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The role of post-translational modifications in the dimerization between the alpha and beta splicing isoforms of Signal Transducer and Activation of Transcription 3 (STAT3) and RelA
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Resumo(s)
Signal Transducer and Activation of Transcription 3 (STAT3) is a pleiotropic transcription factor
that plays key roles in cell survival, differentiation, and immune response. STAT3 has over 80 additional
post-translational modifications (PTMs) and 4 splicing isoforms with yet unclear stoichiometry and
functions. STAT3 interacts directly with the p65 subunit of Nuclear factor kappa B (NF-κB), also known
as RelA, and both pathways are closely connected, regulating similar biological phenomena. The aim
of this work was to elucidate the putative role of PTMs on the subcellular localization of heterodimers
formed by STAT3 proteoforms and RelA, in the absence or presence of stimulating cytokines, leukemia
inhibitory factor (LIF) and tumor necrosis factor α. We first confirmed the genomic alterations that
prevent STAT3 expression in a HeLa STAT3-/-
cell line, recently produced in our laboratory a
CRISPR/Cas9 approach. HeLa STAT3-/-
cells showed impaired proliferation/migration. We then
developed a series of molecular tools to analyze the interaction and dynamics of STAT3α:RelA and
STAT3α:STAT3β dimers in living cells, based on Bimolecular Fluorescent Complementation assays.
We used these tools as templates to produce STAT3 mutants preventing PTMs on K685, Y705 or S701
or mimicking the phosphorylation on S701. Using these tools, we observed that STAT3α and RelA form
a heterodimer that shows the basal subcellular distribution of RelA/NF-κB, but the response to LIF that
is typical of STAT3. The subcellular distribution of different combinations of STAT3α:STAT3β and
STAT3α dimers in the presence or absence of stimuli did not show major differences. The presence of
one modifiable (wild-type) STAT3 monomer was sufficient to accumulate dimers in the nucleus upon
LIF stimulation. Our findings enhance our understanding of the interplay between STAT3 proteoforms
and between STAT3α and NF-κB signalling and could contribute to the unravel the molecular
mechanisms of several pathologies, including cancer.
Descrição
Tese de mestrado, Biologia Molecular e Genética, 2024, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
STAT3 Modificações pós-traducionais RelA Acumulação nuclear Complementação de fluorescência bimolecular Teses de mestrado - 2024