Defense Date

3-11-2016

Graduation Date

Summer 1-1-2016

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

Philip Auron

Committee Member

Kyle Selcer

Committee Member

Michael Seaman

Committee Member

Charles Sfeir

Committee Member

Deborah Galson

Keywords

IRAK1, NF-κB, osteoclastogensis, Sox4, Syntenin, TRAF6

Abstract

TRAF6 is an E3 ubiquitin ligase that is unique among TRAF family proteins in both its structure and protein-protein interaction specificity. It is further distinguished by its ability to transduce a multiplicity of receptors in varied biological systems. Although TRAF6 activity is induced by these receptors, the mechanisms by which TRAF6 is activated under these stimulating conditions remain largely unclear. To address this, standard molecular biological techniques were used to gain insight into the interplay of three proteins with TRAF6: 1) Syntenin-1 (Syn); 2) SRY (sex determining region Y)-box 4 (Sox4); and 3) IL-1 receptor associated kinase 1 (IRAK1). These three proteins physically interact with TRAF6 and affect its signaling activity by modulating subcellular localization, conformation or activation of the NF-κB/Rel family of transcription factors under stimulating conditions in cell lines of various tissue types. While many studies have focused on cytoplasmic TRAF6 and its activation, there are a several reports on the nuclear localization of TRAF6 as well as TRAF3 and TRAF4 in response to either extracellular stimulation or disease. Ectopic Syn or Sox4 was found to not only attenuate TRAF6 signaling, translocates it into the nucleus of hematopoietic and monocytic cells. Syn attenuates TRAF6 activity in an ubiquitin and Sox4 dependent manner. This, and the observation that Syn is impaired in its ability to attenuate a non-ubiquitinated TRAF6 mutein (TRAF6ΔK), led to a hypothesis that Syn associates with TRAF6 in the cytoplasm following activation by IRAK1. Notably, knockdown of Sox4 was found to potentiate TRAF6 activation and abrogates Syn inhibition of TRAF6. Contrary to prior reports, use of domain deletions in both TRAF6 and Syn demonstrate that the full-length molecules are not necessary for their interaction. Instead, mutagenesis or deletion of a newly identified TRAF Interaction motif (TIM) in Syn affects TRAF6 signaling and localization. Interestingly, Syn also localizes to the nucleus when overexpressed with either wild type (WT) or a constitutively active TRAF6 (RZcc). This study demonstrates that Sox4 also attenuates TRAF6 signaling and identifies a unique Sox4 TIM that is necessary for binding TRAF6. TRAF6 and Sox4 colocalize under stimulatory conditions, thus providing a novel mechanism for TRAF6 entry into the nucleus. In sum, TIR and TNF receptor signaling through TRAF6 dependent NF-κB activity is modulated by novel interactions with the Sox4 transcription factor in cooperation with Syn The pair act on TRAF6 nucleocytoplasmic partitioning, conformation and modification state, modulating TRAF6c-dependent ell morphogenesis and intracellular signaling.

Format

PDF

Language

English

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