Protein kinases are allosteric enzymes involved in cell signaling pathways and many pathological diseases (cancer, diabetes, rheumatoid arthritis, cardiomyopathy, and others). In the first grant period, we laid out the basis for understanding allostery in the catalytic subunit of protein kinase A (PKA-C), a ubiquitous kinase that regulates a plethora of vital cellular functions. Using a combination of NMR, X-ray and an array of other bio- physical techniques, we described the intra-molecular allosteric communication of PKA-C, showing how distal mutations affect substrate recognition and product release. Importantly, we defined the structural and dynamic signatures for activated and inhibited states of the kinase. In a recent breakthrough in our laboratory, we were able to reconstitute the full holoenzyme for NMR studies. We are now poised to study regulation and inhibition of PKA-C at the atomic level. In this competitive renewal, we propose to study the allosteric mechanism of regulation of PKA and its dysfunctional states linked to somatic mutations. Specifically, Davide Calebiro at the University of Wrzburgh (Germany) discovered a series of PKA-C mutations correlated with adenoma-associated Cushing's syndrome (AA-CS). Also, Sandy Simon's laboratory at The Rockefeller University discovered a chimera of PKA-C that is implicated in the progression of the fibrolamellar hepatocellular carcinoma (FLHCC). To study the effects of these mutations, we formed a consortium with Susan Taylor (UCSD), and will collaborate with the Calebiro and Simon groups to elucidate the changes in the intra- and inter-molecular signaling generated by these disease mutations using a combination of NMR spectroscopy, thermodynamics and kinetic methods.
In AIM1, we will probe the role of PKA-C in the assembly of the holoenzyme and how the AA-CS mutants affect the R/C complex.
In AIM2, we will analyze how AA-CS mutations affect the regulation of PKA-C by endogenous and exogenous inhibitors.
In AIM3, we will study the structure and the dynamics of the chimeric construct of PKA-C (PKA-CDNAJB1) to understand how the fused chaperone affects pseudo-substrate recognition and regulation, degenerating into FLHCC. Since the ground states of these mutants are identical, the different functions must be encoded in the excited states that only NMR spectroscopy can unveil at the atomic resolution. Understanding the changes in the kinase energy landscape caused by these mutations is key to the elucidation of the molecular mechanisms leading to disease. This information will help the design of new strategies to control kinase activity for innovative therapies.
Phosphorylation is a universal mechanism of cell signaling. Protein kinase A (PKA) cascades are ubiquitous and regulate a plethora of biological events. Mutations in PKA have been linked to dysfunctional phosphorylation, leading to adenoma-associated Cushing's syndrome and fibrolamellar hepatocellular carcinoma. We propose to use a combination of biophysical and biochemical approaches to characterize the dysfunctional behavior of PKA mutants and their aberrant regulation. Understanding how mutations affect allosteric signals and achieving a control of their function will pave the way for innovative therapies to treat these diseases.
|Dahlin, Jayme L; Cuellar, Matthew; Singh, Gurpreet et al. (2018) ALARM NMR for HTS triage and chemical probe validation. Curr Protoc Chem Biol 10:91-117|
|Olivieri, Cristina; Subrahmanian, Manu Veliparambil; Xia, Youlin et al. (2018) Simultaneous detection of intra- and inter-molecular paramagnetic relaxation enhancements in protein complexes. J Biomol NMR 70:133-140|
|Tomasini, Michael D; Wang, Yingjie; Karamafrooz, Adak et al. (2018) Conformational Landscape of the PRKACA-DNAJB1 Chimeric Kinase, the Driver for Fibrolamellar Hepatocellular Carcinoma. Sci Rep 8:720|
|Larsen, Erik K; Olivieri, Cristina; Walker, Caitlin et al. (2018) Probing Protein-Protein Interactions Using Asymmetric Labeling and Carbonyl-Carbon Selective Heteronuclear NMR Spectroscopy. Molecules 23:|
|Wang, Songlin; Gopinath, T; Veglia, Gianluigi (2018) Application of paramagnetic relaxation enhancements to accelerate the acquisition of 2D and 3D solid-state NMR spectra of oriented membrane proteins. Methods 138-139:54-61|
|Xia, Youlin; Rossi, Paolo; Tonelli, Marco et al. (2017) Optimization of 1H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments. J Biomol NMR 69:45-52|
|Xia, Youlin; Rossi, Paolo; Subrahmanian, Manu V et al. (2017) Enhancing the sensitivity of multidimensional NMR experiments by using triply-compensated ? pulses. J Biomol NMR 69:237-243|
|Kim, Jonggul; Ahuja, Lalima G; Chao, Fa-An et al. (2017) A dynamic hydrophobic core orchestrates allostery in protein kinases. Sci Adv 3:e1600663|
|Ahuja, Lalima G; Kornev, Alexandr P; McClendon, Christopher L et al. (2017) Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer. Proc Natl Acad Sci U S A 114:E931-E940|
|Kim, Jonggul; Li, Geoffrey; Walters, Michael A et al. (2016) Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A. Structure 24:353-63|
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