Type Iα PKA as a co-incidence detector

: Integration of cAMP and oxidant signals

Abstract

Protein kinase A (PKA) is activated by elevated intracellular cAMP downstream of β-adrenergic signalling. PKA’s subcellular localisation is determined by its affinity for A-kinase anchoring proteins (AKAPs), scaffolds that sequester PKA proximal to its substrates. The regulatory RIα subunits present in type I PKA can form two intermolecular disulphide bonds, each flanking its AKAP-binding domain, in response to oxidants. Rationally this change in the redox state of PKARIα may alter its affinity for AKAP, with potential for consequential regulation of substrate phosphorylation.

I made the novel observation that co-treatment of cells with H2O2 and cAMP caused increased global PKA substrate phosphorylation compared to cAMP or H2O2 alone in HEK 293 cells. This suggested RIα serves as a co-incidence detector that integrates the two signals to synergistically enhance PKA substrate phosphorylation. Chronic cAMP treatment increased PKARIα expression in HEK 293 cells and PKARIα was also upregulated in a mouse model of hypertrophy, whilst PKARIIα was downregulated. This may represent a switch to an oxidant sensitive PKA signalling phenotype during chronic β-adrenergic stimulation.

AKAP7 was identified as a potential redox-dependent PKARIα AKAP. Examination of the AKAP7 crystal structure revealed putative redox sensitive cysteines adjacent to its A-kinase binding domain, leading to the hypothesis that the redox state of this AKAP may alter its affinity for PKARIα. I developed and validated the ‘PEG-maleimide switch assay’, a method used to identify proteins that are susceptible to reversible oxidative modification. Using the PEGmaleimide switch assay I found AKAP7 to be insensitive to oxidation. Furthermore, I demonstrated that PKARIIα, but not PKARIα, is a binding partner for AKAP7δ and this interaction was redox modulated.

I described, for the first time, that cAMP can modulate PKARIα disulphide dimer levels. DTTdependent PKARIα reduction was potentiated by cAMP, and cAMP-agarose induced PKARIα reduction. Furthermore, a mutant PKARIα that could not bind cAMP formed disulphide-linked complexes under oxidative conditions. These complexes may be cAMP-regulated intermediates that form during a novel oxidant-dependent mechanism of PKARIα targeting to AKAPs. LC-MS/MS identified the novel AKAP tubulin as part of a disulphide linked complex with PKARIα. Consistent with the hypothesis that oxidants target PKARIα to tubulin, phosphorylation of the microtubule associated protein GEF-H1 was enhanced after cotreatment with cAMP and H2O2 compared to cAMP alone. Thus PKARIα is a co-incidence detector at the interface of cAMP and oxidant signalling that is able to integrate both modalities to fine-tune phosphorylation events.

Date of Award	2016
Original language	English
Awarding Institution	King's College London
Supervisor	Metin Avkiran (Supervisor) & Philip Eaton (Supervisor)