An SP/TP motif in the N‐terminal cytoplasmic domain of UT‐A3 as potential activation site of UT‐A3‐mediated urea transport

Abstract

The UT‐A protein variant UT‐A3 possesses in its N‐terminal cytoplasmic domain two SP sites, which are potential phosphorylation sites by proline‐directed kinases. In rat UT‐A3, they are located at positions 15/16 and 35/36. The purpose of the experiments was to test if these sites are involved in the cAMP‐dependent activation of urea influx in UT‐A3‐expressing Xenopus oocytes. By mutating two consensus phosphorylation sites for protein kinase A (S85 and S92 of mouse UT‐A3), Smith and coworkers (Smith CP et al, AJP Cell 287:C1087, 2004) had shown that these PKA sites were not responsible for the observed cAMP dependence of UT‐A3 in oocytes. We prepared N‐terminal deletion constructs of UT‐A3 in which the deleted sequence was replaced by a start codon. Thus these constructs started with a methionine either at positions 18 (del18), 35 (del35) or 53 (del53). When expressed in Xenopus oocytes, UT‐A3‐del18 had the same urea transport activity and cAMP stimulation as wild‐type UT‐A3. However, UT‐A3‐del35 and UT‐A3‐del53, while exhibiting the same unstimulated urea transport activity, were not stimulated by cAMP treatment. To test the hypothesis that the SP35 site is involved in the activation of UT‐A3, we mutated the S15 and S35 either alone or together. As expected, UT‐A3‐S15A behaved like wild‐type UT‐A3 whereas S35A and S15A/S35A were no longer stimulated by cAMP treatment. These data suggest that SP35 is responsible for the activation of UT‐A3 in oocytes. They are also consistent with the findings of the proteomic study of Hoffert et al. (PNAS 103:7159, 2006) who found that S35 was phosphorylated in response to vasopressin in the renal inner medulla. This research was supported by NIH grant DK41707 and the Emory University Research Council