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 Hsp90beta 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.
Chen
Ye
Y
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Schnetz
Michael P
MP
Irarrazabal
Carlos E
CE
Shen
Rong-Fong
RF
Williams
Chester K
CK
Burg
Maurice B
MB
Ferraris
Joan D
JD
D000449:Aldehyde Reductase / Q000235:genetics; D016227:Benzoquinones / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D050485:GABA Plasma Membrane Transport Proteins; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism* / Q000502:physiology; D006360:Heat-Shock Proteins / Q000378:metabolism; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D020365:RNA Helicases / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014162:Transfection; D014882:Water-Electrolyte Balance
D016227:Benzoquinones; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D050485:GABA Plasma Membrane Transport Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D047029:Lactams, Macrocyclic; C118125:NFAT5 protein, human; D011506:Proteins; D014157:Transcription Factors; C072207:betaine plasma membrane transport proteins; D012965:Sodium Chloride; D000449:Aldehyde Reductase; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase; D020365:RNA Helicases; C001277:geldanamycin