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 4

PP1 mediates all effects of IRBIT.

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PP1 mediates all effects of IRBIT. (A and B) Sealed pancreatic ducts wer...
(A and B) Sealed pancreatic ducts were treated with vehicle or with 3 nM tautomycin (Tauto) for 10 minutes, and Na+-HCO3– cotransport activity was measured. B shows mean ± SEM (n = 3, *P < 0.001). (C and D) HeLa cells transfected with NBCe1-B and vector (black), PP1 (red), inhibitor-2 (I-2; green), or inhibitor-2 plus IRBIT (blue). Cells expressing NBCe1-B were also transfected with IRBIT (bars 2 and 6), IRBITI42F44/AA (bar 8), and 3XIRBITI42F44/AA (bar 9) and treated with 3 nM tautomycin for 10 minutes (bars 4 and 6). D shows mean ± SEM of Na+-HCO3– cotransport (n = 4–8, *P < 0.01). (E) HEK cells transfected with NBCe1-B and vector, IRBIT, or IRBITI42F44/AA were used to test recruitment of the native PP1 to NBCe1-B. (F) HEK cells transfected with NBCe1-B and the indicated combinations of IRBIT, PP1, inhibitor-2, and SPAK constructs were labeled with 32P for 2 hours. NBCe1-B was immunoprecipitated and NBCe1-B phosphorylation tested by radiography. (G) Model of regulation of NBCe1-B surface expression and activity by the WNK/SPAK and IRBIT/PP1 pathways and the relationship between them. The WNKs function as scaffolds for SPAK, which phosphorylates NBCe1-B to reduce its surface expression, and IRBIT functions as a scaffold for PP1, which dephosphorylates NBCe1-B to restore surface expression. IRBIT also activates NBCe1-B by preventing autoinhibition by NBCe1-B N-terminus. The black lines represent the probes used to test each component of the pathways. Averages for E and F are given in Supplemental Figure 4A and Supplemental Figure 5A, respectively.