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cGMP Stimulates K48‐linked Poly‐ubiquitination of NKCC2 in Rats Thick Ascending Limb

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter NKCC2. The second messenger cyclic guanosine monophosphate (cGMP) decreases NKCC2 activity by decreasing surface NKCC2 levels. We showed that NKCC2 is constitutively ubiquitinated and degraded by the proteasome, and that cGMP enhances the rate of ubiquitination. Ubiquitination is a post‐translational modification in which the C‐terminus of the ubiquitin is attached to a substrate protein. Poly‐ubiquitination occurs when the C‐terminus of another ubiquitin, is then linked to the previously added ubiquitin, creating a chain. There are 7 lysine (k) in the ubiquitin molecule where poly‐ubiquitination can occur. K48‐linked poly‐ubiquitination on ubiquitin is associated with proteasomal degradation. Therefore, we hypothesized what cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2 in rats TAL. To test our hypothesis we immunoprecipitated K48‐linked poly‐ubiquitin using Tandem Ubiquitin Binding Entities (TUBEs) and western blot against NKCC2 in Sprague‐Dawley rat TAL suspensions. First, we performed a k48‐TUBEs concentration‐response curve against K48‐linked poly‐ubiquitination of NKCC2 to ensure linear relationship. TALs suspension were aliquoted in 4 samples and placed at 37°C for 30 minutes in the presence of the proteasomal inhibitor (MG132 20μM) to stop protein degradation. TALs were lysed at 4°C and excess amount of protein lysate (150 μg) was incubated with increasing concentration of k48‐TUBEs. We obtained a linear increase in K48‐linked poly‐ubiquitination of NKCC2 with increasing concentration of k48‐TUBEs. Next, to study the effect of cGMP on k48‐ubiquitination of NKCC2. The conditions were set to fit within the linear range (80 μg of protein) to ensure complete immunoprecipitation of K48‐linked poly‐ubiquitination of NKCC2. We found that the membrane‐permeant cGMP analogue dibutiryl cGMP (db‐cGMP) stimulates K48‐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). These data indicates that cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2, and suggest a triggering mechanism for NKCC2 degradation by the proteasome. We conclude that cGMP increases K48‐linked poly‐ubiquitination of NKCC2. To our knowledge, this is the first piece of evidence showing that cGMP may trigger a signal for NKCC2 degradation through stimulation of k48‐ubiquitination.