Role of the Stimulatory Proteins Sp1 and Sp3 in the Regulation of Transcription of the Rat Pyruvate Kinase M Gene

Abstract

Site-directed mutagenesis of cis-regulatory elements in the 5' flanking region of the rat pyruvate kinase M gene reveakd that two out of the three GC boxes (-133/-124 and -48/-39) are involved in the stimulation of a core promoter (-3511-46). These two regions were also protected in DNaseI footprinting assays. Spl and Sp3 were identified as binding proteins to all three GC boxes by supershift experiments.Cotransfections in Drosophilu SL2 cells revealed a strong stimulatory function of Spl and a synergistic effect of Sp3 to Spl in the activation of the pyruvate kinase M promoter. No inhibitory effect of Sp3 was detected.These data indicate that binding of Spl at two GC boxes is required for full promoter activity of the pymvate kinase M gene and thus contributes to the observed cell-cycle-dependent expression of this enzyme in proliferating rat thymocytes.Keywords: pymvate kinase M ; rat thymocytes; stimulatory protein 1 ; stimulatory protein 3.The four isozyme types of the glycolytic enzyme pyruvate kinase in rat are encoded by two genes. The M gene codes for both the My and the M, type. Tissue-specific synthesis of the two enzymes is due to differential splicing [ l , 21. L-type and Rtype isoforms are also encoded by a single gene through the use of two distinct promoters [3-51. The 5' flanking region of the M gene was cloned and sequenced by Takenaka et al. [6]. This region contains no TATA box and no CAAT box but putative Spl-binding sites. The reported assays of chloramphenicol acetyltransferase (CAT) revealed that a 0.5-kb fragment (Fig. 1) upstream to the transcription start site is sufficient for expression of the gene in dRLh-84 hepatoma cells, whereas no CAT activity in transfected primary hepatocytes could be detected. The authors discussed a role of Spl for the transcriptional regulation of the pyruvate kinase M (PKM) gene deduced from their sequence data without presenting functional evidence. From transient expression analyses of the promoter in dRLh-84 hepatoma cells, Wang et al. [7] suggest the presence of a cluster of three positive cis-acting regions (A, B, C in Fig. 1) within the sequence -279 to -216 which have a role in the regulation of the PKM gene. The authors, however, concluded that the potential Spl-binding sites in the region between -133/-39 identified by DNA sequencing [6] are not involved in the regulation of the gene.In previous studies [8], we analyzed the cell cycle of rat thymocytes by determining biochemical parameters associated with cell cycle progression. These studies revealed a coincidence of the time courses of the activity profiles of the glycolytic enzymes and the rates of DNA, RNA and protein synthesis. The activity and M,-type-specific mRNA levels of pyruvate kinase increased about 10-fold and !+fold, respectively, in the S-phase of stimulated cells compared to resting cells [9, 101. We could show further that glucose is essential for the induction of glycolytic enzymes that provoke a transition to glycolytic energy production in proliferating rat thymocytes [Ill. It...

Sp1 and Sp3 regulate glucokinase gene transcription in the liver of gilthead sea bream (Sparus aurata)

“…The fact that Sp1 binding sites are found in the insulin-responsive regions of numerous genes suggests that Sp1 is involved in mediating insulin action. In particular, Sp1 plays an important role in the transcription of several glycolytic enzymes, such as pyruvate kinase and aldolase , Netzker et al 1997. Here, we found that insulin increased GCK mRNA levels in the liver of S. aurata.…”

Down-regulation of Sp1 Activity through Modulation of O-Glycosylation by Treatment with a Low Glucose Mimetic, 2-Deoxyglucose

“…Sp1 plays a critical role in the transcription of a number of glycolytic enzymes (55,66,67). Thus, it is conceivable that Sp1 and O-GlcNAc function as glucose sensors that link the metabolic activities of a cell to its nutritional condition.…”

Evidence for a Novel TGF-β1−Independent Mechanism of Fibronectin Production in Mesangial Cells Overexpressing Glucose Transporters

“…Western blotting. For Western blotting studies, 6.0 ϫ 10 6 cells were seeded onto 15-cm culture dishes with 15 ml experimental medium and harvested after 12, 24, or 48 h. Nuclear proteins were prepared as recently described (41). Preparation of total, cytosolic, and membrane fraction and detection of PKC isoforms was recently described (40).…”