Characterization of Novel Substrates of the Tankyrase and RNF146 Destruction Complex and Mechanisms of its Regulation
Poly-ADP-ribose is a post-translational modification that was first described over 50 years ago as a polymer derived from nicotinamide adenine dinucleotide or NAD. Since then, a family of 17 enzymes responsible for generating Poly-ADP-ribosylation or PARylation post translational modification has been identified and characterized. A unique member of this family of enzymes, called Poly-ADP-ribose Polymerases (PARPs), are Tankyrases. There are two mammalian Tankyrases, Tankyrase 1 and 2, whose domain organization include ankyrin repeat clusters which mediate enzyme-substrate interactions and a SAM domain which regulates oligomerization of Tankyrase into large macromolecular complexes. Tankyrases bind to proteins through a ‘RxxxxG’ peptide motif which facilities these proteins to undergo PARylation. A subset of Tankyrase substrates are recognized by an E3 ligase, RNF146, which facilities protein degradation through PARylation dependent ubiquitylation. The studies summarized in this thesis have focused on the identification of new proteins targets involved in the Tankyrase:RNF146 degradation pathway and investigates how Tankyrase PARylation is regulated through FIH dependent hydroxylation. I have identified SH3BP5 and SH3BP5L as new Tankyrase substrates that are targets of RNF146. I have shown that both substrates are guanine exchange factors for the small GTPase Rab11a. I have defined the minimal catalytic core of SH3BP5/L and shown it to be composed of a novel two α-helix GEF domain. My work has demonstrated that SH3BP5 and SH3BP5L are both required for optimal activation of Rab11a in epithelial cells during lumenogenesis and that their activities are repressed by Tankyrase-mediated PARylation. RNF146 regulates Rab11a activity by controlling Tankyrase protein abundance, marking the first time that RNF146 has been described as antagonistic towards Tankyrase function. I have demonstrated that FIH-mediated hydroxylation of Tankyrase affects the ability of substrates to bind to the ankyrin repeat clusters and inhibits autoPARylation and substrate PARylation yet does not affect protein degradation. Since PARylation dependant degradation is unaffected, it suggests that hydroxylation could affect the degradation independent functions of Tankyrase.
ADP-Ribosylation, Poly-ADP-Ribose Polymerases, post-translational modification, Tankyrase
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