AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70

Abstract

Background/Aims: AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. Methods: MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. Results: We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. Conclusion: Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation.

Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

“…In line with findings by Wu et al, our STUB1 (CHIP) knockdown reduced, but did not abolish, AQP2 ubiquitination [56]. Additionally, overexpression of STUB1 does not lead to a complete degradation of AQP2 [57]. Therefore, other E3 ubiquitin ligases are involved.…”

“…The constitutively active form of CDK18 was associated with the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box containing protein 1) also termed CHIP (C-terminal Hsc70 interacting protein) [55] ( Figure 4B). CHIP1 has previously been identified as an E3 ubiquitin ligase of AQP2 [56,57]. Therefore, and as expected, STUB1 knockdown in MCD4 cells decreased the poly-ubiquitination of AQP2 and increased its abundance ( Figure 4C).…”

Green olive leaf extract (OLE) provides cytoprotection in renal cells exposed to low doses of cadmium

“…Mouse cortical collecting duct MCD4 cells derived from M1 cells were stably transfected with the water channel AQP2 [29]. MCD4 cells were cultured as described [29,30]. Briefly, cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100i.u./ml penicillin, 100 μg/ml streptomycin and 5 μM dexamethasone at 37°C, 5% CO 2 .…”

Vasopressin–aquaporin-2 pathway: recent advances in understanding water balance disorders

“…NDI can also be acquired and is a frequent side effect of lithium therapy 55 or other drugs 56– 60 . Hypokalemia or hypercalcemia with associated hypercalciuria also cause acquired NDI 28, 61– 68 .…”