Role of hnRNP K protein in the basal expression of rat ALDH3A1.

摘要

Aldehyde dehydrogenase 3A1 oxidizes hydrophobic aldehydes to their corresponding carboxylic acids. ALDH3A1 is negatively regulated by protein kinase A (PKA) in isolated rat hepatocytes; an ALDH3A1 luciferase reporter gene transfected into HepG2 cells behaved similarly. PKA inhibitors enhanced basal and inducible expression of the native and reporter gene, while activators, forskolin or dibutyryl-cAMP, decreased expression. Sequence analysis of a 66-bp cAMP-responsive region located 1 kB from the promoter revealed no consensus CREB-responsive element. Electrophoretic mobility shift assays (EMSA) demonstrated that DNA-protein complexes with HepG2 nuclear extract increased upon treatment of the cells with forskolin and decreased with H8 (a PKA inhibitor). Binding was specific, since the 66 bp ds-oligonucleotide competed for complex formation. Using EMSA analysis, I identified a 6 bp sequence (CCACCA) capable of forming a DNA-protein complex with rat liver nuclear extracts. EMSAs with oligonucleotides containing the 6 bp sequence did not bind the purified PKA-regulated proteins (CREB, CREM, of ATF-2), but did bind HepG2 or liver nuclear extract. Mutation of this 6 bp sequence resulted in loss of complex formation. Affinity column purification of rat liver nuclear extract using the 6 bp sequence revealed the presence of a single 43 kDa band during SDS-PAGE analysis. Affinity column purified protein was able to form a DNA-protein complex similar in mobility to that obtained from rat liver nuclear extract. Matrix assisted laser desorption ionization time of flight mass spectrometry revealed the presence of the hnRNP K protein. EMSA analysis demonstrated that an oligonucteotide containing the 6 bp sequence did bind purified hnRNP K with similar mobility as rat liver nuclear extract. Our results suggest that the hnRNP K protein bound to the putative HC3 sequence within the 5'-flanking region of rat ALDH3A1 and that this protein is involved in the basal regulation of expression of ALDH3A1. Supported by USPHS grants ES04244.