Finding NEMO: A bifurcated pathway of NF-B activation occurs via the recruitment of NEMO to polyubiquitin scaffolds at TCR
DeRiso, Elizabeth.
2017
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Abstract: Recent advancements in the field of cancer immunotherapy take
advantage of T cell signaling controls, highlighting the importance of fully understanding
basic signaling mechanisms for therapeutic targeted use. The NF-κB essential modulator
protein (NEMO), also known as IKKγ, regulates canonical activation of the pro survival and
growth transcription factor, NF-κB, downstream of the T... read morecell receptor (TCR). Patients with
mutated or deficient NEMO protein generally present with some form of immunodeficiency.
Despite this key role of NEMO in immunity, however, little is known about the specific
mechanisms regulating the sub-cellular localization of NEMO during immunoreceptor
signaling. Using high-resolution, dynamic confocal microscopy, we analyzed the localization
of NEMO in response to TCR ligands in primary human T cells, as well as leukemic Jurkat T
cells. Our model system consists of a glass surface coated with anti-CD3 ligand (OKT3) and
the VLA-4 ligand rhVCAM1, used to mimic the stimulatory surface of an antigen presenting
cell (APC). Using fluorescently tagged chimeric proteins, we can image the interface
between the T cell and the glass to determine where NEMO and other TCR-proximal signaling
proteins localize within the immunological synapse. We demonstrate that NEMO is recruited
into functionally relevant, ubiquitin-dependent microclusters that co-localize with the
TCR-associated kinase Zap70. Recruitment of NEMO into Zap70 clusters occurs within five
minutes of TCR engagement, indicating rapid recruitment of NEMO to the TCR. We also
demonstrate that IKK, the kinase relevant for NF-B activation, is recruited and activated
in the microcluster. Additionally, NEMO is recruited into mobile vesicles and larger, slow
moving, membrane-bound macroclusters. Entrance of NEMO into microclusters is also dependent
on Zap70 and Lck kinase activity, yet is independent of other proximal signaling molecules
such as SLP76 and LAT. Furthermore, we show that NEMO recruitment into microclusters occurs
independent of CBM complex member CARMA1, and did not specifically co-localize with BCL10
polymers. We propose a shift away from the normal antigen stimulated NF-B pathway paradigm
with assumed cytosolic localization of NEMO. Instead, our results suggest that the
mechanisms responsible for NEMO recruitment to the TCR and for the subsequent activation of
NF-κB must diverge downstream of Zap70 before rejoining later at an unknown
point.
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Immunology.
Advisors: Stephen Bunnell, and Alexander Poltorac.
Committee: Joan Mecsas, Marta Gaglia, and Hao Wu.
Keyword: Immunology.read less - ID:
- kk91fz413
- Component ID:
- tufts:23391
- To Cite:
- DCA Citation Guide EndNote
