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

QD Chemistry

Safhi, Mohammed Mohsen A. ; Palmer, Tim

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: A2Aadenosine receptor, soluble IL-6 receptor-α/IL-6, IFNα, leptin, STAT1 and STAT3;   

Others  :  http://theses.gla.ac.uk/310/1/2008MohammedSafhiphd.pdf

来源: University of Glasgow

【 摘 要 】

The A2A adenosine receptor (A2AAR) functions as a key non-redundant suppressor ofinflammatory responses in vivo. However, whether it regulates activation of the JAK-STATpathway utilised by many pro-inflammatory cytokines is unknown. Using avascular endothelial cell model system, I have demonstrated that adenovirus-mediatedexpression 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-dependentreduction in the levels of STAT proteins that was entirely due toproteasomal degradation. In terms of functional consequences, degradation wassufficient to attenuate sIL-6Rα/IL-6-stimulated STAT3-dependent up-regulation ofvascular endothelial growth factor receptor-2 (VEGFR-2) and enhance eNOSexpression. Degradation required JAK activity since A) it was blocked bypreincubation with JAK inhibitor. B) STAT1 but not STAT3 was resistant to bothtyrosine phosphorylation and down-regulation in response to leptin and C) aTyr705→Phe mutated STAT3 was also resistant to cytokine-triggered degradation,suggesting that JAK-mediated phosphorylation of this residue is required to producethe effect. Consistent with this hypothesis, sIL-6Rα/IL-6 treatment of A2AAR-expressingcells resulted in the accumulation of polyubiquitylated endogenous andepitope-tagged recombinant wild-type but not Tyr705→ Phe-mutated STAT3. Inaddition the results show that inhibition of proteasome function was sufficient toblock the inhibitory effect of the A2AAR on STAT3 phosphorylation, demonstratingthat priming of STATs for degradation is the only mechanism responsible for thereduced cytokine-stimulated STAT phosphorylation observed in A2AAR-expressingcells. To date there is only one E3 ligase known for mediating STAT degradationwhich is SLIM protein. However, our results suggest the involvement of another E3ubiquitin ligase in HUVECs, since we have been unable to detect SLIM message orprotein in HUVECs under conditions in which STAT degradation occurs. In addition,while Tyr-phosphorylation is clearly the critical step in targeting STATs fordegradation in A2AAR-expressing cells, it is unclear as to whether it functions simplyas a classical phosphodegron, or whether the nuclear translocation that occurs as aresult of phosphorylation is also important for localising the phosphorylated STATdimer with the relevant E3 ubiquitin ligase.Together, these observations suggest a model whereby expression of the A2AAR inendothelial cells primes JAK-phosphorylated STATs for polyubiquitylation andsubsequent degradation by the proteasome following cytokine treatment, andrepresents a previously unappreciated mechanism by which G-protein-coupledreceptors can negatively regulate responsiveness to specific JAK-STAT-mobilisingadipocytokines acting on the vascular endothelium.