Cyclic AMP‐dependent proteolysis of GATA‐6 expressed in CHO‐K1 cells

Transcription factor GATA binds to a canonical DNA motif (G/A)GATA(A/T), and regulates the expression of various genes required for developmental processes and tissue‐specific functions. When the rat GATA‐6(50) was stably expressed in CHO‐K1 cells, the protein was specifically degraded upon treatment with dibutyryl cAMP or cholera toxin. Inhibitors for protein kinase A and proteasome inhibited the degradation. The full‐length GATA‐6 was also responsive to the induced degradation. The GATA‐6 has a unique serine residue potentially phosphorylated by protein kinase A. However, mutation of this potential site, and deletion of the PEST sequence of GATA‐6 did not abolish the degradation. All these results suggest that cellular factor(s) may play a crucial role in mediating the activation of the cAMP‐dependent process. To examine the cellular mechanism responsible for this specific degradation of GATA‐6, we initially introduced the blasticidin‐S deaminase gene carrying a promoter with GATA motifs. The resulting cell line 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 in these resistant cells was stable in the presence of dbcAMP. 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. Supported in part by grants from MEXT and JSPS.