Our overall objectives are to continue characterizing functional sites that are associated with both the regulatory and catalytic subunits of cAMP-dependent protein kinase. In conjunction with this, a major priority will be to solve the crystal structure of the C-subunit. The homology of the C-subunit with other kinases, in particular oncogenes and growth hormone receptors, makes this objective particularily significant for this will provide a valuable framework for viewing this larger family of protein kinases. A further long range objective will also be to solve the crystal structure of one of the R-subunits. Our specific goals for the catalytic subunit are several fold. We shall use several approaches to further map active site regions. Affinity labeling with 8-N3-ATP will be used to characterize the adenine portion of the ATP binding site in the free C-subunit and in the type I holoenzyme which has a high affinity for ATP. Inhibition with DCCD will be used as a probe for identifying a carboxylic acid group that appears to be important for catalysis. In addition, the in vivo and in vitro phosphorylation sites associated with the C-subunit will be further characterized and correlated with enzymatic activity as well as with protein synthesis. C-subunit that has been labeled with a fluorescent, sulfhydryl-specific reagent, monobromobimane, will be used to characterize R-C interactions.
Our aims for the R-subunits are also several-fold. The cAMP binding sites will be further mapped with affinity analogs of cAMP that are derivatized at the N6 and/or C2 positions and have a preference for site A. DCCD will be used to identify essential carboxylic acid residues that are conformation dependent. Specific interchain disulfide bonds will be identified in order to map specific points of contact between the 2 protomers in the R-dimer. The induction of neural R(II) in PC12 cells will be followed with neural-specific monoclonal antibodies. Finally, the sequencing of the antigenic domains of RI, R(II) heart, and R(II) brain will be completed.
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| 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 |
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| 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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| 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 |
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