Cloning and characterization of a functional promoter of the rat pp120 gene, encoding a substrate of the insulin receptor tyrosine kinase

Abstract

Cloning of the 5'-flanking region of the rat pp120 gene has indicated that it is a housekeeping gene: it lacks a functional TATA box and contains several Sp1 binding sites and multiple transcription initiation sites at nucleotides -101, -71, -41, and -27 spread over a GC-rich area. A fragment between nucleotides -21 and -1609 exhibited promoter activity when ligated in a sense orientation into a promoterless luciferase reporter plasmid and transiently transfected into rat H4-II-E hepatoma cells. 5' progressive deletion and block substitution analyses revealed that the three proximal Sp1 boxes (boxes 3, 5, and 6) are required for basal transcription of the pp120 gene. Promoter activity was stimulated 2-3-fold in response to insulin, dexamethasone, insulin plus dexamethasone, and cAMP. Although unaltered by phorbol esters alone, promoter activity was stimulated 4-5-fold in response to phorbol esters plus cAMP. Several motifs resembling response elements for insulin (in the rat phosphoenolpyruvate carboxykinase gene), glucocorticoids, cAMP, and phorbol esters as well as a number of putative binding sites for activating proteins-1 (Jun/Fos) and -2, and liver-specific factors were detected. The role of these sites in tissue-specific expression of pp120 remains to be investigated.