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The Molecular Architecture of Signal Transduction Complexes
Summary: John Scott is interested in the specificity of signal transduction events that are controlled by anchoring proteins, which facilitate rapid signal transduction by optimally positioning protein kinases and phosphatases in the vicinity of their activating signals and close to their substrates.
My research program focuses on defining the intracellular communication networks that promote specificity in signal transduction events. We have identified a family of A-kinase–anchoring proteins (AKAPs) that target the cAMP-dependent protein kinase (PKA) and other signaling enzymes to specific subcellular sites. AKAPs influence the specificity of second messenger–mediated signal transduction events by targeting enzymes close to their appropriate effectors and substrates. Our lab has made progress on establishing the AKAP model, the functional consequences of PKA anchoring, and the role of AKAP signaling complexes in the coordinate regulation of certain synaptic and cytoskeletal signaling events.
The AKAP Model
The second messenger cAMP is generated following receptor activation by many hormones and neurotransmitters. This leads to increased PKA activity and subsequent phosphorylation of a variety of cellular substrates. Specificity in this signaling pathway is influenced by factors that control where and when PKA becomes active. We have shown that the subcellular localization of PKA occurs through association with AKAPs—a functionally related family of more than 40 distinct proteins classified by their ability to bind to the PKA holoenzyme. Each AKAP contains at least two functional motifs. A conserved amphipathic helix forms a PKA-binding domain that slots into a hydrophobic pocket formed by the amino terminus of the kinase regulatory subunit (RII) dimer. Each AKAP also contains a unique targeting domain that directs the kinase-AKAP complex to distinct intracellular sites. In addition, many AKAPs form multivalent signaling complexes by associating with additional enzymes, including protein phosphatases and other kinases. Such specific combinations of anchored enzymes may allow these complexes to respond to distinct second messenger–mediated signals.
Functional Consequences of PKA Anchoring
The biological relevance of anchoring is underscored by functional studies that have used AKAPs as reagents to manipulate the distribution of PKA inside cells. To date, two approaches have been exploited: (1) cellular disruption of anchoring, using inhibitor peptides derived from AKAPs to block PKA binding, and (2) expression of compartment-specific AKAPs to redistribute the kinase to defined intracellular sites. Many of these studies have focused on rapid cAMP-responsive events, such as modulation of ion channels. Initial studies demonstrated that perfusion of cultured hippocampal neurons with anchoring inhibitor peptides caused a time-dependent "rundown" in AMPA-responsive glutamate receptor currents. Similar approaches suggest that anchored PKA plays a role in the regulation of cardiac and skeletal muscle L-type Ca2+ channels and neuronal N-methyl-D-aspartate (NMDA) receptor channels.
AKAPs also regulate PKA function in more complicated physiological systems, such as hormone-mediated insulin secretion from pancreatic islet β cells. Peptide-mediated disruption of the AKAP-RII interaction inside intact cells reduces insulin secretion, and membrane targeting of the kinase stimulates this process. In part, this effect might result from enhanced Ca2+ entry through L-type Ca2+ channels.
AKAP Signaling Complexes
Although AKAPs have been defined by their ability to bind to PKA, many AKAPs also interact with additional signaling enzymes. By simultaneously binding enzymes with opposing actions, such as kinases and phosphatases, these multivalent anchoring proteins target entire signaling complexes to specific substrates. A prototypic example is the synaptic anchoring protein, AKAP79, that targets PKA, protein kinase C (PKC), and the calcium/calmodulin-dependent phosphatase (PP2B) to sites in the postsynaptic membrane. Since distinct activation signals are necessary to release and activate each enzyme, AKAP79 provides a point of convergence for multiple second messenger signals, such as cAMP, Ca2+, and phospholipids.
AKAP79 itself is recruited into a larger "synaptic transduction unit" that includes PKA effectors such as the β2-adrenergic receptor and is cross-linked to glutamate receptors through a protein bridge formed by MAGUK proteins. Biochemical and electrophysiological studies indicate that this elaborate molecular architecture ensures that PKA and PP2B preferentially modulate the phosphorylation status of serine 845, a regulatory site on the GluR1 subunit of the AMPA channel. Similarly, another AKAP, the NMDA receptor–associated protein yotiao, binds to protein phosphatase I (PP1) as well as PKA. Yotiao holds PP1 in an active state. The functional consequence of this appears to be a limiting of NMDA channel activity. Activation of PKA by cAMP overcomes this inhibition, leading to an enhancement of the channel current. In this way, yotiao directly targets a kinase and a phosphatase with opposing functions to its channel substrate. (A grant from the National Institutes of Health provided support for this project.)
Recently, we have identified AKAPs that organize signaling complexes involved in actin-remodeling events. Gravin, an AKAP that binds both PKA and PKC, accumulates at the filopodia of macrophage-like cells in response to phorbol ester signals. A newly identified AKAP, Scar/WAVE-1, is a member of the Wiskott-Aldrich syndrome (WASP) family of actin-binding proteins that functionally couples the GTPase Rac to cytoskeletal components and the Arp2/3 complex. We have shown that Scar/WAVE-1 is also a multivalent scaffold that recruits PKA and the nonreceptor tyrosine kinase c-Abl to lamellipodia and sites of actin reorganization in response to extracellular signals that activate Rac. Likewise, we have recent data that demonstrate that AKAP Ht31 is a GTP exchange factor for Rho-1 and a member of the LBC family of proto-oncogenes. (A grant from the National Institutes of Health provided support for this project.)
Thus the level of organization achieved by AKAP signaling complexes is more precise than originally was appreciated. Conceivably, different AKAP-enzyme complexes might segregate distinct signaling components/enzymes with different substrates within a single cellular compartment. Such "nanocompartmentalization" might be a major key to specificity in signal transduction and the coordinated regulation of complex biological systems.
Last updated June 01, 2005
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