TY - JOUR
T1 - WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells
AU - Cai, H.
AU - Cebotaru, V.
AU - Wang, Y. H.
AU - Zhang, X. M.
AU - Cebotaru, L.
AU - Guggino, S. E.
AU - Guggino, W. B.
N1 - Funding Information:
This work was supported by American Heart Association 0530222N (HC), National Institutes of Health Grants DK068226-01A1 (HC), and DK32753 (WBG). We thank Drs Xuhang Li, Peying Fong, Anne Fischer, and Deanne Dryciw for their helpful suggestions and discussions in this research projects. We also thank Dr Sang-Ho Kwon for the domain analysis of WNK4.
PY - 2006/6/3
Y1 - 2006/6/3
N2 - Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9±6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9±5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2±11.8% vs 100%, P<0.05, n=6). The reduction of NCC surface expression by WNK4 WT (62.9±3.3% of control group) is not altered by WT dynamin ((61.8±3.7% (P=NS)) or its mutant K44A ((65.4±14.1% (P=NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.
AB - Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9±6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9±5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2±11.8% vs 100%, P<0.05, n=6). The reduction of NCC surface expression by WNK4 WT (62.9±3.3% of control group) is not altered by WT dynamin ((61.8±3.7% (P=NS)) or its mutant K44A ((65.4±14.1% (P=NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.
KW - Cotransporter
KW - Dynamin
KW - Lysosomal pathway
KW - Pseudohypoaldosteronism type II
KW - Sodium chloride
KW - WNK4 kinase
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U2 - 10.1038/sj.ki.5000333
DO - 10.1038/sj.ki.5000333
M3 - Article
C2 - 16688122
AN - SCOPUS:33745076253
VL - 69
SP - 2162
EP - 2170
JO - Kidney International
JF - Kidney International
SN - 0085-2538
IS - 12
ER -