Abstract
Hexokinases catalyze the phosphorylation of glucose and initiate cellular glucose metabolism. Hexokinase II (HKII) is the principal hexokinase isoform in skeletal muscle, heart, and adipose tissue. Isoproterenol and exogenous cyclic AMP (cAMP) increase HKII gene transcription in L6 myotubes. Various segments of the HKII promoter that direct the expression of the chloramphenicol acetyltransferase reporter gene were transfected into L6 myotubes to identify basal and cAMP response elements. The 5'-flanking region that extends 90 base pairs upstream of the transcription start site includes a CCAAT box and a cAMP response element (CRE); both contribute to basal promoter activity and each provides an independent, maximal response to cAMP. An inverted CCAAT motif, or Y box, located just upstream of the CCAAT box, contributes to basal promoter activity but is not involved in the cAMP response. Homo- and heterodimers composed of the CRE-binding protein and activating transcription factor-1 bind specifically to the CRE. The Y box and the CCAAT box specifically bind the factor NF-Y (also known as CBF).
Highlights
The four mammalian hexokinases (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1; designated HKI to HKIV)1 convert glucose to glucose 6-phosphate
Effect of 5Ј Deletion Mutations on the Basal Promoter Activity and protein kinase ␣-catalytic subunit (PKA) Inducibility—We cloned about 5.5 kb of 5Ј-flanking sequence of the rat Hexokinase II (HKII) gene, sequenced the proximal 1176 bp [6], and constructed a series of 5Ј deletion mutants that consist of the HKII promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene
Nonimmune serum had no effect on the complex formation by any of the probes (Fig. 7, control lanes). These results suggest that the HKII CCAAT box and Y box bind the transcription factor NF-Y and that this factor may be important for both the basal and cyclic AMP (cAMP)-inducible expression of the HKII gene
Summary
The four mammalian hexokinases (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1; designated HKI to HKIV) convert glucose to glucose 6-phosphate (for review see Ref. 1). The rate of HKII gene transcription is increased by insulin, catecholamines, and cyclic AMP (cAMP) in L6 myotubes [6, 8], and this induction results in increased HKII mRNA, protein synthesis, and glucose phosphorylation This is interesting in view of observations regarding the regulation of hexokinases in various physiologic circumstances. Skeletal muscle glucose utilization is increased by exercise [9], an effect that can be explained in part by the observation that a single bout of acute exercise increases HKII gene transcription, mRNA, and protein in rat skeletal muscle [10, 71] Other physiologic stimuli, such as cold stress, swimming, or the administration of -adrenergic agonists, increase total hexokinase activity in rat and mouse brown adipose tissue and muscle [11,12,13,14]. Several known transcription factors bind to these sequences, notably CREB and ATF-1 to the CRE and NF-Y to both the Y and the CCAAT boxes
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