Abstract
A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1's role and the mechanism regulating its function in the DCT. We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor-1 (I1), mediates cAMP's effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions. Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation via a protein kinase A (PKA)-dependent pathway. Ex vivo incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC in vitro, and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC. Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA-dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity.
Highlights
Background A number ofcAMP-elevating hormones stimulate phosphorylation of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT)
Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a protein kinase A (PKA)-dependent phosphorylation of I1 and subsequent inhibition of phosphatase 1 (PP1)
This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity
Summary
We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor–1 (I1), mediates cAMP’s effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions. Cells, and Antibodies Unless otherwise stated, reagents were purchased from Sigma Aldrich (Buchs, Switzerland). Calyculin A was purchased from Cell Signaling Technologies (Danvers, MA). The specificity of the antibody was confirmed by immunohistochemistry (Supplemental Figure 1). Rabbit anti-FLAG antibody was purchased from GenScript Rabbit anti-AQP1 antibody was previously described.[32] tNCC and pT58NCC antibodies used to detect calyculin A and endothall stimulation of NCC in MDCK type I cells were previously described (33,34 respectively). Phospho-PKA substrate antibody was purchased from Cell Signaling Technologies Effect of low Cl2 on the stimulation of NCC and SPAK-OSR1 phosphorylation by isoproterenol in kidney slices of WT mouse.
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