Proteomic identification of proteins associated with the osmoregulatory transcription factor TonEBP/OREBP: functional effects of Hsp90 and PARP-1

Hypertonicity ( e. g., high NaCl) activates the transcription factor tonicity- responsive enhancer/ osmotic response element- binding protein ( TonEBP/ OREBP), increasing transcription of protective genes. In the present studies, by stably expressing amino acids 1- 547 of TonEBP/ OREBP in HEK 293 cells and immunoprecipitating it plus associated proteins from the nuclei of cells exposed to high NaCl, we identify 14 proteins that are physically associated with TonEBP/ OREBP. The associated proteins fall into several classes: 1) DNA- dependent protein kinase, both its catalytic subunit and regulatory subunit, Ku86; 2) RNA helicases, namely RNA helicase A, nucleolar RNA helicase II/ Gu, and DEAD- box RNA helicase p72; 3) small or heterogeneous nuclear ribonucleoproteins ( snRNPs or hnRNPs), namely U5 snRNP- specific 116 kDa protein, U5 snRNP- specific 200 kDa protein, hnRNP U, hnRNP M, hnRNP K, and hnRNP F; 4) heat shock proteins, namely Hsp90 beta and Hsc70; and 5) poly( ADP- ribose) polymerase- 1 ( PARP- 1). We confirm identification of most of the proteins by Western analysis and also demonstrate by electrophoretic mobility- shift assay that they are present in the large complex that binds specifically along with TonEBP/ OREBP to its cognate DNA element. In addition, we find that PARP- 1 and Hsp90 modulate TonEBP/ OREBP activity. PARP- 1 expression reduces TonEBP/ OREBP transcriptional activity and the activity of its transactivating domain. Hsp90 enhances those activities and sustains the increased abundance of TonEBP/ OREBP protein in cells exposed to high NaCl.