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Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain

(Requests for offprints should be addressed to I V Baanante; Email: baanantevazquez{at}ub.edu)

To better understand the transcriptional machinery that governs glucokinase (GCK) expression, we have cloned and characterized the proximal promoter region of GCK from gilthead sea bream (Sparus aurata). The 5'-flanking region of GCK was isolated by chromosome walking. SMART RACE-PCR allowed us to locate the transcription start site 98 bp (bp) upstream from the translational start. Transfection analysis in HepG2 cells revealed the presence of a functional promoter in the 1397 bp 5'-flanking isolated fragment (positions –1321 to +76 relative to the transcription start site). Sequential 5'-deletion analysis indicated a core functional promoter for basal transcription within the 288 bp upstream from the transcription start site. Transient transfection experiments performed in HepG2 cells and electrophoretic mobility shift assays denoted that Sp1 binds and transactivates GCK promoter, whereas Sp3 repressed Sp1-mediated activation of GCK by competing for the same binding site. Mutations in the Sp binding site completely abolished the enhancing effect of Sp1. Treatment with insulin stimulated GCK expression, and increased Sp1 levels in S. aurata liver. We propose a new mechanism that involves Sp1 and Sp3 to mediate insulin activation of GCK transcription.

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