cAMP/adenylyl cyclase stimulation promotes 14-3-3 binding to UT-A1 urea transporter.

The 14-3-3 proteins are a group of highly conserved 30-kD regulatory proteins that mainly bind to phosphorylated residues in target proteins. 14-3-3 proteins interact with numerous cellular proteins and influence a large number of cellular processes. UT-A1 urea transporter contains a potential 14-3-3 recognition motif in the large cytoplasmic loop, suggesting a potential role of 14-3-3 in the regulation of UT-A1. To test this model, we examined the expression pattern of seven 14-3-3 protein isoforms in kidney cortex, outer medulla (OM) and inner medulla (IM). Zeta, beta and tau are highly and widely expressed in kidney cortex, OM and IM, as well as in brain, heart and liver. Gamma, eta, and epsilon are found in cortex, OM, and IM, but mainly expressed in IM. There was no detectable sigma in kidney tissue. We then prepared seven 14-3-3 isoform-specific GST fusion proteins for 14-3-3/UT-Al interaction studies. GST pull-down assays with UT-A1-MDCK cell lysate demonstrated that UT-A1 is in a protein complex with selected 14-3-3 isoforms: gamma eta and zeta. R18 peptide, a high-affinity competitive inhibitor of 14-3-3 interactions, prevented UT-A1 from binding to 14-3-3. To examine whether phosphorylation regulates 14-3-3 and UT-A1 binding, UT-A1-MDCK cells were treated with cAMP/adenylyl cyclase stimulator forskolin (10 µM) for 5, 15, and 30 min. Cell lysates were prepared and used for GST 14-3-3 gamma, eta and zeta pull-down assay. Phosphorylation significantly induced UT-A1 binding to 14-3-3. Our study identified 14-3-3 as a novel and potentially important regulator of urea transport activity. [ABSTRACT FROM AUTHOR]