PMC

Overexpression of MEKK5 produces an inhibitory effect in the NEDD4 migration signaling. (A) Overexpression of NEDD4 and MEKK5 upon introducing NEDD4 and MEKK5 cDNA in the cells. (B,C) The effects of overexpression of NEDD4 and MEKK5 on cell migration were determined by the transwell assay. For quantification in the transwell assay, the migrated cells were fixed, stained with crystal violet, and counted under a microscope from three randomly selected fields. ** p < 0.01, *** p < 0.001.

A proposed model of regulation of NEDD4 through interacting with MEKK5. NEDD4 physically interacts with MEKK5 through the WW3 domain. The binding of MEKK5 inhibits the E3 ubiquitin ligase activity of NEDD4, which impairs the NEDD4-mediated ubiquitination and cancer cell migration.

MEKK5 inhibits ubiquitination of substrates by NEDD4 in vitro. (A) Myc-MEKK5 was cotransfected with HA-NEDD4 or the vector into HEK293T cells, immunoprecipitated with an anti-HA antibody and added indicated amount of purified GFP-SQSTM1 protein, and used for the in vitro ubiquitination assay. The ubiquitination of SQSTM1 was detected by immunoblotting with anti-GFP antibody. (B) The lung cancer NCI-H1650 cells were infected by lentiviral vector-loaded MEKK5 shRNA for 24 hours, and endogenous MEKK5 in the cell lysates was detected by immunoblotting with anti-MEKK5. (C) The MEKK5 (MEKK5-WT) or its kinase-dead mutant MEKK5-K709A (MEKK5-KD) was stably expressed in the lung cancer A549 cells using a lentiviral mammalian expression system. The expressed protein level of MEKK5 or its kinase-dead mutant was detected by immunoblotting of the cell lysates with anti-MEKK5. (D) MEKK5 knockdown and overexpression cell lines established in lung cancer NCI-H1650 and A549 cells were immunoprecipitated with an anti-MEKK5 antibody. The indicated amount of purified GFP-SQSTM1 protein was added into immunoprecipitated proteins before the in vitro ubiquitination assay. Ubiquitination of SQSTM1 was detected by immunoblotting with an anti-GFP antibody. * p < 0.05, ** p < 0.01.

MEKK5 specifically interacts with NEDD4. (A) Domain structure of three NEDD4 family E3 ubiquitin ligases and MEKK5. (B) HA-NEDD4, NEDD4-2, WWP1, or vector was coexpressed with Myc-MEKK5 in HEK293T cells. Myc-MEKK5 was immunoprecipitated with anti-Myc antibody; coprecipitated proteins were detected by immunoblotting with anti-HA antibody. (C) NEDD4 was cotransfected with Myc-MEKK5 or the mutants into HEK293T cells. Myc-MEKK5 or the mutants were immunoprecipitated with an anti-Myc antibody, and co-immunoprecipitated HA-NEDD4 was detected by immunoblotting with an anti-NEDD4 antibody. (D) Schematic representation of GST-NEDD4 WW domain constructs. (E) The bead-bound GST or GST-NEDD4-WW domain was incubated with the Myc-MEKK5-expressed HEK293T cell lysates. The coprecipitated MEKK5 was detected by immunoblotting with an anti-Myc antibody. *** p < 0.001.