5' flanking sequence and structure of a gene encoding rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Abstract

The synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis, are catalyzed by 6-phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46), respectively. In liver, these two activities belong to separate domains of the same 470-residue polypeptide. Various mRNAs have been described for this bifunctional enzyme, which is controlled by hormonal and metabolic signals. To understand the origin and regulation of these mRNAs, we have characterized rat genomic clones encoding the liver isozyme, which is regulated by cAMP-dependent protein kinase, and the muscle isozyme, which is not. We describe here a 55-kilobase gene that encodes these isozymes by alternative splicing from two promoters. Each of the putative promoters was sequenced over about 3 kilobases and found to include nucleotide motifs for binding regulatory factors. The two isozymes share the same 13 exons and differ only by the first exon that, in the liver but not in the muscle isozyme, contains the serine phosphorylated by cAMPdependent protein kinase. The gene was assigned to the X chromosome. An analysis ofthe exon limits of6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in relation to its functional domains and to its similarity with other proteins plus its G+C content at the third codon position suggests that this gene originates from several fusion events.Fructose 2,6-bisphosphate is the most potent allosteric stimulator of 6-phosphofructo-1-kinase (EC 2.7.1.11), a key enzyme of glycolysis. The synthesis of fructose 2,6-bisphosphate is catalyzed by 6-phosphofructo-2-kinase (PFK-2; EC 2.7.1.105) and its degradation is catalyzed by fructose-2,6-bisphosphatase (FBPase-2; EC 3.1.3.46). In liver, these two activities belong to separate domains of each subunit (470 amino acids) of the same homodimeric protein. This bifunctional enzyme integrates a number of hormonal and metabolic signals including cAMP. In fibroblasts, PFK-2 activity is stimulated by growth factors, tumor promoters, and tyrosinespecific oncogenic protein kinases (1, 2).Biochemical and immunologic data suggest the existence of distinct PFK-2/FBPase-2 isozymes. Using PFK-2/ FBPase-2 cDNA probes from rat liver (4), we have identified a 2.1-kilobase (kb) mRNA coding for the liver (L) isozyme, a 1.9-kb mRNA coding for the skeletal muscle (M) isozyme, and a 6.8-kb mRNA presumably coding for the heart isozyme (5). The L and M isozymes have a common sequence of438 amino acids and differ only at the N terminus. In the L isozyme, the divergent sequence is 32 residues long and includes the serine (Ser-32) phosphorylated by cAMP-dependent protein kinase. In the M isozyme, the N terminus is 10 residues long and is unrelated to the unique part of the L isozyme. Expression of the L, but not the M, isozyme of PFK-2/FBPase-2 is controlled by diet and insulin (6, 7). This gene is 55 kb long and contains 15 exons, 13 of which are common to the two isozymes. Part of this work has been presented in the form of an abstract (9). MATERIALS...

A rat gene encoding heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase

“…When screel:ing the rat genomic library for gene A [4], four overlapping clones (h2, 3, 15 and 16) detected with probe RLZK-8 yielded restriction fragments that were incompatible with the map of gene A and were no longer seen in Southern blots performed at high stringency. The restriction map ofh3 was established by Southern Cross (Fig.…”

“…In genes A and B, the 3 cysteines that are important for PFK-2 activity and the histidine that is phosphorylated in the FBPase-2 reaction (Fig. 1) are located in the corresponding exons [4]. This conserved region of gene B is followed by a 3' end that contains several interesting features (Fig.…”

“…The rat genomic library was screened as described [4]. Southern blots were performed according to [P] and Southern Cross Restriction Mapping (NEN) according to the manufacturer's instructions.…”

“…Three PFK-2/FBPase-2 isozymes have been identified by biochemical and immunological criteria, namely the liver (L), the muscle (M), and the heart (H) types (reviewed in [l]). We have cloned the L-type [2] and the M-type [3] cDNAs and have characterized a 55-kb rat gene (henceforth called gene A) that encodes the corresponding mRNAs by alternative use of 2 promoters [4]. The elucidatisn of the origin of the H-isozyme [4] is important because it appears to replace the L-isozyme in regenerating liver [6] and in some hepatoma cells [7], Moreover, the three PFK-2/FBPase-2 isozymes differ by their sensitivity to cyclic AMP-dependent protein kinase (PKA).…”

“…We have cloned the L-type [2] and the M-type [3] cDNAs and have characterized a 55-kb rat gene (henceforth called gene A) that encodes the corresponding mRNAs by alternative use of 2 promoters [4]. The elucidatisn of the origin of the H-isozyme [4] is important because it appears to replace the L-isozyme in regenerating liver [6] and in some hepatoma cells [7], Moreover, the three PFK-2/FBPase-2 isozymes differ by their sensitivity to cyclic AMP-dependent protein kinase (PKA). PKA-dependent phosphorylation inactivates PFK-2 and activates FBPase-2 in the L-isozyme, while it activates PFK-2 in the H-isozyme.…”