Isolation of CHO-K1 clones defective in cAMP-dependent proteolysis, as determined by the stability of exogenously expressed GATA-6.
Degradation of the GATA-6(Delta50) protein expressed in a CHO-K1 clone (tc1-17a) is stimulated in the presence of dbcAMP through proteasome without new protein synthesis [FEBS Lett. 408 (1997) 301], whereas the intrinsic GC-box-binding protein was stable. To examine the cellular mechanism responsible for this specific degradation of GATA-6(Delta50), we initially introduced the blasticidin-S deaminase gene carrying a promoter with GATA motifs that are recognized by GATA-6. The resulting cell line (tc2G2) grew in the presence of blasticidin S. However, the presence of both blasticidin S and dbcAMP was lethal due to degradation of GATA-6. Cells resistant to such lethality were isolated by chemical mutagenesis. The GATA-6(Delta50) in these resistant cells was stable in the presence of dbcAMP in contrast to that in the parent tc2G2 cells, as determined by gel-mobility shift analysis and Western blotting. These clones could be beneficial for identification and characterization of the components participating in the signaling pathway for both protein degradation and cAMP-dependent biological processes.
Maeda
Masatomo
M
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan. mmaeda@phs.osaka-u.ac.jp
Ishida
Akiko
A
Ni
Lin
L
Kobayashi
Ayako
A
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003994:Bucladesine / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D050993:GATA6 Transcription Factor; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009705:Nucleosides / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D051381:Rats; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D050993:GATA6 Transcription Factor; D009705:Nucleosides; D011506:Proteins; D014157:Transcription Factors; D003994:Bucladesine; C004500:blasticidin S; D000242:Cyclic AMP