Figure 3. Impact of the p.Y259N mutation on SOX17 mRNA and protein levels. A: Steady-state levels of SOX17 protein are profoundly affected by the p.Y259N mutation. A total of 100, 200, or 300 ng of WT SOX17 or p.Y259N SOX17 expression plasmids were transfected into HEK293T cells and protein lysates were harvested after 48 hr and analyzed by Western blot with an anti-SOX17 antibody, as well as an antitubulin antibody to assess protein loading. Comparison of SOX17-WT and SOX17-p.Y259N protein levels at equal dosage of transfecting DNA (lanes 2 and 5, 3 and 6, 4 and 7) suggests that the mutation increases SOX17 protein stability. Experiment was repeated four times and a representative blot is shown. B: SOX17 mRNA levels measured by quantitative RT-PCR. SOX17 mRNA levels in mock-transfected cells were 0.003 times that measured in cells transfected with 10 ng WT SOX17 (data not shown). N 5 3; P 5 0.001; Error bars represent SEM. C: The effect of the p.Y259N mutation on the transcriptional activity of a SOX17 reporter plasmid. SOX17 expression plasmids were cotransfected with a SOX17-luciferase reporter plasmid. The SOX17-p.Y259N protein showed enhanced activity of the reporter at every transfection dosage, relative to the SOX17-WT protein. Data are shown as ratio of activities of firefly luciferase to control renilla luciferase. N 5 3; Po0.05; Error bars represent SEM. D: The effect of the p.Y259N mutation on the ability of SOX17 to repress Wnt signaling. SOX17 expression plasmids were cotransfected with a TCF-luciferase reporter plasmid (TOP-flash) and an expression plasmid encoding a stabilized form of b-catenin. Repression of Wnt signaling was increased at every dosage with the mutant SOX17 protein, compared to the WT protein. Data are shown as ratio of activities of firefly luciferase to control renilla luciferase. N 5 3; Po0.05; Error bars represent SEM. protein inappropriately suppresses the Wnt signaling pathway during kidney and urinary tract development causing the above- mentioned congenital defects. Discussion A complex mechanism drives the development of the urinary tract in mammals. Both the kidney and urinary tract segments derive from the interaction of two tissue compartments of the embryonic mesoderm, that is, the ureteric bud (UB), derived from the Wolffian duct, and the metanephric mesenchyme (MM), that induces the metanephric mesenchyme to trans-differentiate into nephron epithelia [Vainio et al., 2002; Woolf et al., 2004]. These events are mediated by several soluble factors that act in a cooperative fashion either as pro- or antitubulogenic factors. Among the growing list of such molecules are the members of the fibroblast growth factor (FGF), transforming growth factor (TGF-b), and Wnt families, as well as GDNF, HGF, and EGF [Karihaloo et al., 2005]. b-Catenin is a key component of the canonical Wnt signaling pathway. Several Wnts are expressed during kidney development, and activation of the Wnt pathway can result in localization of b-catenin to the nucleus where it activates a pattern of gene expression that is required for normal nephron development [Karihaloo et al., 2005]. In fact, inhibiting b-catenin/ Wnt signaling in the developing Wolffian duct results in mice with ectopic ureters and renal malformations [Marose et al., 2008]. We report here the identification of a novel heterozygous amino acid substitution in SOX17 in two families and several other subjects with urinary tract malformations. Our results show that the p.Y259N mutation causes an increase in SOX17 protein levels in cultured kidney fibroblast cells. Tyrosine-259 is one of four putative tyrosine-phorphorylation sites, as predicted by analysis of SOX17 primary sequence [Blom et al., 1999]. The interactions between protein phosphorylation and the ubiquitin–proteasome system (UPS) are well documented [Gao and Karin, 2005; Hunter, 2007]. Specifically, a single phosphorylated residue can either target a protein for degradation or prevent its degradation by the UPS, depending on the protein being studied. We therefore HUMAN MUTATION, Vol. 31, No. 12, 1352–1359, 2010 1357