ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2019) 65 OP1.4 | DOI: 10.1530/endoabs.65.OP1.4

Global phosphoproteomics links rapidly induced cytoplasmic signals to transcriptional control

Fiona Leslie1, Stephen Kershaw2, Pauline Pfänder3, Toryn Poolman4, Kathryn McGinnis1, Chiara Frankavilla2, David Ray4, Graham Cook1 & Laura Matthews1

1University of Leeds, Leeds, UK; 2University of Manchester, Manchester, UK; 3German Cancer Research Center, Heidelberg, Germany; 4University of Oxford, Oxford, UK

Glucocorticoids (Gc) are potent anti-inflammatory steroids which mediate their effects by binding the glucocorticoid receptor (GR). Following ligand binding, GR initiates rapid ‘non-genomic’ kinase signals in the cytoplasm, then translocates into the nucleus to mediate ‘genomic’ effects by binding DNA directly or tethering to other DNA bound transcription factors to modulate target gene expression. Little is known about how rapidly induced cytoplasm derived Gc signals might feed forward to modulate the transcriptional response. We have completed global phosphoproteomics following acute (10 min) treatment with three different GR ligands to identify rapidly induced, Gc controlled pathways. In total we identify over 150 Gc regulated phosphoproteins. Of these, we find more than 100 proteins that are controlled by all three GR modulators – suggesting a common signature of GR activation. The identified phosphotargets includes proteins with diverse functions that are localised to the plasma, ER and mitochondrial membranes, the cytoplasm and nucleus. Consistent with our previously published work, we identify phosphoregulation of the lipid raft marker caveolin-1, and the three caveolin partner proteins, cavins 1, 2 and 3. We also identify differential phosphorylation of two G protein coupled receptors which localise to caveolae, thereby linking GR activation with kinase coupled pathways. Functional ontology analysis identifies cyclin-dependent kinase/cyclin mediated phosphorylation of RNA Polymerase II as a key effector pathway following Gc treatment. Using phospho-specific antibodies we demonstrate by immunoblot that the serine phosphorylation signature of Rbp1 C-terminal domain is altered following acute Dex treatment which suggests that rapid Gc signals prime the transcription machinery. We also identify Gc dependent phosphorylation of a number of chromatin re-modellers and transcription factors important for transcription initiation. Collectively our data suggests a common GR-mediated mechanism, whereby rapidly induced Gc signals feed-forward into the nucleus to modulate genomic signals, providing further insight into Gc action in vivo.

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