Identification of a novel mouse hepatic 52 kDa protein that interacts with the cAMP reponse element of the rat angiotensinogen gene

Abstract

To identify the nuclear protein(s) that interact with the putative cAMP response element (CRE) of the rat angiotensinogen (ANG) gene (i.e. nt 806-779 upstream of the transcriptional start site), mouse liver nuclear proteins were prepared for the present studies. The DNase 1 footprinting protection analysis revealed that nt -799/-788 in the 5ʹ-flanking region of the rat ANG gene are protected by the mouse liver nuclear protein. Gel mobility-shift assays revealed that the addition of the unlabelled DNA fragment, ANG nt -806/-779 competed effectively with the binding of the labelled ANG nt -806/-779 to the mouse liver nuclear proteins but the addition of unlabelled mutants of ANG nt -806/-779 were only weakly effective in competing with the labelled ANG nt -806/-779. The addition of unlabelled CRE of the somatostatin (SOM) gene and the CRE of the tyrosine aminotransferase (TAT) gene was also ineffective in competing with the labelled ANG nt -806/-779. Southwestern blot analysis revealed that the labelled ANG nt -806/-779 interacted with two mouse liver nuclear proteins with apparent molecular masses of 52 and 43 kDa, whereas the labelled SOM-CRE, TAT-CRE and the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene interacted with one molecular species of 43 kDa. The binding of the labelled ANG nt -806/-779 to the 52 kDa protein was effectively competed for by the addition of unlabelled ANG nt -806/-779 but not by unlabelled SOM-CRE, TAT-CRE and PEPCK-CRE. Finally, Western blot analysis revealed that polyclonal antibodies against the CRE-binding protein (CREB) interacted with the mouse liver nuclear 43 kDa protein but not with the 52 kDa protein. These studies demonstrate that the CRE of the rat ANG gene (ANG nt -806/-779) interacts with the 43 kDa CREB and a novel 52 kDa protein from mouse liver. The novel 52 kDa protein is immunologically distinct from the 43 kDa CREB. These studies suggest that the 52 kDa protein might have a role in the expression of the hepatic ANG gene.