In resting COS1 cells a dominant negative approach shows that specific, anchored PDE4 cAMP phosphodiesterase isoforms gate the activation, by basal cyclic AMP production, of AKAP-tethered protein kinase A type II located in the centrosomal region
Introduction
It is well appreciated that the cyclic nucleotide, cAMP, plays a pivotal role as a second messenger in controlling a wide range of cellular functions. These include processes as diverse as memory, heart and smooth muscle contraction, water and electrolyte homeostasis, immune responses, key aspects of metabolism, gene expression, differentiation and apoptosis [1], [2], [3].
Analyses of various intracellular messenger systems, namely Ca2+, tyrosyl kinases and inositol phospholipids, have unequivocally established signal compartmentalisation as crucial to normal cellular functioning [4], [5], [6], [7], [8]. Thus the use of optical probes has shown that highly localised gradients of Ca2+ can form in cells. Indeed, it has even been shown that Ca2+ can be funnelled to distinct points within the cell interior by constraining its dispersal through ‘inactivation’ by uptake into spatially constrained groupings of mitochondria [9], [10]. This generates compartmentalised signalling.
The notion that signalling processes are compartmentalised originated from studies analysing the selective activation of protein kinase A (PKA) RI and RII isoforms in cardiac myocytes, by Brunton et al. [11], [12]. Recently, a variety of genetically encoded probes have independently identified intracellular gradients of cAMP occurring in cells, providing additional proof for the concept of compartmentalised cAMP signalling [13], [14], [15], [16], [17], [18], [19], [20]. There is now a good appreciation that PKA sub-populations, which are localised at distinct sites in cells, can sense and act on intracellular gradients of cAMP [2], [3]. A disparate family of PKA anchor proteins, called AKAPs, provide the means of tethering of PKA regulatory (R) subunits to distinct intracellular sites [2], [3]. AKAPs achieve this by interacting with the dimerisation interface of, predominantly, PKA type-II (PKA-RII) [21]. As AKAPs show a unique pattern of intracellular location, they serve to constrain the ability of tethered PKA to be activated by spatially localised intracellular cAMP gradients of appropriate magnitude. Additionally, AKAPs serve as multi-functional scaffold proteins that can recruit specific PKA substrates as well as various regulatory proteins. Thus AKAP-tethered PKA-RII populations are set to ‘read’ specific, spatially localised intracellular cAMP gradients and act accordingly.
The means through which intracellular cAMP gradients are shaped in cells so as to regulate specific AKAP-tethered PKA sub-populations is only just beginning to be appreciated. Adenylyl cyclase family members, located at the plasma membrane, generate cAMP and thereby provide point sources of cAMP generation [1]. The various adenylyl cyclase isoforms show distinct levels of basal activity in resting cells [22], [23], [24]. These enzymes can be activated, however, by specific Gs coupled receptors that are localised to distinct plasma membrane sub-domains and allow spatially distinct point sources of cAMP generation. The only means of degrading cAMP in cells, and thus for establishing and shaping cAMP gradients, is through the action of cAMP phosphodiesterases (PDEs) [25], [26], [27], [28], [29], [30]. A large, multi-gene family encodes a plethora of PDEs able to hydrolyse cAMP [1]. Detailed examination has shown that a feature of many PDE isoenzymes is targeting to specific intracellular sites, as exemplified in particular detail for the PDE4 family [28]. This, coupled with observations that selective inhibitors of particular PDE families can exert distinct actions of cell function, indicates that PDEs are set to play a key role in underpinning compartmentalised cAMP signalling.
Currently there is considerable interest in PDE4 cAMP phosphodiesterases [25], [27], [28]. This is because PDE4 selective inhibitors, which act as anti-inflammatory agents and memory enhancers, are being developed for use in treating asthma, chronic obstructive pulmonary disease (COPD) and depression [31], [32], [33]. Furthermore, the PDE4D gene has been linked to common atherogenic stroke [34]. However, a key feature of many PDE4 isoforms is their ability to be targeted to specific intracellular sites [27], [28]. This was first shown for the PDE4A1 isoform, whose unique, isoform-specific N-terminal region confers exclusive membrane-association [35], [36], [37], [38]. Intracellular targeting of PDE4 isoforms is also achieved through protein–protein interactions, as in the binding of SRC family tyrosyl kinases to PDE4A4/5 [39], [40], of RACK1 to PDE4D5 [41], [42], of myomegalin to PDE4D3 [43], of the immunophilin, XAP2 to PDE4A5 [44] and of βarrestin to various PDE4 isoforms [45], [46]. Thus anchored PDE4 isoforms are eminently poised to shape gradients of cAMP in cells and thence to determine the selective activation of AKAP-anchored PKA.
