Cyclic AMP-dependent proteolysis of GATA-6 was characterized by fusing GATA-6 with the carboxyl-terminal membrane domain of SREBP-2. When the fusion protein was stably expressed in CHO-K1 cells, it was recovered in the ER membrane. This protein was processed in a similar manner to SREBP-2 upon cholesterol starvation, and the GATA-6 moiety moved into the nucleus. The GATA-6 moiety on the membrane became undetectable in the presence of dbcAMP or cholera toxin. However, H-89, K-252a, MG115 and lactacystin inhibited this decrease, suggesting that the cytoplasmic GATA-6 moiety of the fusion protein was degraded by proteasomes though A-kinase upon elevation of the cellular cAMP concentration.

Cyclic AMP-dependent protein kinase, GATA DNA-binding protein, nuclear-cytoplasmic shuttling, regulated protein degradation, stable transfection, cJun N-terminal kinase Transcription factor GATA6 stably expressed in Chinese hamster ovary (CHO)-K1 cells is exported from the nucleus to the cytoplasm and degraded there by proteasome upon treatment with dibutylylcyclic AMP (dbcAMP), which is a membrane-permeable cyclic AMP (cAMP) analogue. The cAMP-dependent proteolysis of GATA6 was characterized by dissection of the GATA6 protein into a zinc-finger domain (Zf) and the surrounding region (ΔZf). These segments were separately expressed in CHO-K1 cells stably, and followed by treatment with dbcAMP. The nuclear localized Zf was degraded by proteasome similarly to the full-length GATA6. Site-directed mutants of nuclear localizing signal (NLS) ( 345 RKRKPK 350 → AAAAPK and AAAAPA) and closely related GATA4 showed the same behavior. Although nuclear-localized ΔZf was degraded by proteasome, the cytoplasmic-located ΔZf was resistant to proteolysis in contrast to the NLS mutants. We also searched for a potential NLS and nuclear export signal (NES) with computational prediction programs and compared the results with ours. All these results suggest that the amino acid sequence(s) of the Zf of GATA6 is responsive to cAMP-dependent nuclear export and proteolysis.

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