The second messenger guanosine monophosphate cycle (cGMP) mediates ubiquitination of specific lysine (K) sites on the mouse Na/K/2Cl cotransporter (NKCC2)

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter (NKCC2). Nitric Oxide and atrial natriuretic peptide decrease NaCl absorption in the TAL by increasing the second messenger cyclic guanosine monophosphate (cGMP). NKCC2 is constitutively ubiquitinated, and cGMP enhances the rate of ubiquitination. We previously showed that the cGMP-dependent increase in NKCC2 ubiquitination is mediated by the CRL (Cullin-Ring-E3 ubiquitin Ligase) complex, which is the largest family of E3-ubiquitin ligase in mammals. Post-translational modifications (like ubiquitination) occur in specific amino acid residues. To date, there is no study that focuses on the role that ubiquitination has on NKCC2 activity nor the lysine involved in NKCC2 regulation. Therefore, we hypothesized that the second messenger cGMP stimulates ubiquitination of NKCC2 in specific lysine residues. Protein quantification and LC-MS/MS combined with Parallel Reaction Monitoring was used to quantify post-translational modifications of peptides by ubiquitin in mice. We first study whether cGMP stimulates ubiquitination at lysine residue K48 is a signal for proteasomal degradation. We found that cGMP produces an increase in the K48-linked poly-ubiquitination on NKCC2 in a concentration-dependent manner (baseline: 100.0%; db-cGMP 100 μM: 115.3 ± 3.7%; db-cGMP 250 μM: 127.8 ± 5.2%; db-cGMP 500 μM: 148.3 ± 18.2% p< 0.05, p< 0.05, n = 6). These data indicates that cGMP is part of the signaling cascade that stimulates NKCC2 ubiquitination and degradation by the proteasome. To study ubiquitination of NKCC2, we generated a synthetic peptide (LIFAGKggQLEDGR) in which a heavy isotope leucine (L) was introduced, which mimics chemically and chromatographically the endogenous tryptic K48 peptide (LIFAGKggQLEDGR). The synthetic peptide function as internal standard control for quantification in TALs using a Parallel Reaction Monitoring (PRM) approach using acute ubiquitin quantification (AQUA). We found that 19 out of the 86 lysines in the NKCC2 sequence are ubiquitinated under baseline conditions. To date, it is unknown whether these ubiquitinated lysine (K) contribute to the activity and stability of NKCC2. We found that cGMP stimulates ubiquitination on the peptide LHESHKDLTTAEKLKRE of mouse NKCC2 (Basal = 100% ± 60.5, db-cGMP 500 µM = 456% ± 119, p< 0.05, n = 4). We conclude that the cGMP-dependent increase in NKCC2 ubiquitination is in part mediated by the ubiquitination of the lysine at LHESHKDLTTAEKLKRE in NKCC2 mouse sequence. To the best of our knowledge, this is the first evidence showing the posttranslational modification in which the second messenger cGMP acts and inhibits NKCC2 trafficking and activity in native TALs. As perspective, NKCC2 has been linked to the development of salt-sensitive hypertension, characterization of new regulatory mechanism for NKCC2 may lead to new therapeutic strategies for the treatment of hypertension. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.