It is generally understood that the key to appreciating the physiological significance of activation of adenylyl cyclase by a Gs coupled receptor is to determine the ratio of PKA activity in stimulated cells compared to that observed in resting cells. In this regard, adenylyl cyclase isoforms vary considerably in their basal activity and are expressed in a cell-type specific fashion [1], [22], [23], implying that controlling basal cAMP levels in resting cells is likely to be of some considerable importance. Indeed it is differences in the levels of basal PKA activity seen in cells (see e.g. [47], [48]) that prompts analysis of PKA activity as an activation ratio [49]. Such basal PKA activity in unstimulated cells will depend on the prevailing [cAMP], which is itself determined not only by the rate of cAMP synthesis by adenylyl cyclase but also its degradation by cAMP phosphodiesterases (PDEs) [50]. We set out here to use a novel strategy to identify whether specific PDE4 isoforms can serve a role in regulating localised PKA activity in unstimulated cells. We have developed a dominant negative strategy to gain insight into the functional role of anchored PDE4s in cells [51]. This involves engineering specific PDE4 isoforms such that they are catalytically inactive. These are overexpressed in cells to such a level that they serve to displace the corresponding, endogenous active PDE4 isoform from its functionally relevant anchor site. In doing this we anticipate altering gradients of cAMP in the locality of such dominant negative PDE4 isoforms, thereby increasing the sensitivity of any nearby PKA to activation. We have employed this dominant negative strategy to demonstrate that the displacement of PDE4D5 from complex with βarrestin prevents β2-agonist challenge from delivering an active PDE4D4/βarrestin complex to the β2-adrenoceptor and lowering localised cAMP levels there [51]. Thus overexpression of dominant negative PDE4D5 enhances the ability of β2-agonist to activate plasma membrane localised PKA and thereby to allow PKA phosphorylation of the β2-adrenoceptor itself. Here we use such a dominant negative strategy to show that distinct PDE4 isoforms regulate the sensitivity of an AKAP-anchored PKA-RII -population to be acted upon by basal levels of cAMP production in COS1 cells. As a reporter of localised PKA action we have used the recombinant PDE4s themselves, as their UCR1 regulatory module provides a target for phosphorylation by PKA [52], [53], [54], [55]. In doing this we identify that catalytically inactive PDE4D3 and PDEC2, but not inactive PDE4A4 and PDE4B1, become phosphorylated by PKA in resting COS cells.
Section snippets
Methods
The COS-1 cell line (ATCC CRL 1650) is an SV40 virus transformed African green monkey derived fibroblast cell line. Restriction enzymes, Complete Protease Inhibitor Cocktail tablets and dithiothreitol were from Roche (Mannheim, Germany). Tris, HEPES, DEAE-dextran (M. wt. 500,000), cytochalasin B, benzamidine hydrochloride, PMSF, aprotinin, pepstatin A, antipain, DMEM, FCS, EDTA, EGTA, cyclic AMP, cyclic GMP, Dowex 1X8-400 (chloride form, 200–400 mesh), 3-isobutyl 1-methylxanthine, snake venom (
Results
Here we set out to displace anchored populations of endogenous, active PDE4 long isoforms in COS1 cells by overexpressing their cognate inactive forms. The aim of this was to see if such an approach elicited the activation of a tethered, PDE4-associated, PKA sub-population and so determine if specific long PDE4 isoforms serve to regulate anchored PKA activity in resting cells. In order to generate inactive PDE4 species we exploited the fact that alanine mutation of a conserved aspartate group
Discussion
PDE4 cAMP phosphodiesterases play a key role in forming and shaping the intracellular gradients of cAMP that are sensed and acted upon by PKA [15], [16], [28]. Indeed, in recent studies done on cardiac myocytes, the use of spatially restricted probes able to detect cAMP dynamically in cells has clearly shown that PDE4 enzymes regulate pools of cAMP that are distinct from those regulated by PDE3 [16], [77]. Underpinning this is the targeting of particular PDEs to distinct intracellular sites. In
Acknowledgements
MDH would like to thank the Medical Research Council (UK) (G8604010) for funding. KT and MDH would like to thank the European Union (QLK3-CT-2002-02149) for funding. TP would like to thank the BBSRC (UK) and Novartis for a CASE research studentship.
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Editorial
2016, Cellular SignallingProtein-protein interactions of PDE4 family members - Functions, interactions and therapeutic value
2016, Cellular SignallingCitation Excerpt :This is associated with an activation of ERK1/2 and thus caused by a local rise in cAMP, PKA activation and G protein switching from Gs to Gi (see Section 2.2) [33]. The dominant negative approach has for example also elucidated that PDE4D3 controls a cAMP microdomain at the β1-adrenoceptor in cardiac myocytes [50] and that PDE4D3 and PDE4C2 control AKAP-anchored PKA activity in the perinuclear region [51]. The dominant negative approach can reveal relevant physiological mechanisms, and contribute to validate a target.
Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains
2015, Cellular SignallingCitation Excerpt :PDE4 isoforms critically define the compartmentalization of cAMP signalling through their ability to be recruited to specific signalling complexes, where they shape cAMP gradients in a temporal and spatial manner [1]. As such, individual isoforms have specific, non-redundant roles acting in defined intracellular compartments; as elucidated through dominant negative, siRNA-mediated knockdown and peptide displacement approaches [19–22]. Their functioning in these distinct locales is dynamically regulated through phosphorylation by kinases such as PKA [23–26], Erk [27–29], MK2 [30,31] and AMPK [30] as well as modification by ubiquitination [32] and sumoylation [33].
- 1
These two individuals contributed equally to this work and should be considered joint first authors.
- 2
Present address: Forschungsinstitut für Molekulare Pharmakologie, Campus Berlin-Buch, Robert-Rössle-Str 10, 13125 Berlin, Germany.