2 mRNA content is associated with posttranscriptional regulation. In parental DU24 cells, MEK2 mRNA content decreased after inhibition of mtDNA transcription by EtdBr and inhibition of translation on mitoribosomes by chloramphenicol (CAM). Cytoplasmic hybrids (cybrids) constructed by fusion of chicken rho0 cells with enucleated parental cells and CEF recovered a basal level of MEK2 expression. The MEK2 protein content is decreased in DUS3 rho0 cells and in parental DU24 rho+ cells treated with EtdBr and CAM for 6 days, while that of MEK1, a closely related kinase, remained unchanged. On the basis of these observations, we propose that mitochondria participate in the mitogenic signal transduction pathway in chicken cells through regulation of MEK2 expression.
Original language | English |
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Pages (from-to) | 15371-15380 |
Number of pages | 10 |
Journal | Biochemistry |
Volume | 36 |
Issue number | 49 |
DOIs | |
State | Published - Dec 9 1997 |
Keywords
- Amino Acid Sequence
- Animals
- Base Sequence
- Cell Line, Transformed
- Chickens
- Cloning, Molecular
- DNA, Complementary
- DNA, Mitochondrial/genetics
- Down-Regulation
- MAP Kinase Kinase 2
- Mitogen-Activated Protein Kinase Kinases
- Molecular Sequence Data
- Protein Serine-Threonine Kinases/genetics
- Protein-Tyrosine Kinases/genetics
- Sequence Homology, Amino Acid