The two forms of bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase result from alternative splicing.
Purified bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) showed two bands with subunit M(r) of 58,000 and 54,000 when analysed by SDS/PAGE. Both the 58,000- and 54,000-M(r) forms were phosphorylated by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) in vitro. Phosphorylation by PKA decreased the apparent Km of PFK-2 for one of its substrates, fructose 6-phosphate, while phosphorylation by PKC did not correlate with any change in PFK-2 activity. The differences between the 58,000- and 54,000-M(r) forms were studied by electroblotting, peptide mapping and microsequencing. Residues 451-510, which correspond to exon 15 in the rat and contain phosphorylation sites for PKA (Ser-466) and PKC (Thr-475), were absent from the 54,000-M(r) form. Peptide mapping after phosphorylation by [gamma-32P]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58,000-M(r) form but not in the 54,000-M(r) form. The fact that the latter form was phosphorylated by PKC and PKA suggests that other phosphorylation sites for PKA and PKC are located outside the region encoded by exon 15. Finally, analysis of RNA from bovine heart showed that the tissue contains two PFK-2/FBPase-2 mRNAs, only one of which was recognized by a probe specific to the region coding for Ser-466 and Thr-475. Taken together, these findings demonstrate that the 58,000- and 54,000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.
Rider
M H
MH
Hormone and Metabolic Research Unit, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Vandamme
J
J
Lebeau
E
E
Vertommen
D
D
Vidal
H
H
Rousseau
G G
GG
Vandekerckhove
J
J
Hue
L
L
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010449:Peptide Mapping; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism; D012326:RNA Splicing; D014357:Trypsin
D007527:Isoenzymes; D010770:Phosphotransferases; D011494:Protein Kinases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin