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 5

Regulation of CFTR by the WNK/SPAK and IRBIT/PP1 pathways.

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Regulation of CFTR by the WNK/SPAK and IRBIT/PP1 pathways. (A and B) Sea...
(A and B) Sealed ducts were treated with scrambled siRNA (black, blue) or WNK (red) siRNA and incubated for 5 minutes with 3 nM tautomycin (blue). CFTR activity was measured with MQAE as NO3–/Cl– exchange activity. B shows mean ± SEM (n = 3, *P < 0.01). (C and D) CFTR current was measured in HEK cells transfected with CFTR and the indicated IRBIT, WNK, and SPAK combinations or treated with 3 nM tautomycin for 5 minutes. D shows mean ± SEM (n = 4–17; *P < 0.01; #P < 0.05 versus control). Currents were normalized to cell capacitance before averaging. (E and F) Mutual interaction of IRBIT and SPAK with CFTR, as measured by the reciprocal coimmunoprecipitation assays. In F, asterisks in lane 4 indicate expression of 4 times excess SPAK relative to IRBIT and in lane 5 expression of 4 times excess IRBIT relative to SPAK. (G) HEK cells expressing CFTR and the indicated combinations of IRBIT, WNK1, WNK4, and SPAK constructs were used to test their effect on surface expression of CFTR by biotinylation. (H) Inhibition of CFTR surface expression by WNK4, WNK1, and WNK11–119 and its reversal by SPAKKD and IRBIT. (I) HEK cells expressing CFTR and the indicated combinations of IRBIT, IRBITI42F44/AA, PP1, SPAK, and inhibitor-2 were used to measure CFTR phosphorylation, as described in Figure 3 for NBCe1-B. (J) HEK cells expressing CFTR and cotransfected with IRBIT or IRBITI42F44/AA were used to show IRBIT-mediated recruitment of the native PP1 to CFTR. Averages for G and H are given in Supplemental Figure 4B and for I in Supplemental Figure 5B.