AKAPs (A-kinase anchoring proteins) are members of a diverse family of scaffold proteins that minimally possess a characteristic binding domain for the RI/RII regulatory subunit of protein kinase A and play critical roles in establishing spatial constraints for multivalent signalling assemblies. Especially for G-protein-coupled receptors, the AKAPs provide an organizing centre about which various protein kinases and phosphatases can be assembled to create solid-state signalling devices that can signal, be modulated and trafficked within the cell. The structure of AKAP250 (also known as gravin or AKAP12), based on analyses of milligram quantities of recombinant protein expressed in Escherichia coli, suggests that the AKAP is probably an unordered scaffold, acting as a necklace on which ‘jewels’ of structure–function (e.g. the RII-binding domain) that provide docking sites on which signalling components can be assembled. Recent results suggest that AKAP250 provides not only a ‘tool box’ for assembling signalling elements, but may indeed provide a basis for spatial constraint observed for many signalling paradigms. The spatial dimension of the integration of cell signalling will probably reflect many functions performed by members of the AKAP family.

Organization of AKAPs (A-kinase anchoring proteins): domains, domains and more domains

The family of scaffold proteins that display a binding domain for the RI/RII subunit of protein kinase A is large (>70 members) and diverse [1,2]. Each member displays multivalency with respect to signalling elements, possessing binding domains in addition to that for the RII subunit of protein kinase A [3]. AKAPs are expressed in a wide range of species from worms, to flies, to fish, to frogs and mammals [4]. With regard to multivalency, AKAP250 displays minimally docking sites for protein kinase A, protein kinase C, protein phosphatases, the non-receptor tyrosine kinase Src, and for a prominent member of the superfamily of GPCRs (G-protein-coupled receptors), the β2-adrenergic receptor (Figure 1) [5,6]. Other AKAPs similarly display a wide range of domains through which signalling elements can assemble and interact.

Schematic view of AKAP250 topological map: location of known and suspected structure–function domains

Figure 1
Schematic view of AKAP250 topological map: location of known and suspected structure–function domains

The major landmarks indicated are the N- and C-termini and the AKAP250-binding domains for protein kinase C (PKC), GPCR (AKAP250/AKAP79) and protein kinase A (PKA). Other structure–function domains are labelled and described in the text.

Figure 1
Schematic view of AKAP250 topological map: location of known and suspected structure–function domains

The major landmarks indicated are the N- and C-termini and the AKAP250-binding domains for protein kinase C (PKC), GPCR (AKAP250/AKAP79) and protein kinase A (PKA). Other structure–function domains are labelled and described in the text.

When one considers the relative low abundance of many signalling molecules (e.g. protein kinase A, protein kinase C and Src), the role of AKAPs in increasing the local concentrations of these signalling elements and the impact of this concentrating effect on the dynamics and temporal character of signalling cannot be overstated. The kinetic character of a biological response that depends on the concerted activation of multiple-signalling pathways also is highly sensitive to the local concentration of those signalling elements. AKAPs provide a multivalent template for cell signalling that overcomes many of the obstacles of organizing protein–protein interactions among low abundance proteins, providing a dynamic, reversible platform for signalling [7].

Structural analysis of AKAP250 and many other AKAPs has been limited largely to analysis of the specific domains, such as the signature RI/II subunit-binding domain found in all AKAPs [8]. The atomic structure has been revealed for the RII-binding site, but little information is available on full-length AKAPs. We have expressed recombinant human AKAP250 in Escherichia coli under conditions that permit isolation of milligram quantities of the essentially pure protein. Unfortunately, conditions suitable for crystal formation of AKAP250 have not been identified, if in fact crystallization of the full-length molecule is possible, and physical analysis of this AKAP in solution have essentially confirmed the results of a thoughtful analysis of the primary sequence suggesting that the AKAP is largely unordered, lacking secondary and tertiary structure except in the domains that act as docking sites for other molecules. We envision this arrangement as an unordered ‘necklace’ into which have been inserted more highly structured domains that appear as ‘beads’ on the necklace. The necklace can provide for dynamic membrane association, targeting of signalling assemblies and the opportunity to traffic these assemblies all together to various regions of the cell.

