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

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Kelch-like protein 3 (KLHL3) and Cullin3 form an E3 ligase complex that ubiquitinates with-no-lysine kinase 4 (WNK4).
• Impaired WNK4 ubiquitination is linked to pseudohypoaldosteronism type II, a hereditary hypertensive disease.

Purpose of the Study:

• To investigate the mechanism of KLHL3-induced WNK4 degradation, particularly whether it involves selective autophagy.
• To explore the role of p62 in the degradation of WNK4 mediated by KLHL3.

Main Methods:

• HEK293T cell culture and treatment with proteasome and autophagy inhibitors (epoxomicin, 3-methyladenine).
• Co-immunoprecipitation assays to detect protein complex formation.
• Western blotting to assess protein levels.
• Immunofluorescent staining to determine protein localization and co-localization with autophagy markers.

Main Results:

• Proteasome inhibition with epoxomicin enhanced WNK4 degradation, suggesting an alternative pathway.
• Autophagy inhibition with 3-methyladenine blocked the epoxomicin-induced decrease in WNK4.
• KLHL3 formed a complex with both WNK4 and p62.
• p62 overexpression decreased KLHL3 and WNK4 levels, while p62 knockdown increased them.
• WNK4 co-localized with KLHL3, p62, and light chain 3 (autophagosome marker) in the cytoplasm.

Conclusions:

• WNK4 degradation is mediated by both the proteasome and p62-KLHL3-dependent selective autophagy.
• This dual degradation mechanism suggests a role for selective autophagy in WNK4 regulation under specific pathophysiological conditions.