The N-terminal fingers of chicken GATA-2 and GATA-3 are independent sequence-specific DNA binding domains.

The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo.     

Characterization of cAMP-dependent proteolysis of GATA-6

Cyclic AMP-dependent proteolysis of GATA-6(Delta50) was characterized using inhibitors for intracellular signaling pathways. Among these kinase inhibitors, only H-89 and K252a inhibited the proteolysis induced by dbcAMP, a membrane permeable cAMP analogue, others such as PD98059, SB203580, calphostine C, PP1, and KN-93 did not do so. These results suggest that A-kinase, but not C-kinase, MEK, P38 MAP-kinases or Src kinase, could participate in the observed phenomenon. We further demonstrated that an inhibitor for ubiquitin isopeptidase (Delta12-PGJ2) inhibited the degradation of GATA-6(Delta50) in the presence of dbcAMP, suggesting that the cAMP-dependent proteolysis could be mediated through the ubiquitin-proteasome pathway, although proteasome activity did not change significantly during dbcAMP treatment. The full-length GATA-6 was also responsive to the induced degradation. Furthermore, mutation of a potential phosphorylation site (Ser-290-->Ala) for A- and C-kinases, 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.