The AKAP Model
Generation of the second messenger cAMP following receptor activation leads to an increase in PKA activity and the subsequent phosphorylation of a variety of cellular substrates. Where and when PKA becomes active determines the specificity of a given response. We have shown that the subcellular targeting of PKA occurs through association with AKAPs—a functionally related family of more than 50 distinct proteins defined by their ability to bind to the PKA holoenzyme. Each AKAP contains at least two functional motifs. A conserved amphipathic helix which slots into a hydrophobic pocket formed by the amino terminus of the PKA regulatory subunit (RII) dimer. Also each AKAP contains a unique targeting domain that directs the kinase-AKAP complex to distinct intracellular sites.
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. Two approaches that have been exploited are the (1) disruption of anchoring using peptides derived from AKAPs to interfere with PKA binding, and (2) expression of anchoring deficient forms of the AKAP. Early 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 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 Ca++ entry through L-type Ca++ channels. Ongoing studies on the role of PKA anchoring in insulin homeostasis using knockout mice in which a particular AKAP has been knocked down or in which the knockout animal has been rescued with an anchoring deficient mutant AKAP are addressing these questions.
AKAP Signaling Networks
Many AKAPs are also multivalent scaffold proteins that associate with numerous signaling enzymes including other kinases, protein phosphatases, phosphodiesterases and substrates to form signaling complexes. Specific combinations of anchored enzymes allow these complexes to respond to distinct second messenger–mediated signals. Indeed, studies are underway to examine if AKAPs interact with other multivalent scaffold proteins to allow convergence of signaling pathways in a context specific manner.
By simultaneously binding enzymes with opposing actions, such as kinases and phosphatases, AKAPs target entire signaling complexes to specific substrates. A prototypic example is the neuronal anchoring protein, AKAP79, that targets PKA, protein kinase C (PKC), and the calcium/calmodulin-dependent phosphatase (PP2B) to sites in the 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, Ca++, and phospholipids. Furthermore, AKAP79 can assemble distinct macromolecular complexes within different cellular contexts. In hippocampal neurons AKAP79 forms a complex with AMPA receptors in which PKA phosphorylation and Ca++/calmodulin regulated PP2B dephosphoryation work together to regulate channel activity. However, in superior cervical ganglion neurons the same AKAP interacts with M-channels and it is the AKAP79-bound PKC that is the binding partner that regulates suppression of the currents. Furthermore, recent work has shown that interaction with AKAP79 protects this kinase from inhibition with certain ATP competitive inhibitors. Therefore, intracellular binding partners not only couple regulation of individual molecular events in a cell signaling process, but can also change the pharmacological profile of a protein kinase.
In the heart, the anchoring proteins mAKAP and AKAP-Lbc play critical roles in organizing signaling complexes in response to fluctuations in cAMP, oxygen depletion, elevation in catecholamines and cardiac hypertrophy. In addition to synchronizing a cAMP-feedback loop by anchoring both PKA and a PDE, the mAKAP complex includes the cAMP-responsive guanine nucleotide exchange factor Epac-1, the kinase ERK5, and the transcription factor HIF-1α.
A key cellular response to a state of reduced oxygen tension (hypoxia) involves induction of genes by hypoxia-inducible factor 1α (HIF-1α). Under normoxic conditions HIF-1α is kept low through ubiquitin mediated proteasomal degradation. However, when oxygen drops the myocytes enter a hypoxic state and the destruction of HIF-1α halts. This allows the protein to form a stable complex with HIF-1β to initiate transcription of genes to promote cell survival in response to a sudden ischemic event. mAKAP organizes several ubiquitin E3 ligases that managed the stability of HIF-1α and optimally positioned it close to its site of action inside the nucleus. Compartmentalization of oxygen-sensitive signaling components may influence the fidelity and magnitude of the hypoxic response. These findings infer a link between hypoxia and hypertrophic signaling pathways. Indeed, mAKAP may provide such a link as mAKAP levels are increased in response to hypertrophic stimuli and it anchors two signaling enzymes that act sequentially to influence hypertrophy and the stability of HIF-1α. One of these, the cAMP-responsive guanine nucleotide exchange factor Epac-1, is the upstream element in a signaling pathway that modulates the activity of ERK5, a protein kinase that augments the hypertrophic response and influences the stability of HIF-1α. Thus, mAKAP complexes may create cellular microenvironments in which cAMP signals can feed into oxygen-responsive transcriptional activation pathways.
Elevated catecholamines in the heart evoke transcriptional activation of the Myocyte Enhancer Factor (MEF) pathway to induce a cellular response known as pathological myocardial hypertrophy. The anchoring protein AKAP-Lbc is one of the genes upregulated in hypertrophic myocytes. This AKAP functions as a scaffolding protein for PKA and PKC to mediate activation of a third enzyme in the complex, protein kinase D (PKD). Activated PKD is released from the complex and moves into the nucleus where it phosphorylates the histone deacetylase HDAC5 to promote its nuclear export. Finally, the concomitant reduction in nuclear histone deacetylase activity favors MEF2 transcription and the onset