Over the tenure of this Merit Award we have built a molecular understanding of the catalytic (C) subunit of cAMP-dependent protein kinase (PKA). Using the crystal structure as a starting point, we have defined the subdomains and conformational states as the enzyme shuttles through its catalytic cycle. We have combined biochemistry with structural studies and over the past five years, in particular, have developed methods that allow us to monitor the dynamic properties of the C-subunit in solution. The resulting description of PKA is the most comprehensive for any protein kinase and has established PKA as a prototype for understanding the entire protein kinase superfamily. We have also come to appreciate that the catalytic subunit is not simply a catalyst but also a scaffold on which other proteins dock. The evolution of the kinase family is very advanced and includes not only conservation of the active site cleft but also the surface which is where the family members display remarkable diversity. How PKA recognizes its inhibitors and how it is activated by cAMP is fundamental to understanding its biological function. During this next granting period, having just solved the structure of an Rlalpha:C complex, we are poised for the first time to understand the molecular basis for cAMP activation of PKA. It reflects the convergence of two major signaling pathways. Our attention will focus on the surface of the large lobe where we will try to achieve a comprehensive and quantitative understanding of how molecular recognition is achieved and how cAMP activates PKA using the extensive Rlalpha:C interface as a template. In addition, we shall map the functional roles and dynamic states of the activation loop, focusing specifically on the structural and functional importance of Thr197 phosphorylation and on the role of Cys199 as a sensor for oxidation. In parallel with our biochemical studies, we shall use solution methods, specifically fluorescence approaches and hydrogen/deuterium exchange coupled with mass spectrometry (H/DMS) to monitor the dynamic features of the enzyme and to comprehensively map the surface. Finally, we shall continue to use crystallography to obtain high resolution structures of the various conformational states that we define by our biological studies.
| Raju, Saravanan; Whalen, Daniel M; Mengistu, Meron et al. (2018) Kinase domain dimerization drives RIPK3-dependent necroptosis. Sci Signal 11: |
| Meng, Yilin; Ahuja, Lalima G; Kornev, Alexandr P et al. (2018) A Catalytically Disabled Double Mutant of Src Tyrosine Kinase Can Be Stabilized into an Active-Like Conformation. J Mol Biol 430:881-889 |
| Meharena, Hiruy S; Fan, Xiaorui; Ahuja, Lalima G et al. (2016) Decoding the Interactions Regulating the Active State Mechanics of Eukaryotic Protein Kinases. PLoS Biol 14:e2000127 |
| Zhang, Ping; Kornev, Alexandr P; Wu, Jian et al. (2015) Discovery of Allostery in PKA Signaling. Biophys Rev 7:227-238 |
| Taylor, Susan S; Shaw, Andrey S; Kannan, Natarajan et al. (2015) Integration of signaling in the kinome: Architecture and regulation of the ?C Helix. Biochim Biophys Acta 1854:1567-74 |
| Bastidas, Adam C; Wu, Jian; Taylor, Susan S (2015) Molecular features of product release for the PKA catalytic cycle. Biochemistry 54:2-10 |
| Hu, Jiancheng; Ahuja, Lalima G; Meharena, Hiruy S et al. (2015) Kinase regulation by hydrophobic spine assembly in cancer. Mol Cell Biol 35:264-76 |
| Niyitegeka, Jean-Marie V; Bastidas, Adam C; Newman, Robert H et al. (2015) Isoform-specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A: significance in acute and chronic kidney injury. Am J Physiol Renal Physiol 308:F56-68 |
| Knape, Matthias J; Ahuja, Lalima G; Bertinetti, Daniela et al. (2015) Divalent Metal Ions Mg²? and Ca²? Have Distinct Effects on Protein Kinase A Activity and Regulation. ACS Chem Biol 10:2303-15 |
| Kornev, Alexandr P; Taylor, Susan S (2015) Dynamics-Driven Allostery in Protein Kinases. Trends Biochem Sci 40:628-647 |
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