Cloning of cDNA Encoding for a Novel Isozyme of Fructose 6-Phosphate,2-Kinase/Fructose 2,6-Bisphosphatase from Human Placenta

Abstract

Two independent cDNA clones encoding fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase were isolated from a human placental cDNA library. The deduced amino acid sequences showed that one of the clones, 2K-1, was almost identical to the rat testis isozyme and the other, 2K-3, was different from any known isozymes expressed in mammalian tissues. The results of Southern blot analysis suggested that clones 2K-1 and 2K-3 were encoded as single copy genes and located in different parts of the genome. Since open reading frames of the cDNA clones were not complete, we obtained the 5'-end of the clone 2K-3 cDNA using the 5'-rapid amplification of cDNA end method. The entire cDNA (HP; 1,756 bp) had a coding capacity of 519 amino acids (M(r) = 59,410), and putative phosphorylation sites for protein kinases A and C on the C terminus. Northern blot analysis using a fragment of the HP as a probe showed that a major band of 5.4 kb, significantly different in size from known isozyme mRNAs such as liver (2.1 kb), muscle (1.9 kb), heart (4.0 kb), and testis (2.0 kb), was present in poly(A)+RNA preparations of human first trimester and term placentae. These results strongly suggested that this 5.4 kb mRNA codes a novel isozyme of fructose 6-phosphate,2-kinase/fructose 2,6-bisphosphatase.

An inducible gene product for 6-phosphofructo-2-kinase with an AU-rich instability element: Role in tumor cell glycolysis and the Warburg effect

“…1). The amino acid sequence deduced from the ORF was found to share 98% identity with the sequence predicted from a shorter length cDNA clone obtained recently from human placenta (19) and 66% identity with human liver PFK-2 (12). The major difference between the regions encoded by the AU-containing PFK-2 cDNA and the placental PFK-2 sequence was in the length and composition of the carboxy terminus, suggesting that the two cDNA clones may represent alternatively spliced variants of one gene.…”

“…AF056320). (B) Predicted amino acid sequence and alignment of the PFK-2 cDNA with PFK-2 sequences deduced from a human placental (19) and a human liver (12) cDNA clone.…”

Ras transformation requires metabolic control by 6-phosphofructo-2-kinase

“…The enzymes derived from PFKFB1, PFKFB2 and PFKFB4 were originally found to be expressed by cells of the liver/muscle, kidney/heart and testes, respectively, and all display nearly equal kinase: phosphatase ratios (Okar and Lange, 1999). The PFKFB3 gene encodes for the inducible PFK2/FBPase (Chesney et al, 1999;Atsumi et al, 2002;Mahlknecht et al, 2003), which is rapidly induced by inflammatory stimuli (Chesney et al, 1999) and hypoxia (Minchenko et al, 2002;Minchenko et al, 2004) and has also been termed placental PFK2 (Sakai et al, 1996;Sakakibara et al, 1999), ubiquitous PFK2 (Manzano et al, 1998;Kessler and Eschrich, 2001;Navarro-Sabate et al, 2001) and PGR1 (Hamilton et al, 1997)). The PFKFB3 protein product was recently documented to be overexpressed in the neoplastic cells of human solid tumors, including lung, breast, prostate and colon tumors and to display minimal phosphatase activity (kinase:phosphatase ratio 740:1), suggesting a constitutively pro-glycolytic activity (Sakakibara et al, 1997;Chesney et al, 1999;Chesney and Bucala, 2001;Atsumi et al, 2002).…”

Tissue-specific Structure/Function Differentiation of the Liver Isoform of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase

“…An elevated cellular concentration of Fru-2,6-P 2 increases glycolytic flux, whereas a lowered concentration of Fru-2,6-P 2 results in a net increase in gluconeogenic flux. Because the liver is the major organ for vertebrate glucose homeostasis, regulation of the activity in the liver isoform of 6-Fru(P)-2-kinase/Fru-2,6-P 2 ase and the resulting Fru-2,6-P 2 regulates glucose homeostasis in an organism (1)(2)(3)(4)(5). Reflecting the diversity of tissues and their functions, at least six different tissue-specific isoforms from five distinct genes express varying ratios of kinase to bisphosphatase activity.…”

“…Reflecting the diversity of tissues and their functions, at least six different tissue-specific isoforms from five distinct genes express varying ratios of kinase to bisphosphatase activity. A single isoform generally predominates in each distinct tissue (1,4).…”