INTERACTION OF THE TRANSCRIPTION FACTOR SP1 WITH THE NUCLEAR-PORE PROTEIN P62 REQUIRES THE C-TERMINAL DOMAIN OF P62

The transcription factor Spl plays an important role in the expression of many cellular genes. In studies of proteins that associate with Sp l, a 62-kDa glycoprotein was found in immunoprecipitates of Spl. This protein was detected in these immunoprecipitates by the monoclonal ant ibody, RL2, which was originally raised against nuclear pore proteins but was subsequently found to recognize an epitope that contains O-lin ked N-acetylglucosamine (O-GlcNAc). The association of this protein wi th Spl could be blocked by SDS denaturation of the protein complex. We stern blot analysis of the Sp1 immunoprecipitate using antibodies to p 62 nucleoporin indicated that this nuclear pore protein associates wit h Spl. Furthermore, immunoprecipitation of p62 nucleoporin resulted in the coprecipitation of Spl. Recombinant p62, expressed as a GST-fusio n protein using a vaccinia virus system, also interacted with both rec ombinant and native Spl. This interaction between p62 and Sp I require d the C-terminus of p62 and the C-terminus was able to bind Spl, albei t less efficiently than native p62. A mammalian two-hybrid interaction assay was devised in which p62 was fused to the Gal4 DNA-binding doma in. This system also indicated that p62, through its C-terminus, inter acts with Spl in the living cell. We propose that this interaction of a nuclear pore protein With Sp1 may reflect the nuclear organization r equired to bring transcribable DNA in contact with the transcription f actors. (C) 1998 Wiley-Liss, Inc.