IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway
Dongki Yang, Qin Li, Insuk So, Chou-Long Huang, Hideaki Ando, Akihiro Mizutani, George Seki, Katsuhiko Mikoshiba, Philip J. Thomas, Shmuel Muallem
Dongki Yang, Qin Li, Insuk So, Chou-Long Huang, Hideaki Ando, Akihiro Mizutani, George Seki, Katsuhiko Mikoshiba, Philip J. Thomas, Shmuel Muallem
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Research Article

IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway

Abstract

Fluid and HCO3– secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO3– in secretory glands is fueled by Na+/HCO3– cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl–/HCO3– exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP3) receptor–binding protein released with IP3 (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO3– secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase–regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.

Authors

Dongki Yang, Qin Li, Insuk So, Chou-Long Huang, Hideaki Ando, Akihiro Mizutani, George Seki, Katsuhiko Mikoshiba, Philip J. Thomas, Shmuel Muallem

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Figure 3

The WNK/SPAK pathway inhibits and IRBIT reverses inhibition of NBCe1-B surface expression without dissociating the NBCe1-B–SPAK complex.

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The WNK/SPAK pathway inhibits and IRBIT reverses inhibition of NBCe1-B s...
(A and B) HEK cells were transfected with GFP–NBCe1-B and the indicated WNK and Flag-SPAK constructs. Note that in the last two lanes in A, WNK11–491 was coexpressed with SPAKKD and with IRBIT; and in lanes 5, 6 and 7, 8 in B, WNK1KD and WNK4KD were coexpressed with Flag-SPAKKD and Flag-IRBIT, respectively. In lane 9 Flag-SPAK was coexpressed with Flag-IRBIT. The cells were used to determine total (lower blots) and surface expression (upper blots) of GFP–NBCe1-B by biotinylation. (C and D) HEK cells were transfected with GFP–NBCe1-B and with empty vector, Flag-IRBIT, Flag-SPAK, or Flag-IRBIT+Flag-SPAK and were used to determine the effect of IRBIT and SPAK on their interaction with GFP–NBCe1-B by coimmunoprecipitation assays. Note that IRBIT and SPAK do not affect their mutual interaction with NBCe1-B. NBCe1-B was detected with anti-GFP and SPAK and IRBIT with anti-Flag antibodies. Analysis of multiple experiments and averages are given in Supplemental Figure 3.