The two forms of bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase result from alternative splicing

Abstract

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.

Evidence for new phosphorylation sites for protein kinase C and cyclic AMP‐dependent protein kinase in bovine heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase

“…The bovine heart PFlGYFBPase-2 preparation (WOE(~) was incubatrxi in a Rnal volumcof0.2 ml with 5OyM &-'zP]MgATPand 1 mU of either PKA or PKC for 60 min as described in [7]. Proleins were precipitated by 10% (w/v) trichloroacetic acid and the mixture was Fractions (1 ml) were collected and those containing radioactivity were lyophilised.…”

“…In rat liver PFK-YFBPase-2, the phosphorylation Bovine heart PFK-2IFBPasc-2, PKA, nnd PKC were purified as described previously [7,8]. One unit of enzyme activity ia the amount which catalyses the formation of 1 ,umol of producbmin under the assay conditions.…”

“…Phosphorylation by PKA has different effects on the activities of the L-and H-type isozymes: phosphorylation of the L-type leads to PFK-2 inactivation (increase in K",fP for fructose 6-phosphate (Fru-6-P) and decrease in V,,,J and FBPase-2 activation (increase in V,,,,) [4,5], whereas phosphorylation of the H-type results in a small activation of PFK-2 (decrease in &fP for Fru-6-P) with no effect on FBPase-2 activity [6,7]. H-Type PFK-2/FBPase-2 is also a substrate of protein kinase C (PKC), but phosphorylation has no effect on its enzyme activities [7,8].…”

“…H-Type PFK-2/FBPase-2 is also a substrate of protein kinase C (PKC), but phosphorylation has no effect on its enzyme activities [7,8].…”

“…Two forms of PFK-2/FBPase-2 have heen identified in bovine heart with subunit M,s of 58,000 and 54,000 [6,7,11], wllich result from alternative splicing of the same primary transcript [7]. The 54,000 M, form of bovine heart PFK-UFBPase-2 differs from the 58,OW M, form in that it lacks a C-terminal peptide (residues 451-510), which is encoded by exon 15 in the rat gene [12] and which contains the phosphorylation sites for PKA and PKC; however, since the SS,ooO JJr form is also phosphorylated by both PKA and PKC [7], phosphorylation sites other than Ser-466 (WA) and Thr-475 (PKC) should be present in this form. In this paper we present evidence for new phosphorylation sites for PKA and PKC in bovine heart PFK-2/FBPase-2.…”