Regulation of the bHLH transcription factor E2A in epithelial cells by interaction with the Na/K-ATPase beta1 subunit.
The bHLH transcription factor E2A controls proliferation and differentiation in many cell types including kidney epithelial cells. To identify regulatory binding partners of E2A in the kidney, a yeast two-hybrid assay with a human adult kidney cDNA library was performed. Results demonstrated E2A interactions with other HLH proteins including Id1-3 and Pod1 and the Na/K-ATPase beta1 subunit. The specificity of beta1 subunit binding was confirmed by co-immunoprecipitation of E2A and beta1 subunit deletion constructs in HEK cells demonstrating E2A binding to the cytoplasmic tail of the beta1 subunit. Immunofluorescence and Western analysis of HEK cells co-transfected with GFP-beta1 subunit and E2A demonstrated E2A membrane binding and increased beta1 subunit membrane localization. Increased beta1 subunit expression resulted in decreased nuclear E2A expression and protein half-life and reduced E2A induced gene expression. These results suggest that E2A and Na/K-ATPase beta1 subunit expression in epithelial cells are regulated by interactions between these proteins.
Plotkin
Matthew
M
New York Medical College, Renal Research Division, BSB C06, 95 Grasslands Road, Valhalla, NY 10595, USA. matthew_plotkin@nymc.edu
Pelger
Lex
L
D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques
D051792:Basic Helix-Loop-Helix Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017931:DNA Primers; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase; C503551:ATP1B1 protein, human; C493956:CDKN1A protein, human; C407909:TCF3 protein, human