Cloning and characterization of cDNAs encoding chicken mitogen-activated protein kinase kinase type 2, MEK2: downregulation of MEK2 in response to inhibition of mitochondrial DNA expression.

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 post-transcriptional 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.

Chicken acidic ribosomal phosphoprotein PO: isolation and molecular characterization of cDNA clones.

Differential screening of a cDNA library prepared from chicken mitochondrial DNA-less cells led to the isolation of the acidic phosphoprotein PO, as inferred from similar sequences from human, rat and mouse deposited in databases. The chicken PO protein is highly conserved among vertebrates. Nucleotide identities between the chicken and mammalian cDNA sequences are greater than 94%. Amino acid sequence identities greater than 92% are observed between the chicken protein and its mammalian counterparts, and when changes to conservative amino acids are considered, similarities range from 99.4 to 100%. The gene evolved mainly by silent transitions occurring at the third codon position. The highly antigenic carboxy-terminal sequences of mammalian PO proteins are identical to that of the chicken. Southern blot analysis of restricted DNA from chicken embryo fibroblasts suggests that only one gene encoding PO exists in the chicken genome. By Northern analysis, the PO probe detects a major RNA species 1.1-kilobases long, and a minor species 4.0-kilobases long which has no equivalent thus far in mammals.

Glucose metabolism in dog inner medullary collecting ducts.

The adenosine triphosphate (ATP) generating pathways of dog inner medullary collecting ducts (IMCD) were examined in vitro using suspensions of dog IMCD tubules incubated under aerobic and anaerobic conditions. Glucose is always the preferred substrate for this tissue, even if lactate can be oxidized under aerobic conditions. The metabolism of glucose proceeds largely towards lactate accumulation in the presence or absence of oxygen. Glycogen is also consumed and more markedly so during anoxia. The pentose shunt represents a minor pathway for glucose metabolism in this tissue. Under aerobic conditions, the net oxidation of glucose to CO2 contributes significantly to the cell energetics, mitochondrial and cytoplasmic mechanisms sharing equally the ATP synthesis. In the absence of oxygen, only the cytoplasmic routes of ATP synthesis are used, but the apparent ATP turnover is markedly reduced. A marked inhibition of the activity of the Na-K-ATPase during anoxia explains this observation. The utilization of glucose for osmolyte synthesis is a minor process and appears to be suppressed under anaerobic conditions. It is concluded that the ATP turnover is low in dog IMCD cells as compared with that of other nephron segments and is largely dependent upon glucose availability under aerobic or anaerobic conditions.

Glycogen metabolism in dog inner medullary collecting ducts.

The regulation of glycogen degradation and synthesis in canine inner medullary collecting ducts (IMCD) was investigated using IMCD tubules suspensions prepared from dog papilla. A small but significant amount of glycogen was found in dog IMCD. Under aerobic condition and especially when no exogenous substrate is available, glycogen breakdown can support IMCD glycolysis for a short period of time. Increasing concentration of exogenous glucose but not lactate was able to reduce and even to suppress (20 mM glucose) the glycogen breakdown. A net synthesis of glycogen was observed only when the endogenous glycogen pool was previously partially or totally depleted. Under anaerobic condition, glycogenolysis was stimulated. The addition of up to 20 mM glucose now reduced but never suppressed this process. Glycogen metabolism responded to variation in the cells energy needs, since the net glycogen breakdown was diminished and glycogen synthesis increased when the cellular ATP turnover was reduced. The reverse effects were observed when the ATP turnover was increased. At all times, glycogen metabolism correlated well with changes in tissue glucose 6-phosphate concentration. The energy requirement of IMCD cells and the availability of alternative energy sources (active mitochondria, exogenous glucose) are therefore capable of eliciting an integrated and appropriate response of glycogen phosphorylase and synthase in IMCD tubules in suspension studied in vitro.