Jason H. Yang

Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP

Understanding how specific cAMP signals are organized and relayed to their effectors in different compartments of the cell to achieve functional specificity requires molecular tools that allow precise manipulation of cAMP in these compartments. Here we characterize a new method using bicarbonate-activatable and genetically targetable soluble adenylyl cyclase (sAC) to control the location, kinetics and magnitude of the cAMP signal. Using this live-cell cAMP manipulation in conjunction with fluorescence imaging and mechanistic modeling, we uncover the activation of a resident pool of PKA holoenzyme in the nuclei of HEK-293 cells, modifying the existing dogma of cAMP-PKA signaling in the nucleus. Furthermore, we show that phosphodiesterases (PDE) and A-Kinase Anchoring Proteins (AKAP) are critical in shaping nuclear PKA responses. Collectively, our data suggests a new model where AKAP-localized PDEs tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.

Computational Models Reduce Complexity and Accelerate Insight Into Cardiac Signaling Networks

Cardiac signaling networks exhibit considerable complexity in size and connectivity. The intrinsic complexity of these networks complicates the interpretation of experimental findings. This motivates new methods for investigating the mechanisms regulating cardiac signaling networks and the consequences these networks have on cardiac physiology and disease. Next-generation experimental techniques are also generating a wealth of genomic and proteomic data that can be difficult to analyze or interpret. Computational models are poised to play a key role in addressing these challenges. Computational models have a long history in contributing to the understanding of cardiac physiology and are useful for identifying biological mechanisms, inferring multiscale consequences to cell signaling activities and reducing the complexity of large data sets. Models also integrate well with experimental studies to explain experimental observations and generate new hypotheses. Here, we review the contributions computational modeling approaches have made to the analysis of cardiac signaling networks and forecast opportunities for computational models to accelerate cardiac signaling research.

PKA catalytic subunit compartmentation regulates contractile and hypertrophic responses to β-adrenergic signaling.

β-Adrenergic signaling is spatiotemporally heterogeneous in the cardiac myocyte, conferring exquisite control to sympathetic stimulation. Such heterogeneity drives the formation of protein kinase A (PKA) signaling microdomains, which regulate Ca(2+) handling and contractility. Here, we test the hypothesis that the nucleus independently comprises a PKA signaling microdomain regulating myocyte hypertrophy. Spatially-targeted FRET reporters for PKA activity identified slower PKA activation and lower isoproterenol sensitivity in the nucleus (t50=10.6±0.7 min; EC50=89.0 nmol/L) than in the cytosol (t50=3.71±0.25 min; EC50=1.22 nmol/L). These differences were not explained by cAMP or AKAP-based compartmentation. A computational model of cytosolic and nuclear PKA activity was developed and predicted that differences in nuclear PKA dynamics and magnitude are regulated by slow PKA catalytic subunit diffusion, while differences in isoproterenol sensitivity are regulated by nuclear expression of protein kinase inhibitor (PKI). These were validated by FRET and immunofluorescence. The model also predicted differential phosphorylation of PKA substrates regulating cell contractility and hypertrophy. Ca(2+) and cell hypertrophy measurements validated these predictions and identified higher isoproterenol sensitivity for contractile enhancements (EC50=1.84 nmol/L) over cell hypertrophy (EC50=85.9 nmol/L). Over-expression of spatially targeted PKA catalytic subunit to the cytosol or nucleus enhanced contractile and hypertrophic responses, respectively. We conclude that restricted PKA catalytic subunit diffusion is an important PKA compartmentation mechanism and the nucleus comprises a novel PKA signaling microdomain, insulating hypertrophic from contractile β-adrenergic signaling responses.

Phospholemman is a negative feed-forward regulator of Ca2+ in β-adrenergic signaling, accelerating β-adrenergic inotropy

Sympathetic stimulation enhances cardiac contractility by stimulating β-adrenergic signaling and protein kinase A (PKA). Recently, phospholemman (PLM) has emerged as an important PKA substrate capable of regulating cytosolic Ca(2+) transients. However, it remains unclear how PLM contributes to β-adrenergic inotropy. Here we developed a computational model to clarify PLMs role in the β-adrenergic signaling response. Simulating Na(+) and sarcoplasmic reticulum (SR) Ca(2+) clamps, we identify an effect of PLM phosphorylation on SR unloading as the key mechanism by which PLM confers cytosolic Ca(2+) adaptation to long-term β-adrenergic receptor (β-AR) stimulation. Moreover, we show that phospholamban (PLB) opposes and overtakes these actions on SR load, forming a negative feed-forward loop in the β-adrenergic signaling cascade. This network motif dominates the negative feedback conferred by β-AR desensitization and accelerates β-AR-induced inotropy. Model analysis therefore unmasks key actions of PLM phosphorylation during β-adrenergic signaling, indicating that PLM is a critical component of the fight-or-flight response.

Systems Analysis of Small Signaling Modules Relevant to Eight Human Diseases

Using eight newly generated models relevant to addiction, Alzheimer’s disease, cancer, diabetes, HIV, heart disease, malaria, and tuberculosis, we show that systems analysis of small (4–25 species), bounded protein signaling modules rapidly generates new quantitative knowledge from published experimental research. For example, our models show that tumor sclerosis complex (TSC) inhibitors may be more effective than the rapamycin (mTOR) inhibitors currently used to treat cancer, that HIV infection could be more effectively blocked by increasing production of the human innate immune response protein APOBEC3G, rather than targeting HIV’s viral infectivity factor (Vif), and how peroxisome proliferator-activated receptor alpha (PPARα) agonists used to treat dyslipidemia would most effectively stimulate PPARα signaling if drug design were to increase agonist nucleoplasmic concentration, as opposed to increasing agonist binding affinity for PPARα. Comparative analysis of system-level properties for all eight modules showed that a significantly higher proportion of concentration parameters fall in the top 15th percentile sensitivity ranking than binding affinity parameters. In infectious disease modules, host networks were significantly more sensitive to virulence factor concentration parameters compared to all other concentration parameters. This work supports the future use of this approach for informing the next generation of experimental roadmaps for known diseases.

Simulating complicated human birth for research and training

We report on the design, testing and implementation of a novel birthing simulator developed specifically to research the delivery process and improve clinical training in uncommon but inevitable complicated human births. The simulator consists of a maternal model and an instrumented fetal model, used in conjunction with an existing force-sensing system and a data-acquisition system. The maternal model includes a bony, rotatable pelvis, flexible legs, and a uterine expulsive system. The fetal model, which can be delivered repeatedly through the maternal model, is instrumented with potentiometers to measure neck extension, rotation and flexion during delivery. Simulation of the brachial plexus within the model fetal neck allows measurement of stretch in those nerves at risk for injury during difficult deliveries. Wooden elements mimic the properties of neonatal bone and can break either spontaneously or purposely. Two methods for measuring clinician-applied force during simulated deliveries provide trainees with real-time assessment of their own traction force and allow researchers to correlate fetal neck motion and nerve stretch parameters with clinician-applied traction. Preliminary testing indicates the system is biofidelic for the final stages of the birthing process, and can be used for training and research in obstetrics.

Science careers: where does advocacy fit?

In his Presidential address (“A global perspective on science and technology,” 24 October 2008, p. [544][1]), D. Baltimore warned against erosion of U.S. leadership in the biological sciences, acknowledging the entire scientific communitys lack of involvement and personal responsibility in our