Regulation of NKCC2 Ubiquitination by the Skp1‐Cullin1‐FBox Complex in Thick Ascending Limbs

Abstract

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 thick ascending limb (TAL) by increasing the second messenger cyclic guanosine monophosphate (cGMP). We recently showed that NKCC2 is constitutively ubiquitinated, and that cGMP enhances the rate of ubiquitination. Ubiquitination is a post‐translational modification mediated by the ubiquitin‐activating enzyme (E1), the ubiquitin‐conjugating enzyme (E2), and the ubiquitin ligase (E3). The SCF (Skp1, Cullin‐1, F‐box protein) complex, is the largest family of E3 ubiquitin ligases in mammals. Therefore, we hypothesized that the SCF complex mediates the cGMP‐stimulated NKCC2 ubiquitination in rats TAL. To test our hypothesis we first tested whether we could inhibit neddylation of cullin‐1 with the inhibitor MLN4924 (1 μM). TALs samples were incubated for 60 minutes at 37°C in the presence or absence of the inhibitor, samples were lysed and we measured neddylated cullin‐1 by Western blot. As expected, neddylation of cullin‐1 was blunted in MLN4924 treated samples (baseline: 100%; MLN4924: 11.0 ± 3.5%, p<0.05). We then study whether inhibition of cullin‐1 inhibits the cGMP‐stimulated NKCC2 ubiquitination. TALs suspension from Sprague‐Dawley rats were aliquoted in 4 samples in the presence of the proteasomal inhibitor (MG132 20μM) to stop protein degradation. Then aliquots were incubated at 37°C in the presence or absence of MLN4924 for 10 minutes, and treated with vehicle or db‐cGMP 500 μM at 37°C for an extra 50 minutes. We found that blocking neddylation of cullin‐1, inhibits the cGMP‐stimulated NKCC2 ubiquitination (baseline: 100%; db‐cGMP 500μM: 162.5 ± 10.3%; MLN4924: 75.1 ± 20.2%; MLN4924 + db‐cGMP 500μM: 96.8 ± 19.6%, p<0.05). To further test our hypothesis, we generated a peptide containing the conserved sequence of F‐Box proteins, called the F‐Box domain, to compete for the binding site between native F‐Box proteins to the SCF complex. We used a chariot to facilitate the delivery of the peptide into the TAL cells. We found that the F‐Box peptide binds to the SCF complex by the pull down of cullin‐1, and inhibits the cGMP‐stimulated NKCC2 ubiquitination (F‐Box peptide: 100%; F‐Box peptide + db‐cGMP 500μM: 89.8 ± 3.9%, n.s.). Our data indicates that blocking neddylation of cullin‐1 or impairment the binding of the native F‐Box proteins to the SCF complex inhibits the cGMP‐dependent increase in NKCC2 ubiquitination. We conclude that the cGMP‐dependent increase in NKCC2 ubiquitination is mediated by the SCF complex. To our knowledge, this is the first evidence pointing to the E3 ubiquitin ligase complex involved in the ubiquitination of NKCC2 in native TALs.Support or Funding InformationInstitutional Mentored Grant Program‐Henry Ford Health SystemThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.