Date of Award

2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Besim Ogretmen

Second Advisor

Christopher Davies

Third Advisor

Steven Rosenzweig

Fourth Advisor

L. Ashley Cowart

Fifth Advisor

Dennis Watson

Abstract

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase with tumor suppressor function. PP2A holoenzyme contains numerous combinations of its various isoforms of scaffolding (A), regulatory (B), and catalytic (C) subunits. In many cancer types, an endogenous PP2A inhibitor, inhibitor 2 of PP2A (SET) oncoprotein, is overexpressed, resulting in PP2A inhibition, leading to enhanced cell growth, and attenuation of cell death. It is known that targeting SET with bioactive sphingolipid ceramide or sphingolipid analogue drug FTY720 leads to the reactivation of PP2A, leading to necroptosis. However, structural details of the interaction between SET and FTY720 (or ceramide) with regards to mechanism of the PP2A activation have been unknown. Here, we report the first in solution examination of SET-sphingolipid complex by NMR spectroscopy. Data revealed that FTY720 binding may result in a structural shift in the N-terminal region of SET, which prevents its oligomerization. This then leads to the release of SET from the catalytic subunit of a specific PP2A holoenzyme, which comprises PP2AAβ, PP2A-B56γ, and PP2ACα subunits, for increased PP2A activity, while SET remains associated with the PP2A-B56γ. The activation of this specific PP2A holoenzyme by SET-FTY720 complex then regulates a number of downstream effector proteins involved in various biological functions, such as tumor suppressor non-muscle myosin IIA. Attenuation of FTY720-SET association by point mutations enhances SET- PP2A inhibitory complex, leading to resistance to PP2A activation, which is recapitulated by R71A and E111A SET mutations.

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