Priming of STAT1 and STAT3 for cytokine-triggered degradation by the proteasome upon A2Aadenosine receptor (A2AAR) expression

Safhi, Mohammed Mohsen A. (2008) Priming of STAT1 and STAT3 for cytokine-triggered degradation by the proteasome upon A2Aadenosine receptor (A2AAR) expression. PhD thesis, University of Glasgow.

Abstract

The A2A adenosine receptor (A2AAR) functions as a key non-redundant suppressor of
inflammatory responses in vivo. However, whether it regulates activation of the JAK-STAT
pathway utilised by many pro-inflammatory cytokines is unknown. Using a
vascular endothelial cell model system, I have demonstrated that adenovirus-mediated
expression of the human A2AAR conferred an ability of IFNα, leptin and a soluble IL-
6 receptor-α/IL-6 (sIL-6Rα/IL-6) trans-signalling complex to promote a time-dependent
reduction in the levels of STAT proteins that was entirely due to
proteasomal degradation. In terms of functional consequences, degradation was
sufficient to attenuate sIL-6Rα/IL-6-stimulated STAT3-dependent up-regulation of
vascular endothelial growth factor receptor-2 (VEGFR-2) and enhance eNOS
expression. Degradation required JAK activity since A) it was blocked by
preincubation with JAK inhibitor. B) STAT1 but not STAT3 was resistant to both
tyrosine phosphorylation and down-regulation in response to leptin and C) a
Tyr705→Phe mutated STAT3 was also resistant to cytokine-triggered degradation,
suggesting that JAK-mediated phosphorylation of this residue is required to produce
the effect. Consistent with this hypothesis, sIL-6Rα/IL-6 treatment of A2AAR-expressing
cells resulted in the accumulation of polyubiquitylated endogenous and
epitope-tagged recombinant wild-type but not Tyr705→ Phe-mutated STAT3. In
addition the results show that inhibition of proteasome function was sufficient to
block the inhibitory effect of the A2AAR on STAT3 phosphorylation, demonstrating
that priming of STATs for degradation is the only mechanism responsible for the
reduced cytokine-stimulated STAT phosphorylation observed in A2AAR-expressing
cells. To date there is only one E3 ligase known for mediating STAT degradation
which is SLIM protein. However, our results suggest the involvement of another E3
ubiquitin ligase in HUVECs, since we have been unable to detect SLIM message or
protein in HUVECs under conditions in which STAT degradation occurs. In addition,
while Tyr-phosphorylation is clearly the critical step in targeting STATs for
degradation in A2AAR-expressing cells, it is unclear as to whether it functions simply
as a classical phosphodegron, or whether the nuclear translocation that occurs as a
result of phosphorylation is also important for localising the phosphorylated STAT
dimer with the relevant E3 ubiquitin ligase.
Together, these observations suggest a model whereby expression of the A2AAR in
endothelial cells primes JAK-phosphorylated STATs for polyubiquitylation and
subsequent degradation by the proteasome following cytokine treatment, and
represents a previously unappreciated mechanism by which G-protein-coupled
receptors can negatively regulate responsiveness to specific JAK-STAT-mobilising
adipocytokines acting on the vascular endothelium.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: A2Aadenosine receptor, soluble IL- 6 receptor-α/IL-6, IFNα, leptin, STAT1 and STAT3
• Subjects: Q Science > QD Chemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Palmer, Dr Tim
• Date of Award: 2008
• Depositing User: M Mohammed Mohsen A. Safhi
• Unique ID: glathesis:2008-310
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 26 Jun 2008
• Last Modified: 07 Apr 2015 08:14
• URI: https://theses.gla.ac.uk/id/eprint/310