Analysis of Protein Kinase Autophosphorylation Using Expressed Protein Ligation and Computational Modeling

Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, United States
Journal of the American Chemical Society (Impact Factor: 12.11). 05/2008; 130(17):5667-9. DOI: 10.1021/ja711244h
Source: PubMed


Protein kinases represent a family of enzymes that are critical in cell signaling. One mechanism by which protein kinases are regulated is via autophosphorylation. In the studies described here, we have examined the mechanism of autophosphosphorylation at serine 338 in the regulation of protein kinase A (PKA). Expressed protein ligation allowed for the covalent linkage of an ATP moiety to a Ser mimic at this phosphorylation site. Using a combination of size exclusion chromatography, fluorescence nucleotide binding, kinase measurements, and limited proteolysis assays on this semisynthetic ATP-linked protein, we have obtained unique evidence for an intramolecular autophosphorylation mechanism in PKA regulation. Computational analysis provided a plausible model for a PKA conformation consistent with intramolecular phosphoryl transfer. This approach could be applied to other autoprocessing enzymes by exploiting appropriate transition state analogue motifs in the context of protein semisynthesis.

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Available from: Sidhartha Chaudhury, Jan 10, 2014
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