Analysis of mDRA (Mouse Down-Regulated in Adenoma)-PP2A Bδ interaction in the yeast two-hybrid system

Abstract

[한글]

[영문]

The expressed recombinant cDNA of mouse Down-Regulated in Adenoma (mDRA) originally proposed as a candidate tumor suppressor mediates DIDS-sensitive, Na^+-independent, electroneutral Cl^-/HCO₃^- exchange (34). Several potential

regulatory sites of nuclear localization signals and numerous putative phosphorylation sites within the COOH-terminus of the deduced amino acid sequence of mDRA prompted us to investigate mDRA-associated regulatory proteins using yeast-based two-hybrid system. Two candidate clones containing mouse cDNA insert of

PAX 3 or PP2A B regulatory subunit delta isoform (PP2A Bδ) were verified to interact with the COOH-end of mDRA. Like rat PP2A Bδ (46), mouse PP2A Bδ homolog mRNAs (2.4 kb) were abundantly expressed in testis and colon, whereas PAX 3 transcripts (6.4 kb) were expressed at high levels in heart. Patterns of tissue

distribution of mouse PP2A Bδ homolog and PAX 3 were similar to those of mDRA. The functional relevance of PP2A Bδ to mDRA as a negative regulator of tumorigenesis led us to clone the full-length of mouse PP2A Bδ homolog cDNA. The full-length open reading frame as well as 31 nucleotides of 5'-untranslated and 565 nucleotides of 3'-untranslated were obtained using an reverse transcription-polymerase chain reaction (RT-PCR) of mouse colon cDNA. The mouse PP2A Bδ homolog cDNA encodes a protein of 453 amino acids with a calculated Mr of 51,954 (accession number

AF366393). An amino acid alignment of mouse PP2A Bδ reveals 96％ identity with rat PP2A Bδ. The heterologous functional expression of this full-length PP2A Bδ cDNA along with the core subunits of A and C in the mDRA stable transfectants will allow

us to study how PP2A holoenzyme binding to mDRA can modulate mDRA function.

Taken all together, the identification and characterization of the associating proteins with mDRA, suggestive of potential regulatory proteins can be the essential study to elucidate the regulatory mechanism of mDRA function.