Involvement of selective autophagy mediated by p62/SQSTM1 in KLHL3-dependent WNK4 degradation.

We reported that kelch-like protein 3 (KLHL3)-Cullin3 E3 ligase ubiquitinates with-no-lysine kinase 4 (WNK4) and that impaired WNK4 ubiquitination causes pseudohypoaldosteronism type II, a hereditary hypertensive disease. However, we also found that KLHL3-induced WNK4 degradation could not be inhibited completely by a proteasome inhibitor. Rather, on exposure, for 24 h, of HEK293T cells expressing WNK4 and KLHL3 to a proteasome inhibitor, epoxomicin, the WNK4 protein level was further decreased. As proteasome inhibition is known to activate p62-mediated selective autophagy, we investigated whether WNK4 degradation induced by KLHL3 is also mediated by such an autophagic mechanism. 3-Methyladenine, an autophagy inhibitor, blocked the epoxomicin-induced decrease in WNK4. Co-immunoprecipitation assays revealed that KLHL3 formed a complex not only with WNK4 but also with p62 via its kelch repeat domain. Under proteasome inhibition, p62 overexpression decreased KLHL3 and WNK4 protein levels, and p62 knockdown dramatically increased KLHL3 and WNK4 protein levels. Based on immunofluorescent staining, transiently overexpressed WNK4 showed punctate localization in the cytoplasm where it co-localized with KLHL3, p62 and light chain 3, a marker of autophagosomes. Thus, WNK4 was degraded not only by proteasomes but also by p62-KLHL3-mediated selective autophagy, which may be involved in WNK regulation under certain pathophysiological conditions.