Identification of trans‐acting factors interacting with the positive regulatory element spanning −148 to −124 region of c‐jun.

Abstract

c-Jun is one of the major component of the transcription factor AP-1 and involved in regulation of the expression of genes indispensable for cell proliferation and differentiation. Earlier reports from our laboratory have identified a positive regulatory element located between −148 to −124 (Jun25) region of the c-jun that is differentially recognized in normal and proliferating rat liver. The factor interacting with this region is present in the cytosol and undergoes post-translational modification and nuclear translocation upon growth stimulus. To identify the factors interacting with this region, EMSA using rat liver cytosolic extract (RCE) and nuclear extract (RNE) was performed. A slow-migrating complex was obtained with RCE when compared with that obtained with RNE. DNA-affinity purification of factors from the RCE and RNE, and their identification by MALDI were carried out. Two protein bands of ~ 42 and 70 kDa in the affinity purified RCE were identified to beγactin and HSP70, respectively. Western blot analysis using anti-actin antibody confirmed the ~42 kDa band to be actin. A single band of ~40 kDa obtained in the affinity purified RNE was identified to be nuclear actin. Southwestern blot analysis of the affinity purified RCE showed that only ~42 kDa band was recognized with the Jun-25 oligonucleotides. Thus, actin directly interacts with the positive regulatory element Jun25 of the c-jun and is likely to be involved in its positive regulation.