HECTD1 modulates centrosome duplication, cytokinesis, and ciliogenesis through HAX1

HECTD1, also known as E3 ubiquitin ligase with affinity to the inhibin receptor (EULIR), has been demonstrated to play a critical role in embryonic development. When HECTD1 mutated, neural tube closure defects occur and placenta development is hampered. Ongoing research demonstrated the many involvements of HECTD1 in cell spreading, cell migration and in Wnt signaling transduction. Defect of HECTD1 accelerates cell spreading/migration and invasion through inhibiting IQGAP1 degradation and excess Hsp90 secretion. HECTD1 ubiquitylates adenomatous polyposis coli (APC), and promotes APC/Axin interaction, which negatively regulates the Wnt signaling pathway. Although all these functions of HECTD1 have already been reported elsewhere, many other functions of HECTD1 remain unknown.
The involvement of HECTD1 in mitosis and cytokinesis was noticed in my previous research. Immunofluorescence staining of HECTD1 in different cell lines demonstrated that HECTD1 is localized at different subcellular sites, such as the centrosome, the basal body of cilia, the intercellular bridge, cytoplasm and in the nucleus. These findings promoted me to further investigate into the function of HECTD1 in the centrosome, in ciliogenesis and in cytokinesis.
First, by using HECTD1 mutated (HECTD1r/r) mouse embryonic fibroblasts (MEF) and HECTD1 short-hairpin RNA (shRNA) down-regulated HeLa cells, centrosome numbers were examined. The results revealed that loss of HECTD1 induced centrosome over-duplication. Centrosome abbreviations sequentially affect ciliogenesis both in HECTD1r/r and HECTD1-KD cells. To study the mechanism how HECTD1 controls centrosome numbers, I studied the relationship of HECTD1 with its interacting partners. HAX1 (HCLS1-associated protein X1), one of HECTD1 interacting partners previously screened by yeast two-hybrid assay, was regulated through HECTD1 ubiquitination. HAX1 is also located in the centrosome and becomes accumulated in centrosome during the S phase of the cell cycle when HECTD1 is down-regulated. Over-expression HAX1 phenocopied centrosome over-duplication as cells with defected HECTD1. Furthermore, by knockdown of HAX1, HECTD1-induced over-duplication of the centrosomes was abolished. HAX1 works as a scaffold protein to recruit proteins of the Hippo pathway, SAV1 and MOB1a, to the centrosome in order to control centrosome duplication. Thus, we propose the HECTD1-HAX1-SAV1-MOB1a axis to control centrosome duplication numbers.
Besides amplification of the centrosome and impaired ciliogenesis, defects in the completion of cytokinesis were also found in HECTD1-KD cells. Down-regulation of HECTD1 resulted in elongation of intercellular bridges and missed abscission. HAX1 formed ring structure in the midbody and its ectopic expression resulted in similar defects as in HECTD1-KD cells. Knocdown of HECTD1 or ectopic localization of HAX1 resulted in mislocalization of microtubules binding proteins, such as MKLP1 (Mitotic kinesin-like protein), PRC1 (Protein required for cytokinesis) and Kif4 in the midbody. Depletion of HAX1 could partly rescue the defects in cytokinesis caused by knockdown of HECTD1, by shortening the length of the intercellular bridges, while the abscission site assembly was recovered with Plk1 inhibitor, BI2536.
Finally, to further investigate the function of HECTD1 cell cycle regulation, epidermal growth factor (EGF) was employed. The EGF/EGF receptor (EGFR) endocytic and signaling pathways were explored. Knockdown of HECTD1 inhibited the degradation of EGFR and promoted the recycling of EGF/EGFR through APPL1/Rab4. Furthermore, knockdown of HECTD1 sustained phosphorylated Akt and MAPK protein expression after EGF stimulation, without increasing cell proliferation rates. In addition, SNAIL, the key regulator of epithelial-mesenchymal transition (EMT), protein expression was significantly increased in HECTD1-KD cells. SNAIL and HECTD1 protein-protein interaction was determined with immunoprecipitation. Cell morphology and EMT associated protein expression levels changed between control and HECTD1-KD cells, consistent with the notion that mutations in the HECTD1 gene are detected in various human cancers. This finding implies that HECTD1 may be involved in the metastasis of cancer and may be associated with clinical outcome of cancer.
Taken all these findings together, HECTD1 is multi-functional protein depending on its substrates, controlling centrosome duplication, ciliogenesis, and cytokinesis through HAX1, determining the EGFR trafficking from sorting endosome to early or recycling endosome via APPL1, and interacting with SNAIL modulating EMT.