THE 2 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 58000 and 54000 when analysed by SDS/PAGE. Both the 58000- and 54000-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 K(m) 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 58000- and 54000-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 54000-M(r) form. Peptide mapping after phosphorylation by [gamma-P-32]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58000-M(r) form but not in the 54000-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 58000- and 54000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.