WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells
WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells
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DOI：
10.1038/sj.ki.5000333
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发表时间：
2006-06-01
影响因子：
19.6
通讯作者：
Guggino, W. B.
中科院分区：
文献类型：
--
作者：
Cai, H.;Cebotaru, V.;Guggino, W. B.
假性醛固酮减少症II型（PHA II）是由两个WNK（不含赖氨酸(k)）激酶家族成员的突变引起的。WNK4野生型（WT）在爪蟾卵母细胞中表达可抑制氯化钠共转运体（NCC）的活性和表面表达。在这里，我们通过表面生物素化、Western blot、共免疫沉淀（Co-IP）和免疫染色，研究了在WNK4 WT及其突变体E562K和R1185C存在或不存在的情况下，哺乳动物细胞中NCC蛋白的加工。WNK4 WT显著降低Cos-7细胞中NCC表面表达（58.9 +/- 6.8% vs 100%, P < 0.001, n=6），而其突变体E562K对NCC表面表达无显著影响（92.9 +/- 5.3% vs 100%， P=NS, n=6）。另一个突变体R1185C仍然部分降低了NCC的表面表达（76.2 +/- 11.8% vs 100%, P < 0.05, n=6）。WNK4 WT（对照组的62.9 +/- 3.3%）对NCC表面表达的降低没有被WT动力蛋白（61.8 +/- 3.7% (P=NS)）或其突变体K44A （65.4 +/- 14.1% (P=NS)）改变。一项联合ip研究表明，WNK4 WT和WNK4 E562K都与NCC相互作用。此外，质子泵抑制剂巴菲霉素A1部分逆转了WNK4 WT对NCC表达的抑制作用。我们的数据表明，WNK4 WT显著抑制NCC表面表达，这不是由于网格蛋白介导的NCC内吞作用的增加，而可能是由于通过溶酶体途径增强了NCC的降解。
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.