Date of Graduation
5-2016
Document Type
Dissertation (PhD)
Program Affiliation
Experimental Therapeutics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Jian Kuang
Committee Member
Gary E. Gallick
Committee Member
Sue-Hwa Lin
Committee Member
Pierre D. McCrea
Committee Member
Zahid H. Siddik
Abstract
The ESCRT (endosomal sorting complex required for transport) is an evolutionary conserved membrane remodeling machinery that performs membrane invagination and abscission. ALIX is a widely expressed adaptor protein that is critically involved in three classical ESCRT-mediated processes, including MVB (multivesicular body) sorting, cytokinetic abscission and retroviral budding. Previous studies have demonstrated that ALIX involvement in these ESCRT-mediated processes requires ALIX interaction with the ESCRT-III component CHMP4 as well as a cargo protein. However, the native form of ALIX contains a default intramolecular interaction between N-terminal Bro1 domain and C-terminal PRD (proline-rich domain), leading to a closed conformation of ALIX that cannot interact with CHMP4. This predicts that ALIX involvement in ESCRT-mediated processes requires an activation step that relieves the intramolecular interaction of ALIX.
The objective of my dissertation research is to identify the regulatory mechanisms that activate the ESCRT function of ALIX in the three classical ESCRT-mediated processes. Whether ALIX is critically involved in MVB sorting of ubiquitinated receptors has been controversial. By examining the effects of ALIX on the level of activated EGFR in the lumen of MVB and the level of EGF-induced phosphorylated ERK1/2, which is the downstream signaling of activated EGFR, I showed that CHMP4-bound ALIX dimer plays an essential role in MVB sorting and silencing of activated EGFR. However, MVB sorting of activated EGFR is not required for its degradation. My studies thus identify ALIX as an important regulator for signaling output of the activated EGFR. I further demonstrated that ALG-2 (apoptosis-linked gene 2 product) is responsible for generating ALIX in open conformation that supports MVB sorting and timely silencing of activated EGFR. My studies thus identify ALG-2 as a potential negative regulator for the signaling transduction of activated EGFR and provide a new understanding for the role of ALG-2 in apoptotic induction.
While ALG-2 is important for ALIX-mediated MVB sorting, I found that ALG-2 is not important for ALIX-mediated cytokinetic abscission or retroviral budding. Thus, I further identified mechanism that activates ALIX in these two ESCRT-mediated processes. By studying mitotic phosphorylation of ALIX, I found that phosphorylation at S718 and S721 residues in the PRD domain of ALIX relieves the intramolecular interaction of ALIX. This mechanism is required for generating ALIX in open conformation that supports cytokinetic abscission and retroviral budding. These findings provide a new understanding for the regulation of cytokinesis and retroviral budding and may provide new strategies for inhibiting cell division and retroviral budding.
In summary, my dissertation studies have identified two regulatory mechanisms that relieve the intramolecular interaction of ALIX in the three classical ESCRT-mediated membrane remodeling processes. These studies provide insights into the regulation of ESCRT-mediated membrane remodeling processes and suggest that ALIX may be a potential target for anti-cancer and anti-viral therapy.
Keywords
ALIX, MVB sorting, EGFR, ALG-2, intramolecular interaction, CHMP4, cytokinetic abscission, retroviral budding