Signalling of cAMP at the centrosome.
PhD thesis, University of Glasgow.
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The compartmentalisation of cAMP/PKA signalling pathway within specific regions of the cell plays a critical role to achieve the specificity of response. Adenylyl cyclases (AC) are localised at discrete regions of the plasma membrane and phosphodiesterases (PDEs), the only enzymes that degrade cAMP, have been shown to be pivotal in generating spatially restricted pools of cAMP, therefore underpinning spatial control of this second messenger signal. In addition, A-kinase anchoring proteins (AKAPs) are of key importance as they anchor PKA in proximity of its specific targets, thus favouring target selective phosphorylation. Such organisation leads to local activation of PKA subsets through the generation of confined intracellular gradients of cAMP.
Interestingly it has been shown that AKAP450 localises to the centrosome, the major microtubule-organising centre, where it functions as a ‘multi-scaffolding’ protein by simultaneously associating PKA with PDE4D3 as well as other kinases and phosphatases. Beside this a large body of evidence suggests that the centrosome is essential for the regulation of the cell cycle progression by acting as a scaffold protein for a network of signalling pathways which in turn trigger cellular division.
In the past few years the development of FRET-based sensors has allowed the study of cAMP dynamics with high spatial-temporal resolution. By using this approach it is now possible to monitor real-time fluctuations of cAMP and PKA activity in distinct subcellular compartments and to investigate their physiological role.
The aim of the research presented in this dissertation is to exploit FRET-based sensor to investigate the signalling of cAMP at the centrosome and to define the role of PDE4D3 anchored to AKAP450 in shaping a cAMP pool in such specific compartment. The centrosomal AKAP450/PKA/PDE4D3 macromolecular complex may play a role in the control of cell cycle progression.
To this purpose a CHO clone stably expressing the FRET sensor based on PKA was generated. As expected fluorescence microscopy analysis of this clone indicated that the sensor anchors to endogenous centrosomal AKAPs. Further real-time imaging of basal cAMP provided evidence that the centrosome is a domain with lower cAMP concentration as compared to the bulk cytosol and that PDE4D3 activity is required to maintain a low cAMP level in the centrosomal area.
Interestingly the same cells challenged with the cAMP raising agent forskolin show a larger FRET change at the centrosome as compared to the bulk cytosol.
By using the unimolecular FRET EPAC-based sensor for cAMP, targeted to the centrosome, it was possible to exclude that the level of cAMP generated at the centrosome by forskolin was higher than the level of cAMP generated in the cytosol. Thus, it has been hypothesised that anchoring of PKA to AKAP450 lowers the activation constant of the enzyme leading to a higher FRET change at the centrosome as compare to the bulk cytosol. This hypothesis has been confirmed by expressing in the cytosol the fragment of AKAP450 that anchors PKA and by showing that binding of PKA to the cytosolic fragment also results in increased sensitivity of the enzyme to cAMP. Eventually analysis of PKA activity, by using a FRET-based A-kinase activity reporter (AKAR), indicated that anchoring of PKA to the cytosolic fragment of AKAP450 accounts also for an increased PKA activity.
The molecular mechanism involved in the increased sensitivity of PKA-bound to AKAP450 was also investigated. Interestingly anchoring of PKA to AKAP450 increases the auto-phosphorylation of PKA. Generation of a non-phosphorylatable version of PKA-RII subunit (mutRII) and further generation of a CHO clone stably expressing the mutPKA FRET based sensor strongly indicates that the high sensitivity of PKA bound to AKAP450 is mediated by the auto-phosphorylation site and more specifically the binding of PKA to AKAP450 seems to favour the auto-phosphorylation of PKA.
Finally the role of AKAP450/PKA/PDE4D3 macromolecular complex in the regulation of cell cycle progression was analysed. Displacement of endogenous PDE4D3 from the centrosome by over-expression of a catalytically dead version of PDE4D3 (dnPDE4D3), results not only in the abolishment of difference in cAMP concentration between centrosome and cytosol, but also in an altered cell cycle progression, suggesting that PDE4D3 plays a key role in the regulation of the cell cycle.
In conclusion this study provided evidence for a novel mechanism by which anchoring of PKA to AKAPs modulate the activation constant of the enzyme, thereby providing a mean to regulate enzyme activity locally.
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