Compartmentation of cAMP Signaling

The idea of compartmentalized pools of PKA originated in the late 1970s when Corbin et al. (1977) showed the existence of both soluble and particulate fractions of PKA activity in the rat heart. Later experiments demonstrated that the two pools of PKA were differentially activated. While both particulate and soluble PKA were stimulated by P-adrenergic receptor agonists, ligand binding to prostaglandin receptors only activated soluble PKA (Hayes et al. 1979, 1980). Moreover, while both the P-AR agonist isoproterenol and prostaglandin E1 increased cAMP concentrations, only isoproterenol increased glycogen metabolism and phosphorylation of troponin I (Brunton et al. 1979). These results illustrate that even though different hormones may act through the same second messenger, they can stimulate different pools of PKA and mediate distinct physiological responses.

More recent experiments have allowed the visualization of cAMP compart-mentalization within a living cell. Elegant electrophysiological experiments by Jurevicius and Fischmeister (1996) revealed that local stimulation of P-adrenergic receptors on one side of a cardiac myocyte results in local cAMP production and the restricted stimulation of adjacent calcium channels. Zaccolo (2004) have extended these observations using a fluorescence resonance energy transfer (FRET) cAMP sensor based on the PKA holoenzyme. They found that P-adrenergic agonists preferentially generated cAMP at the myocyte transverse tubule and junctional sarcoplasmic reticulum membranes, while the adenylyl cyclase activator forskolin produced global increases in cAMP levels. Their results suggest that in the cardiac myocyte cAMP could be restricted to compartments as small as 1 |im across. Remarkably, activation of the cAMP sensor by P-adrenergic agonists was dependent on it being anchored by AKAPs and was enhanced by phosphodiesterase inhibition. Later, Mongillo et al. (2004) found that type-4 phosphodiesterases (PDE4) were specifically responsible for modulating the amplitude and duration of P-adrenergic-induced cAMP signaling, while PDE3 controlled other pools of cAMP. The results of these and an increasing number of other recent studies support the existence of distinct cAMP microdomains that control specialized PKA signaling. These signaling domains are, in part, due to AKAPs.

2 AKAPs

AKAPs are a family of over 50 functionally related proteins defined by their ability to bind PKA. The PKA holoenzyme is a tetramer consisting of two catalytic (C) subunits held in an inactive conformation by a regulatory (R) subunit dimer (Scott 1991). Binding of cAMP to the R subunits leads to the dissociation of the holoenzyme and release of active C subunits. The C subunits are multifunctional serine/threonine kinases that phosphorylate a number of downstream targets. To date, three C-subunit genes (Ca, Cß, Cy) and four R-subunit genes (RIa, RIß RIIa, and RIIß) have been identified (Taylor et al. 1990; Scott 1991). While the C-subunits display similar kinetic and physiological properties, the R-subunits exhibit distinct cAMP-binding affinities and sub-cellular localizations (Taylor et al. 1990).

AKAPs bind the R-subunits of PKA via an amphipathic helix motif within the AKAP (Carr et al. 1992; Newlon et al. 2001). The molecular details of this association are well characterized and will be discussed further in a subsequent chapter. Type-II PKA (containing RII subunits) binds to AKAPs with high affinity (Kt = 10-9M) (Carr et al. 1992; Alto et al. 2003). While there are a few AKAPs that bind with high affinity to either type-I PKA alone or both type-I and type-II PKA, most AKAPs bind type-I PKA with a 1,000-fold lower affinity than type-II PKA (Stokka et al. 2006). As a result, type-I PKA is predominately cytosolic, whereas type-II

Fig. 1 Diagram depicting the four properties of AKAPs. AKAPs are defined based upon their ability to bind the R-subunit of PKA. Unique targeting domains contained within the individual AKAP determine their subcellular placement. One of the most intriguing properties of AKAPs is their ability to incorporate multiple signaling pathways, while the association with additional adaptor proteins allows AKAPs to integrate into multi-protein networks

Fig. 1 Diagram depicting the four properties of AKAPs. AKAPs are defined based upon their ability to bind the R-subunit of PKA. Unique targeting domains contained within the individual AKAP determine their subcellular placement. One of the most intriguing properties of AKAPs is their ability to incorporate multiple signaling pathways, while the association with additional adaptor proteins allows AKAPs to integrate into multi-protein networks

PKA is typically associated with cellular structures and organelles. The importance of AKAPs is underscored by the fact that half of the kinase activity in the mammalian heart is associated with the particulate fraction (Corbin et al. 1977).

AKAPs exhibit several common properties (Fig. 1). By definition, all AKAPs bind PKA R-subunits and can mediate the immunoprecipitation of PKA catalytic activity from cells. The second property allows for the specific subcellular location of each AKAP. Distinct binding regions in each AKAP participate in protein/protein or lipid/protein interactions, allowing for the subcellular distribution of the AKAP (Fraser et al. 1998; Dodge and Scott 2000; Westphal et al. 2000). The third and most intriguing characteristic of AKAPs is that they coordinate the integration of enzymes from multiple signaling networks onto a specific substrate. These additional components may be almost any protein in the signal transduction toolbox, including protein kinases, phosphatases, phosphodiesterases, and adenlyl cyclases, as well as G-protein-coupled receptors and ion channels (Coghlan et al. 1995; Klauck et al. 1996; Fraser et al. 1998, 2000; Westphal et al. 1999; Dodge et al. 2001). Lastly, AKAPs are recruited into much larger multiprotein complexes through the interactions with other adapter molecules, such as PDZ and SH3 domain-containing proteins (Colledge et al. 2000; Westphal et al. 2000). These four properties of AKAPs allow the scaffolding proteins to integrate multiple signaling pathways, allowing for the convergence of signals onto a common target.

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