AKAP localization: creating functional compartmentation in cells

AKAPs are expressed in many tissues and appear to localize preferentially to specific cellular compartments [4]. AKAP75/AKAP79/AKAP150, for example localize to the plasma membrane in nervous tissues [911], found in very high abundance at postsynaptic densities. AKAP82, on the other hand, seems to be expressed only in testis, more specifically in the fibrous sheath of the sperm tail [12]. Yotiao localizes largely to the neuromuscular junction, organizing protein kinase A and phosphoprotein phosphatase PP1 to NMDA (N-methyl-D-aspartate) receptors [13]. AKAP250, in contrast, is expressed widely in tissues [14] and can be seen to occupy the cytoplasmic and perinuclear regions of cells (Figure 2).

Agonist-stimulated trafficking of AKAP250 to the cell membrane

Figure 2
Agonist-stimulated trafficking of AKAP250 to the cell membrane

Confocal images of A431 cells stably transfected with haemagglutinin-tagged human AKAP250. The cells were serum-deprived overnight and stimulated with β-adrenergic agonist (10 μM isoprenaline, +Iso) for 30 min at 37°C. Cells were fixed and stained for haemagglutinin antigen (rat monoclonal antibody, 1:2000; Sigma) and secondary antibodies (Alexa Fluor 647 goat anti-rat IgG, 1:400; Molecular Probes). The fluorescent labelling was examined by the use of a confocal laser-scanning microscope (Zeiss LSM 510 inverted microscope) equipped with a krypton–argon laser. Original magnification, ×63 (oil immersion). Scale bar, 10 μm. Yellow arrows are provided to highlight dense fluorescent label accumulation in the vicinity of plasma membrane.

Figure 2
Agonist-stimulated trafficking of AKAP250 to the cell membrane

Confocal images of A431 cells stably transfected with haemagglutinin-tagged human AKAP250. The cells were serum-deprived overnight and stimulated with β-adrenergic agonist (10 μM isoprenaline, +Iso) for 30 min at 37°C. Cells were fixed and stained for haemagglutinin antigen (rat monoclonal antibody, 1:2000; Sigma) and secondary antibodies (Alexa Fluor 647 goat anti-rat IgG, 1:400; Molecular Probes). The fluorescent labelling was examined by the use of a confocal laser-scanning microscope (Zeiss LSM 510 inverted microscope) equipped with a krypton–argon laser. Original magnification, ×63 (oil immersion). Scale bar, 10 μm. Yellow arrows are provided to highlight dense fluorescent label accumulation in the vicinity of plasma membrane.

At the cellular level, AKAPs display localization that is selective (if not specific) and dynamic. Many AKAPs (e.g. Ezrin and AKAP250) are found in close association with the actin cytoskeleton [2]. AKAP-KL also associates with the actin cytoskeleton, but when expressed in epithelial cells this association is confined predominantly to the apical rather than basal region of the cell [15]. AKAP350, in contrast, localizes to the Golgi apparatus and centrosome [1618], whereas AKAP149 localizes to the outer membrane of mitochondria in the flagellar cytoskeleton [19]. Thus AKAPs act as targeting devices that assemble signalling elements on a scaffold that itself targets to microdomains in cells. Microdomains, such as the actin cytoskeleton Golgi apparatus and plasma membrane, are made accessible by virtue of AKAP-binding domains targeting these molecules. N-myristoylation and the presence of domains in AKAPs that reversibly interact with the GPCRs embedded in the lipid bilayer probably address AKAP250/AKAP79 to their proper microdomains in cells, i.e. the plasma membrane [2]. This particular cellular localization of AKAP250/AKAP79 enables cell signalling to occur within defined boarders, creating spatially distinct compartments. The compartmentation is achieved by overcoming the problems of protein–protein interactions among low abundance proteins, while ensuring that the generation of signals (e.g. changes in intracellular concentrations of cyclic nucleotides, Ca2+ and inositol phosphates) will be local, achieving functional levels only at points in close proximity to the scaffold. Furthermore, it has been shown that mutation of the RII-binding domain for protein kinase A on AKAP250 provokes the inability of that AKAP molecule and the GPCR to which it binds from being substrates for protein kinase A, even in cells in which the amount of wild-type and mutant AKAP expressed is equivalent [20]. The conclusion drawn from such results is that the AKAP brings in its own ‘tool box’, and those protein kinases, phosphoprotein phosphatases and adaptor molecules act ‘cis’ and not ‘trans’ with respect to the AKAP-based signalling complex and the functions provided by the molecules bound to the AKAP domains [20].

