Image_2_Epithelial Na+ Channel: Reciprocal Control by COMMD10 and Nedd4-2.TIFF

<p>Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney’s long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2’s ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.</p

Image_9_Epithelial Na+ Channel: Reciprocal Control by COMMD10 and Nedd4-2.TIFF

<p>Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney’s long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2’s ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.</p

Image_8_Epithelial Na+ Channel: Reciprocal Control by COMMD10 and Nedd4-2.TIFF

<p>Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney’s long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2’s ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.</p

Image_4_Epithelial Na+ Channel: Reciprocal Control by COMMD10 and Nedd4-2.TIFF

<p>Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney’s long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2’s ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.</p

Image_3_Epithelial Na+ Channel: Reciprocal Control by COMMD10 and Nedd4-2.TIFF

<p>Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney’s long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2’s ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.</p

Image_6_Epithelial Na+ Channel: Reciprocal Control by COMMD10 and Nedd4-2.TIFF

<p>Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney’s long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2’s ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.</p

Nedd4-2 and Nedd4 WW-domains interact with SGK <i>in vitro</i>.

<p>(A) ΔN60SGK-HA and Nedd4-2_C821A–FLAG were transiently co-expressed, or expressed alone as indicated, in COS7 cells. Complexes were immunoprecipitated with anti-HA antibody. Immunoprecipitates were analyzed by 10% SDS-PAGE and sequential western blotting (IB) with anti-FLAG antibody (Nedd4-2) and anti-HA antibody (ΔN60SGK). Expression of the proteins (5% input) is shown in the bottom panel, n = 3. Either ΔN60SGK-HA (B, E) or SGK-FLAG (C, D) was transiently expressed in COS7 cells. Cells were lysed 48 h after transfection with TBS +1% Triton-X-100. The lane labelled “lysate” shows expression of SGK in whole cell lysate. COS7 cell lysates were incubated with the indicated Nedd4-2 or Nedd4 WW-domain GST-fusion proteins, or GST alone, to test for interaction with SGK. Bound SGK was analyzed by immunostaining with anti-FLAG/HA antibody. The bands indicate that WW-domains 2 and 3 of Nedd4-2, and WW-domains 1 and 3 of Nedd4 interact with SGK, n = 7. The two SGK bands observed in some experiments are likely to be unphosphorylated and phosphorylated forms of SGK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012163#pone.0012163-Wang1" target="_blank">[8]</a>.</p

Binding affinities of the WW-domains of Nedd4 and Nedd4-2 to PY motif peptides from SGK and βENaC.

<p>Values are mean ± S.E, n = 3–4.</p

SGK_S422D binding to Nedd4-2 and stimulation of ENaC is enhanced compared to wildtype SGK.

<p>(A) SGK-FLAG or SGK_S422D-FLAG were expressed in COS7 cells. After 48 hr cells were lysed, pre-cleared with GST bound to glutathione-Sepharose beads and then incubated with 50 µg of GST fusion proteins WW-domain 2, WW-domain 3, WW-domains 2 and 3 of Nedd4-2, or GST alone (left) as indicated. After washing the beads, the bound proteins were eluted and analyzed by SDS-PAGE and western blotting with anti-FLAG. The right panel shows interaction of SGK with the WW-domains, and also the interaction of SGK_S422D with the WW-domains. (B) The resulting bands were quantified by densitometry and normalized to total expression of either SGK or SGK_S422D. Bars represent mean +/− S.E. (n = 9 for WW2 and WW3, n = 6 for WW2–3). (C) Relative amiloride-sensitive short-circuit sodium current in FRT cells after transfection with α-, β- and γENaC together with SGK-HA or SGK_S422D-HA under serum (FCS) or serum-free conditions, n = 4. ***indicates <i>P</i>&lt;0.001, **indicates <i>P</i>&lt;0.01 and *indicates <i>P</i>&lt;0.05, analysis of variance.</p

The SGK PY motif is necessary for SGK to inhibit Nedd4-2.

<p>Amiloride-sensitive short-circuit sodium current (relative to 0 µg of Nedd4-2) in FRT cells 48 hr after transfection with α-, β- and γENaC and SGK (closed circles) or SGK_Y298A (open circles) or GFP (closed squares), in the presence of increasing amounts of Nedd4-2, n = 18. Nedd4-2 is expressed as a fraction of the amount of ENaC cDNA. Expression of SGK and SGK_Y298A was reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012163#pone-0012163-g001" target="_blank">Figure 1B</a> of Snyder <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012163#pone.0012163-Snyder5" target="_blank">[19]</a>.</p