The where's and when's of kinase anchoring

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Kinase anchoring has gained acceptance as a means to synchronize spatial and temporal aspects of cell signaling. A-kinase anchoring proteins (AKAPs) are a diverse group of functionally related proteins that target protein kinase A and other enzymes to coordinate a range of signaling events. Recent advances in this field have shown that incorporating phosphodiesterases into AKAP signaling complexes exerts local control of cAMP metabolism, that phosphorylation of some AKAPs potentiates downstream signaling events, that anchoring of distinct enzyme combinations functions as a mechanism to expand the repertoire of cellular events controlled by a single AKAP, and that fluorescent biosensors can be used to visualize dynamic aspects of localized cAMP signaling.

Introduction

A half-century of work has proclaimed protein phosphorylation as a principal means of reversibly controlling biochemical events. It began when Fischer and Krebs [1] demonstrated that conversion of inactive muscle phosphorylase b into active phosphorylase a requires ATP and is catalyzed by a phosphorylase b kinase. They then showed that phosphorylase b kinase itself is controlled by a serine kinase that is responsive to the second messenger cAMP. Together with their colleagues, they subsequently discovered protein kinase A (PKA) and the concept of a ‘kinase cascade’ [2].

From these humble beginnings the field of protein kinase research was born, paving the way for the discovery of phosphotyrosine by Hunter and co-workers [3], the identification of Src tyrosine kinase by Brugge and Ericsson et al. [4], the seminal work of Pawson and associates on phosphotyrosine recognition motifs [5], and the resolution of a crystal structure of the PKA catalytic subunit by Taylor and colleagues [6]. Consequently, we now recognize that the ∼518 members of the human kinome represent a superfamily of enzymes that participate in all aspects of cellular regulation [7].

Protein kinase research is now focusing on how these enzymes are organized in relation to their effectors and substrates within the three dimensions of the cell. Interestingly, the study of PKA is shedding new light on the complexity of these protein–protein interactions.

A-kinase anchoring proteins (AKAPs) have emerged as important signal-organizing components for PKA and other kinases. Here, we discuss their role in the spatio-temporal control of cAMP signaling and other transduction events.

Section snippets

In the beginning*

It was clear from early physiology experiments that stimulation of cAMP synthesis by different agonists produces distinct physiological outputs, even within the same tissue. It was subsequently suggested that the occupancy of particular G-protein-coupled receptors (GPCRs) favored the activation of PKA pools located in different subcellular compartments. Conclusive evidence supporting this concept was not obtained until the early 1980s, however, when it was shown that adrenergic stimulation

AKAP signaling complexes

Another biological role of AKAPs became apparent when it was discovered that AKAP79 (the human ortholog of AKAP75 and AKAP150) not only anchored PKA but also bound the protein phosphatase PP2B [28]. This finding changed the way in which we think about AKAPs because it suggested that signals controlling phosphorylation and dephosphorylation of a single substrate can pass through the same AKAP signaling complex.

The notion of multivalent AKAPs continued to evolve when subsequent studies showed

AKAPs and phosphodiesterases: compartmentalization of cAMP action

Spatiotemporal control of cAMP flux requires the concerted action of two enzyme classes: adenylyl cyclases, which synthesize cAMP; and compartmentalized pools of phosphodiesterases (PDEs), which locally metabolize cAMP into 5′-AMP. The laboratories of Houslay (http://www.gla.ac.uk/ibls/BMB/mdh) and Conti (http://www-med.stanford.edu/profiles/Marco_Conti) have identified several PDE-binding proteins that target distinct isozymes to specific cellular microenvironments [34]. A prototypical example

Phosphorylation in AKAP complexes

Soon after AKAPs were discovered, it was postulated that they that would be the preferred substrates for their associated kinases [48]. Although experimental evidence supporting this prediction has been slow in coming, three recent examples suggest that it might be correct.

First, the AKAP Yotiao associates with various ion channels, including the KCNQ1 subunit of K+ channels responsible for IKS currents that shape the duration of cardiac action potentials in response to β-adrenergic agonists

Combinatorial assembly of distinct AKAP signaling complexes

A basic premise of AKAP action is that signaling specificity is obtained when enzymes are targeted towards selected substrates. This use of distinct enzyme combinations provides a way in which to expand the repertoire of cellular events that can be modulated by a given AKAP. This concept is illustrated by work published in 2005 by Hoshi et al. [59] showing that AKAP79/150 coordinates different enzyme combinations to modulate the activity of two distinct neuronal ion channels: AMPA-type

Time: the final frontier

Although we have identified most, if not all, of the proteins that make up the cAMP signal transduction cascade, we still face the challenge of resolving the mechanics of their action in real time. Fluorescent probes that report the activation dynamics of cAMP effector proteins such as PKA, cyclic-nucleotide-gated (CNG) ion channels and Epac-GEFs have become the methods of choice to visualize the dynamics of cAMP signaling inside cells. Pioneering work by Roger Tsien's laboratory (//www.tsienlab.ucsd.edu/

Concluding remarks

We anticipate that these cutting-edge imaging technologies will be central to obtaining precise definitions of the spatial and temporal patterns of anchored kinase function in the next decade. The information gained could be used in conjunction with computational models of signal transduction to predict the effects of perturbing the system. Ultimately, a balance of both approaches might contribute to the development of therapeutics that target ‘signaling diseases’ such as heart failure, asthma,

Acknowledgements

We thank Debbie Willoughby, Dermot Cooper and Jin Zhang for providing unpublished data for use in Figure 4; Joe Soughayer for the FRET images in Figure 4; and all members of the Scott Laboratory for helpful discussions. J.D.S. was supported in part by a grant from the National Institutes of Health (DK54441).

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