An additional benefit provided by the ability of the AKAP to act as a scaffold for multivalent signalling is that, at least in some cases (e.g. AKAP250), the scaffolds are truly ‘mobile’ [5]. For the well-known activation, desensitization, internalization, resensitization and recycling of GPCR-based signalling complexes, it was of interest to define where the scaffold localizes after activation of the receptor with an agonist (Figure 2). For AKAP250, activation of the β2-adrenergic receptor-based signalling complex results in enhanced association of the scaffold with the plasma membrane, probably reflecting the essential role of this AKAP in receptor resensitization and recycling [20]. Suppression of AKAP250 expression leads to a disregulation of GPCR resensitization and recycling [6,21]. This particular AKAP provides a binding domain for at least one phosphoprotein phosphatase involved in resensitization of the β2-adrenergic receptor [27]. Other AKAPs have been shown to provide a binding domain for phosphodiesterase activity that would also contribute to the dampening of an agonist-stimulated increase in the local concentration of cyclic nucleotides [22,23]. Analysis of the localization of AKAP250, the receptor and other components by confocal microscopy clearly shows the scaffold as mobile, trafficking with the internalized receptor and several other key molecules bound to the scaffold as agonist-induced desensitization/internalization proceeds [5].

AKAP250, a necklace for GPCR signalling decorated with beads of structure–function

Although first disappointed by the apparent unordered structure of the purified rAKAP250 scaffold, we have developed a new working hypothesis about this scaffold that may have relevance towards the understanding of the function and evolution of AKAPs. The unordered structure of AKAP250 we envision as a necklace, decorated with beads that are domains conferring structure–functions to the scaffold in toto. We can elaborate on the nature and organization of these ‘beads’, although the analysis of this important molecule is still in its infancy. Starting from the N-myristoylation site and concluding with the C-terminal region beyond the RII-binding domain for protein kinase A (Figure 1), we encounter more than 12 domains of structure–function. The N-myristoylation site provides for increasing the relative concentration of AKAPs in the vicinity of the cell membrane by 10–100-fold. Unlike palmitoylation, which firmly embeds a local region of a modified protein into the lipid bilayer, the C-10 extension of the myristoyl group penetrates the lipid bilayer reversibly, providing an ideal ‘clasp’ for the AKAP necklace.

The next domains encountered moving C-terminally in AKAP250 are three domains that may overlap with each other and resemble the MED (membrane effector domain) of the MARCKS (myristoylated alanine-rich C-kinase substrate) protein (Figure 3) and have homology to a Ca2+/calmodulin-binding site that provides regulatable membrane association [24,25], subject to reversible phosphorylation by protein kinases A and C, two resident ligands of AKAP250. The binding domain for protein kinase C is C-terminal to the MEDs and has embedded within it both a putative F-actin-binding domain, as well as a PXXP domain to which the non-receptor tyrosine kinase Src can associate. Src is a well-known regulator of GPCR-signalling complexes [26]. Abutting the protein kinase C-binding domain is a domain with high homology to sites that bind phosphoprotein phosphatases, such as calcineurin [27]. Thus AKAP250 displays binding of two serine/threonine protein kinases (protein kinases A and C), a non-receptor tyrosine kinase (Src) and a prominent protein phosphatase (calcineurin).

Association of the MARCKS protein-like MED found in AKAP250 with the lipid bilayer

Figure 3
Association of the MARCKS protein-like MED found in AKAP250 with the lipid bilayer

AKAP250 displays an N-terminal region that is highly analogous to the MED of the MARCKS protein. The proposed interaction of the positively charged (boldface), polar (dark grey) and hydrophobic (light grey) residues of this domain with the negatively charged phospholipids constituting the lipid bilayer is shown. The model displays polar head groups of residues embedded by electrostatic charges into the inner leaflet of the bilayer. Experimentally, the interaction of the MED with the lipid bilayer can be largely neutralized by increases in Ca2+ and the concentration of calmodulin (CaM) or by the action of protein kinase C (PKC). The authors propose that the same combination of physical forces and regulation by Ca2+, CaM and PKC may be operating in the regulation of AKAP250–lipid bilayer interactions. (Adapted with permission from [25]. © (2004) Biophysical Society.

Figure 3
Association of the MARCKS protein-like MED found in AKAP250 with the lipid bilayer

AKAP250 displays an N-terminal region that is highly analogous to the MED of the MARCKS protein. The proposed interaction of the positively charged (boldface), polar (dark grey) and hydrophobic (light grey) residues of this domain with the negatively charged phospholipids constituting the lipid bilayer is shown. The model displays polar head groups of residues embedded by electrostatic charges into the inner leaflet of the bilayer. Experimentally, the interaction of the MED with the lipid bilayer can be largely neutralized by increases in Ca2+ and the concentration of calmodulin (CaM) or by the action of protein kinase C (PKC). The authors propose that the same combination of physical forces and regulation by Ca2+, CaM and PKC may be operating in the regulation of AKAP250–lipid bilayer interactions. (Adapted with permission from [25]. © (2004) Biophysical Society.

The association of AKAP250 with the plasma membrane is also influenced by a unique sets of sites located near the midpoint of the protein. This region has been shown to harbour a domain that supports reversible binding of the scaffold to the heptahelical GPCR, β2-adrenergic receptor. Agonist activation of the signalling complex leads to enhanced association of the scaffold with this GPCR, enhanced phosphorylation of the scaffold as well as the receptor, and movement of the mobile scaffold with the receptor to vesicles that traffic to an intracellular compartment [20]. This domain has been found in AKAP79 and AKAP250, each scaffold demonstrating reversible agonist-driven association to the β2-adrenergic receptor. To what extent AKAPs generally target to the plasma membrane through association with a GPCR remains an open question. In the region C-terminal to the AKAP250/AKAP79 domain, studies are concentrating on defining possible sites of association of phosphodiesterase(s). This work on AKAP250 is in its infancy and will evolve slowly due to the plethora of possible phosphodiesterase partners expressed in most cells [28]. Intuitively, the presence of a phosphodiesterase activity seems attractive, providing for another spatial constraint on the accumulation or diffusion of cyclic nucleotides away from the site of activation of the signalling complex.

Most intriguing is the presence of a 600+ amino acid sequence of AKAP250 found in the second half of the molecule for which no partner-binding sites have been identified. In comparison, the first 900 amino acid N-terminal reach of AKAP250 has more than ten domains, organized like beads on a necklace. The next 600+ residues do not display any obvious binding motifs, so that analysis of this region by yeast two-hybrid screening would seem in order. In the C-terminus, last quarter of the sequence, we find a second PXXP domain, discussed above, and the binding site for the RII subunit of protein kinase A. The crystal structure of the RII subunit-binding site of AKAPs has been solved, representing another bead of structure function in the AKAP necklace of G-protein signalling. The remaining C-terminal region of the AKAP also has no known binding sites or activities.

Concluding remarks

The overall structure envisioned of unordered stretches of AKAP decorated with beads of structure–function might well explain the ability of AKAPs to act as integrators of multivalent signalling, to undergo regulatable localization, and to create spatial constraints of cell signalling. AKAPs have been shown to be essential in cell signalling from rapid activation of ion channels, cyclases and phosphodiesterases, to name but a few of the effectors. This localization of protein kinases to the proximity of ion channels probably facilitates regulation of the rapid kinetics of membrane conductance. In other circumstances, the AKAPs appear to provide a scaffold that facilitates longer-term kinetics of activation, desensitization and resensitization. Finally, the AKAP provides an excellent example of a scaffold protein, which assembles a tool box of interesting signalling molecules, traffics them to unique subcellular microdomains, and provides a mobile platform capable of supporting the cycles of activation, desensitization and resensitization necessary for proper cell signalling by GPCRs.

Signalling Outwards and Inwards: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by J. Challiss (Leicester, U.K.), A. Harwood (University College London, U.K.), M. Humphries (Manchester, U.K.), C. Isacke (Institute of Cancer Research, London, U.K.), R. Liddington (Burnham Institute, La Jolla, CA, U.S.A.), T. Palmer (Glasgow, U.K.), K. Siddle (Cambridge, U.K.), C. Sutherland (Dundee, U.K.), H. Wallace (Aberdeen, U.K.) and M. Welham (Bath, U.K.).

Abbreviations

     
  • AKAP

    A-kinase anchoring protein

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • MARCKS

    myristoylated alanine-rich C-kinase substrate

  •  
  • MED

    membrane effector domain

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