E exact same surface of the propeller and are identified to interact with target proteins in related paralogs (14). CUL3 mutations are all heterozygous, predominantly de novo mutations, and all outcome in skipping of exon 9, major to an inframe 57aa deletion (five). In the observation that recessive mutations in KLHL3, dominant mutations in KLHL3, and dominant mutations in CUL3 all result in phenocopies from the same illness, we inferred that all ofAuthor contributions: S.S. and R.P.L. made investigation; S.S., J.Z., and J.P. performed research; S.S., J.Z., K.L.S., and R.P.L. analyzed information; and S.S. and R.P.L. wrote the paper. The authors declare no conflict of interest. Freely out there on the internet by way of the PNAS open access selection. See Commentary on web page 7535.To whom correspondence need to be addressed. Email: [email protected] short article contains supporting data on the web at www.pnas.org/lookup/suppl/doi:ten. 1073/pnas.1304592110//DCSupplemental.www.pnas.org/cgi/doi/10.1073/pnas.these mutations likely create loss of ubiquitination of certain proteins typically targeted by KLHL3.1698378-64-1 manufacturer The identity of these proteins was unknown. We report herein that WNK4 is a direct target for ubiquitination by CUL3 LHL3 CRL complexes and that this ubiquitination leads to degradation and decreased levels of WNK4. We show that diseasecausing dominant mutations in either KLHL3 or WNK4 inhibit binding, ubiquitination, and degradation of WNK4, resulting in greater WNK4 levels in mammalian cells and in vivo. This enhance in WNK4 level is enough to enhance inhibition of one of WNK4’s recognized targets, ROMK. These findings demonstrate that CUL3 LHL3 and WNK4 act together in the similar biochemical pathway and give a molecular explanation for the mechanism of dominant KLHL3 and WNK4 mutations. ResultsKLHL3 Binds to CUL3, WNK1, and WNK4. To achieve insight into theWe subsequent tested whether or not WNK4, that is not endogenously expressed in COS7 cells, could also interact with this ubiquitin ligase complex by expressing fulllength WNK4 tagged using the HA epitope in the C terminus (WNK4HA) in COS7 cells with or with no FLAGKLHL3. Inside the presence of FLAGKLHL3, IP of WNK4HA with antiHA pulled down each FLAGKLHL3 and CUL3 (Fig. 1D). The reciprocal experiment demonstrated that IP of FLAGKLHL3 with antiFLAG pulled down CUL3 and WNK4 (Fig.1350518-27-2 In stock 1E).PMID:33752514 The capacity of antiHA (WNK4) to pull down CUL3 was dependent upon coexpression of KLHL3, constant with a physical interaction amongst KLHL3 and WNK4. Collectively, these results demonstrate that WNK4 is often identified inside a complex with KLHL3 and CUL3, constant with all the thought that a CUL3 LHL3 complex may well target WNKs for binding and ubiquitination.PHAII Mutations in KLHL3 and WNK4 Inhibit Their Interaction. Our findings raise the question of regardless of whether dominant PHAIIcausing mutations in KLHL3 and WNK4 might avoid binding of WNK4 to KLHL3. Accordingly, we separately expressed constructs bearing WT FLAGKLHL3 and FLAGKLHL3 with an R528H missense mutation, which has been located recurrently in unrelated individuals with dominant PHAII (5). In parallel, we expressed WT WNK4HA. We then incubated WT or mutant FLAGKLHL3 with WNK4HA and performed IP with antiFLAG, followed by Western blotting with the resultant protein with antiHA to detect WNK4. The results demonstrated robust binding of WT KLHL3 to WNK4, but virtually full loss of binding of KLHL3R528H to WNK4 (Fig. 2). We subsequent performed the analogous experiment using WT FLAGKLHL3 and either of two dominant WNK4.
