My research program focuses on defining the intracellular communication networks that promote specificity in signal transduction events, and we have identified key molecular players in this process, a family of A-kinase–anchoring proteins (AKAPs). AKAPs target the cAMP-dependent protein kinase (PKA) and other signaling enzymes to specific subcellular sites. By doing so, these anchoring proteins bring enzymes proximal to their appropriate effectors and substrates. This provides a molecular mechanism to spatially and temporally regulate a variety of vital physiological processes, including synaptic transmission, heart rate, and glucose homeostasis. In recent years we have established the functional consequences of PKA anchoring and discerned a broader role of AKAP signaling networks in the coordinate regulation of cellular signaling processes that pertain to disease.
The AKAP Model
Generation of the second messenger cAMP following receptor activation leads to an increase in protein kinase 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 40 distinct proteins defined by their ability to bind to the PKA holoenzyme. Each AKAP contains at least two functional motifs. A conserved amphipathic helix slots into a hydrophobic pocket formed by the amino terminus of the PKA type II regulatory subunit (RII) dimer. Also, each AKAP contains a unique targeting domain that directs the kinase-AKAP complex to distinct intracellular sites.
Although considerable information has been amassed on the physiological consequences of anchored phosphorylation events, much less is known about the mechanism of action. This is due to limited knowledge on the macromolecular organization, topology, and stoichiometry of AKAP-PKA complexes. Nuclear magnetic resonance spectroscopy and x-ray crystallographic studies show that the type I or type II regulatory subunits of PKA (RI and RII) homodimerize through a four-helix-bundle docking and dimerization interface, creating a high-affinity binding groove for AKAPs. An amphipathic helix forms the reciprocal binding surface on each AKAP. Currently, structural details on PKA anchoring are limited to this protein-protein interface, in part because most AKAPs are large, intrinsically disordered macromolecules without recognized structural domains. Therefore, we have employed alternative approaches, including protein mass spectrometry and single-particle fluorescence imaging, to establish the composition and stoichiometry of selected AKAP complexes. We have used electron microscopy to evaluate the architectural arrangement of the type IIα PKA holoenzyme when anchored to AKAP18. Structure-function studies reveal an unanticipated role for intrinsically disordered regions of RII that define the range of motion and radius of action of the anchored kinase. One mechanistic ramification of our structural analyses is that flexibility within the PKA complex could permit precise orientation of the anchored catalytic subunit toward substrates. These data show that AKAPs can be thought of as catalysts that physically bring the reactants together, and the flexibility within the anchored PKA holoenzyme allows for the precise orientation of the enzyme and substrate required for optimal reactivity.
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. Studies are under way to determine whether 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 , which 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, Ca2+, 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 Ca2+/calmodulin-regulated PP2B dephosphorylation 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. Our 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.
A point of intersection between our molecular, cellular, and integrative analyses is the investigation of AKAP signaling in age-related and chronic disorders. AKAP-mediated control of glucose homeostasis is one example in which failure of localized signaling can contribute to extrapancreatic complications of diabetes, including hypertension, cardiac hypertrophy, and visual impairment due to cataract. Three elements of our research program align with this theme. AKAP79/150 complexes underlie aspects of vascular smooth muscle hypertension, AKAP-Lbc networks coordinate pathological cardiomyocyte hypertrophy, and AKAP2 maintains PKA-dependent fluid circulation in the lens of the eye.
- In arterial myocytes, AKAP150-associated PKC has been implicated in recurrent gating of L-type Ca2+ channels (CaV1.2) to enhance vascular tone. AKAP150-/- mice do not develop angiotensin II–induced hypertension, suggesting that local modulation of CaV1.2 channels by AKAP150-associated PKC contributes to changes in blood pressure. Ongoing collaborations with Fernando Santana (University of Washington) show that AKAP150∆PKC knockin mice are resistant to angiotensin II–induced hypertension, whereas AKAP150∆PP2B and AKAP150∆PKA animals are fully responsive to this vasoconstrictive hormone.
- Another hallmark of diabetic progression is myocyte hypertrophy and cardiac remodeling. We have shown that AKAP-Lbc is upregulated in hypertrophic cardiomyocytes. This anchoring protein evokes catecholamine-induced transcriptional activation of the myocyte enhancer factor pathway to initiate transcriptional reprogramming events. Live-cell imaging, fluorescent kinase activity reporters, and RNA interference techniques showed that AKAP-Lbc couples activation of protein kinase D with the phosphorylation-dependent nuclear export of the class II histone deacetylase HDAC5. This revealed that increased expression of AKAP-Lbc amplifies a mitogenic signaling pathway that drives cardiac myocytes toward a pathophysiological outcome. A follow-up study revealed that AKAP-Lbc and the scaffolding protein kinase suppressor of Ras (KSR-1) form the core of a signaling network that efficiently relays mitogenic signals through a three-tier RAF/MEK/ERK1/2 kinase cascade. Thus, AKAP-Lbc has the capacity to coincidently regulate two different aspects of the hypertrophic response.
- High blood sugar in diabetes often causes the lens of the eye to swell and can lead to early-onset cataract. Further lens deterioration can occur as a consequence of defective water and nutrient circulation. A key element of the lens circulatory system is the aquaporin-0 (AQP0) water channel. We have found that products of the AKAP2 gene form a stable complex with AQP0 to sequester PKA with the channel. This permits PKA phosphorylation of Ser235 within a calmodulin (CaM)–binding domain of AQP0 (Figure).
The additional negative charge introduced by phospho-Ser235 perturbs electrostatic interactions between AQP0 and CaM to favor water influx through the channel. In mouse lenses, displacement of PKA from the AKAP2-AQP0 complex restricts water flow because calmodulin binding shuts down the channel. Defective fluid circulation in the lens ensues, and cellular damage results in cortical cataracts. The major implication of our study is that anchored PKA modulation of water channels may be a homeostatic mechanism to maintain lens transparency. These findings also explain why a previously identified mutation in AQP0 that alters the PKA recognition motif for Ser235 phosphorylation is cataractogenic from birth. This new work has been well received because it establishes the first link between anchored PKA phosphorylation of AQP0 and maintenance of lens clarity.
Grants from the National Institutes of Health provided partial support for these projects.
As of July 17, 2014