Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
In liver, the 470-residue bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyses the synthesis and degradation of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. In rat hepatoma (HTC) cells, this enzyme has kinetic, antigenic, and regulatory properties, such as insensitivity to cyclic AMP-dependent protein kinase and lack of associated FBPase-2 activity, that differ from those in liver. To compare the sequence of the HTC enzyme with that of the liver enzyme, we have cloned the corresponding fully-coding cDNA from HTC cells. This cDNA predicts a protein of 448 residues in which the first 32 residues of liver PFK-2/FBPase-2 including the cyclic AMP target sequence have been replaced by a unique N-terminal decapeptide. The rest of the protein is identical with the liver enzyme. An N-terminally truncated recombinant peptide of 380 residues containing the PFK-2 and FBPase-2 domains was expressed in Escherichia coli as a beta-galactosidase fusion protein. It was recognized by anti-PFK-2 antibodies but its enzymic activities were barely detectable. In contrast, a cDNA fully-coding for the HTC enzyme could be expressed in E. coli as a beta-galactosidase-free peptide that exhibited both PFK-2 and FBPase-2 activities. This peptide had those PFK-2 kinetic properties of the HTC enzyme that differ from the liver enzyme. These data, together with immunoblot experiments, suggest that the lack of associated FBPase-2 activity in HTC cells results from a post-translational modification of the enzyme rather than from the difference in amino acid sequence. As well as this peculiar type of PFK-2/FBPase-2 mRNA, HTC cells also contained low concentrations of the liver-type mRNA. Unlike in liver, neither mRNA was induced by dexamethasone in these cells.
Crepin
K M
KM
International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Darville
M I
MI
Michel
A
A
Hue
L
L
Rousseau
G G
GG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002951:Citrates / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005994:Glycerophosphates / Q000378:metabolism; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D008969:Molecular Sequence Data; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D010770:Phosphotransferases / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D002951:Citrates; D005994:Glycerophosphates; D011994:Recombinant Proteins; D003907:Dexamethasone; D004247:DNA; D010770:Phosphotransferases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases