Defense Date

12-18-2009

Graduation Date

2009

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

Philip Auron

Committee Member

Jana Patton-Vogt

Committee Member

Bruce Sneddon

Committee Member

Lawrence Kane

Keywords

E3 ubiquitin ligase, Intramolecular interaction, NF-kappaB activity, Sequestosome, TRAF6, Ubiquitination

Abstract

The Tumor Necrosis Factor (TNF) Receptor Associated Factor 6 (TRAF6) is an intracellular signal transducers, being responsible for mediating many of the activation events initiated by TNF receptor (TNFR) and Toll-like/Interleukin-1 and 18 receptor (TIR) families, in which TRAF6 plays central roles in numerous biological processes including innate and adaptive immunity, osteoclastogenesis and bone development, CD40 signaling, neuronal cell development, and cancer cell progression.

Acting as an E3 ubiquitin ligase, TRAF6 catalyzes lysine 63 linked poly-ubiquitination of itself and many other signal transducers upon association with upstream effectors possessing a short TRAF Interaction Motif (TIM) peptide sequence in the NF-KappaB signal transduction pathway. Ectopic over-expression of TRAF6 acts as a dominant-positive. However, the mechanism of TRAF6 activation by upstream activators or over-expression is unclear. This motivated our enthusiasm to study the role played by ubiquitination for TRAF6 in NF-KappaB signaling.

We now demonstrate that two critical regions of TRAF6, the MATH domain required for TIM activator binding and the RING-Zinc region for downstream signaling, mutually interact and render the molecule structurally closed and inactive. Our results implicate that auto-ubiquitination disrupts such interaction, thus providing a means of sustaining the open conformation necessary for downstream signaling. However, excessive ubiquitination induced by TRAF6 over-expression results in formation of large cytoplasmic sequestosomes and its inactivation. Furthermore, the inferred cis nature of TRAF auto-ubiquitination is now demonstrated to act in trans and is regulated via its RING-Zinc and coiled-coil domains. We also demonstrate that both the RING-Zinc region and MATH domain of TRAF6 can be targeted for ubiquitination, but trans-ubiquitination of TRAF6 muteins is incapable of activating the NF-KappaB pathway, suggesting that ubiquitination, alone, is insufficient for activity.

Format

PDF

Language

English

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